The bh TCSPC Handbook
Time-Correlated Single Photon Counting Modules
SPC-130 EM SPC-134 EM
Detectors and peripheral devices
SPCImage Data Analysis
Dr. Wolfgang Becker
Tel. +49 / 30 / 787 56 32
FAX +49 / 30 / 787 57 34
Becker & Hickl GmbH
Nunsdorfer Ring 7-9
Tel. +49 / 30 / 787 56 32
FAX +49 / 30 / 787 57 34
7th Edition, September 2017
This handbook is subject to copyright. However, reproduction of small portions of the material in scientific papers or other non-commercial publications is considered fair use under the copyright law. It is requested that a complete citation be included in the publication. If you require confirmation please feel free to contact Becker & Hickl GmbH.
Time-correlated single photon counting (TCSPC) is an amazingly sensitive technique for recording low-level light signals with picosecond resolution and extremely high precision. TCSPC originates from the measurement of excited nuclear states and has been used since the late 60s [711, 1123]. For many years TCSPC was used primarily to record fluorescence decay curves of organic dyes in solution. Due to the low intensity and low repetition rate of the light sources and the limited speed of the electronics of the 70s and 80s the acquisition times were extremely long. More important, classic TCSPC was intrinsically one-dimensional, i.e. limited to the recording of the waveform of a periodic light signal.
Light sources ceased to be a limitation when the first mode-locked Argon lasers and synchronously pumped dye lasers were introduced. For the recording electronics, the situation changed with the introduction of the SPC-300 modules of Becker & Hickl in 1993. Due to a new analog-to-digital conversion principle these modules could be used at photon count rates almost 100 times higher than the classic TCSPC devices. Moreover, the modules were able to record the photons of a large number of detectors simultaneously. They were thus able to record a photon distribution not only versus the time in a fluorescence decay but also versus a spatial coordinate or the wavelength of the photons. Multi-dimensional TCSPC was born.
Within a few years, more dimensions were added to multidimensional TCSPC. Fast sequential recording was introduced with the SPC-430 in 1995, fast scanning with the SPC-535 in 1997. Time-tag recording was introduced with the SPC-431 in 1996; multi-module TCSPC systems followed in 1999. Since then, the Becker & Hickl TCSPC systems became bigger, faster and more flexible. Recent TCSPC modules, like the SPC-830, can be configured for sequential recording, imaging, or time-tag recording by a simple software command. Multi-module systems, like the SPC-134 and SPC‑154, can be used for scanning at unprecedented count rates and acquisition speeds.
Nevertheless, TCSPC still has the reputation to be an extremely sluggish technique unable to record any fast changes in the fluorescence or scattering behaviour of a sample. The multi-dimensional features of modern TCSPC are not commonly understood. Thus, many users do not make efficient use of their SPC modules. However, if appropriately used, multi-dimensional TCSPC techniques not only deliver superior results but also solve highly sophisticated measurement problems.
This handbook is an attempt to help existing and potential users understand and make use of the advanced features of modern TCSPC.
After an introduction into the bh TCSPC devices and associated detector, laser, and experiment control modules the principles of advanced TCSPC techniques are described. These include multi-detector TCSPC, multiplexed TCSPC, sequential recording techniques, scanning techniques, parameter-tag recording, and multi-module TCSPC techniques. The next chapter describes the architecture of the bh SPC modules. A chapter about detectors gives a review of detector principles and of the parameters used to characterise detectors. It describes a number of detectors commonly used for TCSPC and gives advice about obtaining best performance from them.
The implementation of bh SPC devices is described in the next part of the handbook. It includes principles and wiring diagrams for typical experiments, guidelines for first system setup, and advice for system optimisation. It describes dead-time, counting loss, and pile-up effects, detector effects, and effects related to the optical system.
The next chapter of the handbook is dedicated to TCSPC applications. The first part of this chapter describes the measurement of fluorescence and anisotropy decay curves, multi-spectral lifetime experiments, recording of transient fluorescence lifetime phenomena, and measurements of phosphorescence decay curves. The second part of the chapter is dedicated to time-resolved laser scanning microscopy. It contains sections on a wide variety of fluorescence-lifetime imaging (FLIM) experiments and procedures, such as FLIM with various excitation principles, excitation sources, and detection principles, high-speed and time-series FLIM, Z-stack FLIM, simultaneous fluorescence and phosphorescence lifetime imaging (FLIM/PLIM), fluorescence lifetime-transient scanning (FLITS), and FLIM with special microscope configurations. A third part contains FLIM background knowledge: Signal-to-noise ratio, acquisition time, the effect of counting loss and pile-up, photobleaching, and fluorescence depolarisation on the recorded data.
The book contains a large chapter on TCSPC applications, most of them in Biology. It contains sections on FLIM of molecular environment parameters in tissue, FLIM-based FRET measurements in cells, autofluorescence FLIM of biological tissue, plant physiology, and clinical FLIM applications. A section about diffuse optical tomography (DOT) by NIRS techniques includes breast imaging, static and functional brain imaging, perfusion measurement in the human brain, diffuse tissue spectroscopy, and small-animal imaging. Picosecond photon correlation, fluorescence correlation spectroscopy, burst-integrated fluorescence lifetime techniques, and photon counting histogram techniques are reviewed in the next sections. The last part of the application chapter gives an review of non-biological TCSPC applications like positron lifetime measurement, measurement of barrier discharges, remote sensing, metrological applications, and characterisation of detectors. The application chapter also includes practical hints about optical systems, detectors, and other technical aspects of the applications described.
Another large chapter describes the SPCM operating software of the bh SPC modules. It describes the various user interface configurations, operation modes, the system and control parameters, the handling and display of the multidimensional data recorded by the modules, and the associated data file structure.
The current edition also contains a chapter on the SPCImage fluorescence decay and FLIM data analysis software. It describes the general principles of fluorescence decay analysis, the calculation of fluorescence decay parameters and lifetime images by various decay models, pseudo-global analysis, multi-wavelength FLIM analysis, batch-processing of FLIM series, and analysis of PLIM data.
The handbook ends with a list of more than 1100 references of publications related to TCSPC, most of them being applications of the bh SPC devices.
Overview about bh TCSPC Instrumentation
General Features of the bh TCSPC Devices
TSPC Module Types
Simple-Tau TCSPC Systems
Simple-Tau II System
Power-Tau TCSPC Systems
Other Time-Resolved Photon Counting Devices
Measurement Modes of the bh TCSPC Modules
bh DCS‑120 Confocal Scanning FLIM Systems
FLIM Systems for Zeiss LSM 510 NLO Multiphoton Microscopes
FLIM Systems for Zeiss LSM 710, 780, 880 Family Microscopes
FLIM Systems for Leica SP2, SP5, and SP8 MP Laser Scanning Microscopes
FLIM Systems for Olympus FV 300 and FV 1000
FLIM Systems for Nikon C1, A1, A2 Microscopes
FLIM Systems for Sutter MOM Microscopes
FLIM Systems for Abberior STED System
PZ-FLIM-110 Piezo-Scanning FLIM System
WF-FLIM-101 Wide-Field FLIM System
Picosecond Diode Lasers
Laser Control Electronics
SPCM 64-bit Instrument Software
Data Analysis Software
SPCImage FLIM Data Analysis
Burst Analyzer Single-Molecule Data Analysis
Classic Time-Correlated Single Photon Counting
Principle of Time-Correlated Single Photon Counting
The Classic TCSPC Architecture
Principle of Data Acquisition
Architecture of Multi-Dimensional TCSPC
Multi-Dimensional TCSPC Implementations
Multi-Detector and Multi-Wavelength TCSPC
Fluorescence Lifetime Imaging (FLIM)
FLIM with Excitation Wavelength Multiplexing
Phosphorescence Lifetime Imaging (PLIM)
Recording Parameter-Tagged Photon Data
Fluorescence Correlation Spectroscopy (FCS)
Calculation of FCS from Parameter-Tag data
Using the Micro-Time of TCSPC in FCS
Single-Molecule Burst Analysis
Multi-Module TCSPC Systems
Architecture of the bh TCSPC Modules
CFD and SYNC circuits
Detailed Description of Building Blocks
CFD in the Detector (Start) Channel
CFD in the Synchronisation (Stop) Channel
ADC Error Correction
Variable ADC Resolution
Detectors for TCSPC
Channel and Microchannel PMTs
Single-Photon Avalanche Photodiodes
Single Electron Response
Pulse Height Distribution
Signal Transit Time
Transit Time Spread and Timing Jitter
Dark Count Rate
Description of Selected Detectors
R3809U MCP PMTs
Hamamatsu R5600 and R7400 Miniature PMTs
Hamamatsu H5783 and H5773 Photosensor Modules
H11900 / H11901 and H10720 / H10721 Photosensor Modules
PMH-100, PMC-100, PMZ‑100
PMC-150 PMT Modules
Hamamatsu R10467 Hybrid PMT
HPM‑100 Hybrid Detector Modules
Detection Efficiency Comparison
PML-16C and PML‑16C GaAsP Multichannel Detector Heads
Single-Photon Avalanche Photodiodes
SPAD-8 Eight-Channel SPAD Module
Id 220 InGaAs SPAD
id 230 InGaAs SPAD
Superconducting NbN Detectors
Preamplifiers and Detector Control
The DCC-100 Detector Controller
Safety Recommendations for Using Detectors
Installation of the bh TCSPC Modules
Computer and Operating System
Installing DLL and Lab View Libraries
Installing New Software Components
Removing the TCSPC Package
Hardware Installation - Single SPC Modules
Hardware Installation - Multi-Module Systems
Module Test Program
Starting the SPC Software without an SPC Module
Implementation of the bh TCSPC Technique
The Classic TCSPC Implementation
Synchronisation to Random Signals
Using the Experiment Trigger
Resolving Cable Conflicts
Cable Diagrams of FLIM Systems
DCS-120 Confocal Scanning FLIM System
DCS-120 System Connections
Setup and Training Service
Multi-Wavelength Systems - F(xyt) Mode
Multi-Wavelength FLIM Systems
Multi-Wavelength FLIM by Mosaic FLIM Function
Setup Procedure for Other TCSPC Applications
Detector Operating Conditions
CFD and SYNC Inputs
DCC Gain and CFD Threshold
Dependence of the Time Resolution on the Detector Gain
Maximum Count Rate of the Detector
CFD Zero Cross Level
Detector Operation at High CFD Threshold
Adjusting the SYNC and CFD Signal Path Length
Avoiding Pile-Up Distortions
Multiplexing of Lasers
Optimising a Photomultiplier
Dark Count Rate
Checking the SER of PMTs
Quick Test of a PMT
Absorptive Colour Filters
Monochromators and Polychromators
Baffles and Aperture Stops
Avoiding Optical Reflections
Re-Absorption and Re-Emission
Non-Scanning TCSPC Applications
Measurement of Luminescence Decay Curves
Recording Fluorescence Decay Curves with a FLIM System
Multi-Spectral Lifetime Experiments
Multi-Wavelength Tissue Lifetime Spectrometers
Fibre-Based TCSPC Systems
Multi-Wavelength Micro Spectrometers
Measurement of Time-Resolved Fluorescence Anisotropy
Transient Fluorescence Phenomena
Stopped Flow Techniques
Continuous Flow Mixing Techniques
Simultaneous Measurement of Fluorescence and Phosphorescence Decay
Time-Resolved Laser Scanning Microscopy
The Advantage of Scanning
The Laser Scanning Microscope
Picosecond Diode Lasers
Confocal FLIM with Diode Laser Excitation
Confocal FLIM with Tuneable-Excitation
Multiphoton NDD FLIM
Live Cell and Tissue Imaging
NADH FLIM with Ultra-Fast Detectors
Confocal Multispectral FLIM
Multiphoton Multispectral NDD FLIM
Applications of Multi-Spectral FLIM
Excitation Wavelength Multiplexing
Confocal NIR FLIM
NIR Multiphoton NDD FLIM with OPO Excitation
High-Speed Parallel-Channel FLIM
Z Stack FLIM
Z Stack FLIM by Record-and-Save Procedure
Z Stack FLIM by Mosaic Recording
Temporal Mosaic FLIM
FLITS: Fluorescence Lifetime-Transient Scanning
Ca2+ Transients in Neurons
Fast Online FLIM
Linearisation of the FLIM Intensity Scale
PLIM / FLIM: Simultaneous Phosphorescence and Fluorescence Lifetime Imaging
FLIM with Special Scanning Techniques
FLIM of Macroscopic Objects
Imaging in the Primary Image Plane of a Scanner
Combination of Beam Scanning with a Motorised Sample Stage
Scanning through Endoscopes
Multi-Beam Scanning Techniques
Scanning Systems with Piezo Stages
Stimulated Emission Depletion Microscopy (STED)
Time-Resolved Optical Near-Field Microscopy (NSOM)
Spatial and Temporal Resolution
FLIM Background Knowledge
What is important to FLIM of Biological Objects?
Signal-to-Noise Ratio of FLIM
Why does FLIM need more photons than ‘normal’ imaging?
Acquisition Time of TCSPC FLIM
Counting Loss and Pile-Up Effects
Binning of Decay Data
Multi-Exponential Decay Functions
Effect of Fluorescence Depolarisation on Measured Decay Curves
Biological FLIM Applications
Why use the Fluorescence Lifetime?
Measurement of Local Environment Parameters
Other Environment Parameters
Förster Resonance Energy Transfer (FRET)
Single-Exponential FLIM FRET
Double-exponential FLIM FRET
Using information from the acceptor decay function
FRET between Endogenous Fluorophores
Practical Hints for FRET Measurements
Autofluorescence FLIM of Cells and Tissue
NAD(P)H and FAD Lifetime Imaging
Review of NAD(P)H / FAD FLIM Literature
Unmixing NAD(P)H / FAD Fluorescence Components
Two-Photon FLIM of Mammalian Skin
FLIM of Other Endogenous Fluorophores
Diffusion of Nanoparticles in Skin
Autofluorescence of the Ocular Fundus
Oxygen Sensing by Phosphorescence Lifetime Measurement
Oxygen Sensing by PLIM
Simultaneous Recording of Oxygen and NAD(P)H Images
Delayed Fluorescence of PpIX
Other FLIM Applications
Clinical Applications of FLIM
Multiphoton Tomography of Human Skin
Heidelberg Engineering FLIO System
Results and Applications
Diffuse Optical Tomography: DOT, NIRS and fNIRS
Static Brain Imaging
Functional Brain Imaging
Perfusion Measurements by ICG Boli
Perfusion Measurement by Diffuse Optical Correlation (DCS)
DOT Experiments at Small Source-Detector Distance
Fibre-Based DOT Endoscopes
Fibre-Based System for Brain Activation Measurement in Mice
Tissue Scanning System with Laser Wavelength Multiplexing
Muscle and Bone Studies
Fluorescence Lifetime Detection in DOT
Technical Issues of DOT
Picosecond Photon Correlation
Fluorescence Correlation Spectroscopy
Calculation of FCS curves from the TCSPC data
Using Micro-Time Information in Combination with FCS
FCS System Parameter Setup
FCS with Laser Scanning Microscopes
FCS with the LSM 510
Zeiss LSM 710 / 780 / 880
Zeiss LSM 880
FCS with the DCS‑120 confocal FLIM System
FCS in Live Cells
Time-Resolved Single-Molecule Spectroscopy
Burst-Integrated Fluorescence Lifetime (BIFL) Techniques
Multi-Parameter Single-Molecule Burst Analysis
Single-Molecule FRET Experiments
Identification of Single Molecules
Correlation of Lifetime Fluctuations
Photon Counting Histograms
Other TCSPC Applications
Two-Photon Excitation by Picosecond Diode Lasers
Scanning with Galvanometer Mirror
Positron Lifetime Measurements
Characterisation of Detectors
Spatially Resolved Characterisation of SPADs
Light emission from SPADs
Measurement of Absolute Detector Quantum Efficiency
Photon-Number Sensitive TCSPC
Configuration of the SPC Main Panel
Changing Between Different Instrument Configurations
Display and Trace Parameters
Resizing and Positioning the Display Windows
Cursors in the Display Windows of the Main Panel
System Parameter Settings
Module Select (Multi-SPC Systems)
Data Export and Import: Convert Functions
Link to SPCImage Data Analysis
Continuous Flow Mode
Scan Sync Out Mode
Sequential Recording in the Scan Sync Out mode
Scan Sync In Mode
Scan XY Out Mode (SPC-700/730 only)
FIFO Imaging Mode
Display of FIFO Imaging Data
Delay Correction for 16-Channel Detectors
Control Parameters (Photon Distribution Modes)
Stop Condition and Overflow Handling
Cycles and Autosave
Display after each step and each cycle
Add / Sub Signal
Measurement Timing Control Parameters
Dead Time Compensation
Parameter Management for Multi-Module SPC Systems
Trigger Master and Clock Master Function
General Display Parameters
2D Display Parameters
3D Display Parameters
Displaying Subsets of Multidimensional Data
Display Parameters for Different Display Windows
2D Trace Parameters
Trace Parameters for 2D Curve Mode
Trace Parameters for 2D Block Mode
Export of Trace Data
3D Trace Parameters
Routing X and Y Windows
Scan X and Y Windows
Auto Set Functions
2D Data Processing
Display of Multi-Dimensional Data
Cursors and Zoom Function
3D Data Processing
Starting and Stopping a Measurement
Operation of Experiment Control Devices from the SPCM Software
Scanner Control via the GVD‑120 Card
DCC-100 Detector Controller Card
Control of the Zeiss Axio Observer Z1
DB-32 SYNC delay box
DDG‑210 Pulse Generator Card
Ti:Sa Laser and AOM Control
SPCImage Data Analysis Software
Analysing FLIM Data with SPCImage
SPCImage Software Panel
Loading of FLIM Data
Calculating the Lifetime Image
Saving the Data
Export of Data
Analysing Single-Curve Data
Initial ‘Hot Spot’ Selection
Region of Interest (ROI) Selection
Lines of Interest
Fit Definition Parameters
General Model Parameters
Model Options and Fit-Control Parameters
How Many Exponential Components Are Needed?
Calculation of Multi-Wavelength Lifetime Images
Mosaic FLIM Data
Analysis of Phosphorescence Lifetime Images
Display of Lifetime Images
Selection of Decay Parameter
Instrument Response Function
Synthetic IRF: Calculating an IRF from fluorescence decay data
IRF from scattering data
IRF from SHG data
Two-Dimensional Histograms of Decay Parameters
TCSPC Data file structure
Data of the Photon Distribution Modes
Measurement Description Blocks
FIFO Data Files (SPC-600/630, 4096 Channel Mode)
FIFO Data Files (SPC-600/630 256 Channel Mode)
FIFO Data Files (SPC-130, SPC-140, SPC‑150, SPC-160, SPC-830)
How to Avoid Damage
Testing the Module by the SPC Test Program
Test for General Function and for Differential Nonlinearity
Test of Time Resolution
Frequently Encountered Problems
Assistance through bh
Routing and Control Signals
SPC-700/730 and SPC-830
SPC‑130EM/134EM, SPC‑140, SPC-144, SPC‑150, SPC‑154, SPC-160
DCC‑100 Detector Controller
SPC-130EM / 134EM
DCC-100 Detector Controller
Absolute Maximum Ratings (for all SPC modules)
This handbook applies to the bh SPC‑130/134, SPC‑130EM/134EM, SPC‑150/154, SPC‑150N, SPC-150NX, SPC-160, and SPC‑830 time-correlated single photon counting modules, to the bh Simple-Tau TCSPC systems, and their combination with detectors, experiment control modules, picosecond diode lasers, and optical systems provided by bh. General information provided in this book applies also to older SPC modules, such as the SPC-730 or the SPC-630.
The bh SPC modules are using a multi-dimensional TCSPC process. Unlike classic TCSPC devices, they record photon distributions not only over the time in the excitation pulse period, but also over additional parameters that can be associated to the individual photons. This can be the wavelength of the photons, the spatial location where they come from, the time from the start of an experiment, the time within the period of a stimulation of the sample, the time within the period of a modulation of the excitation laser, or any other parameters that are determined during the recording process. As a result, the bh SPC modules can be used for the classic fluorescence decay applications as well as for fluorescence lifetime imaging (FLIM), multi-wavelength fluorescence decay measurement, multi-wavelength FLIM, simultaneous fluorescence and phosphorescence decay measurement, simultaneous FLIM and PLIM, fast time-series fluorescence decay recording, fast time-series FLIM, fluorescence correlation (FCS), single-molecules experiments, anti-bunching experiments, and many other multi-dimensional photon recording tasks.
The bh TCSPC devices use a high-speed high-resolution TAC/ADC principle. The internal timing jitter is on the order of 2 to 3 ps (rms) for the standard modules, and 1.6 ps (rms) for the SPC-150NX boards, see Fig. 1. The minimum time channel width for the standard boards is 810 fs, and 405 fs for the SPC-150NX board. The bh TCSPC modules deliver a far better time resolution for fast detectors than any other TCSPC product. The full-width-half-maximum instrument response width for fast hybrid detectors, MCP PMTs and superconducting NbN detectors is in the sub-20 ps range. A few examples are shown in Fig. 2.
Fig. 1: Electrical time resolution of SPC-150 (left) and SPC-150NX (right)
Fig. 2: Instrument response functions for fast hybrid detector, MCP PMT, and superconducting NbN detector.
FLIM images can be recorded at a maximum data size of 4096 x 4095 pixels x 64 time channels, 2048 x248 pixels x 256 time channels, or 1024 x 1024 pixels x 1024 time channels. Several such images can be recorded in parallel. An example is shown in Fig. 3.
Fig. 3: FLIM images with 2048 x 2048 pixels and 256 time channels per pixel, recorded by a dual-channel SPC‑160pcie system.
The bh SPC modules contain complete electronics for recording fast light signals by multi-dimensional TCSPC on single PC boards. All the building blocks, such as constant-fraction discriminators, time-to-amplitude converter, analog-digital converter, data processing logics, data memory and data buffer, and the associated control circuitry are integrated.
Fig. 4: SPC-150N TCSPC/FLIM module. The complete electronics for recording multi-dimensional TCSPC data is integrated on a single circuit board.
Up to four bh TCSPC modules of similar type can be operated in parallel and controlled by the device software. Up to eight modules can be operated in an extension box connected to one computer.
Decay curves, multi-wavelength decay data, FCS curves, photon counting histograms, intensity images, and FLIM and PLIM images are displayed online. Intermediate results are displayed in programmable time intervals. For multi-dimensional data two-dimensional sub-sets can be defined and displayed during the measurement.
The boards have saturated count rates on the order of 10 to 12.5 MHz, and can be used at count rates in excess of 5×106 photons/s. Thus, fluorescence decay curves or other optical waveforms can be recorded at acquisition times in the ms range. The speed can further be increased by operating several TCSPC modules in parallel. This way, saturated count rates of 40 MHz can be reached; experiments can be run at count rates of 20 MHz.
The boards can be operated both in a hardware accumulation mode, in a software accumulation mode, or in a single-photon parameter-tag mode. In the hardware-accumulation mode, the boards build up photon distributions directly in the device memory. These can be single fluorescence decay curves or other optical waveforms, anti-bunching curves, multi-wavelength decay data, time-series of decay curves, or FLIM data. Since the data acquisition occurs in the hardware of the TCSPC module the recording process does not require any computer interaction during the measurement. Photons can thus be recorded up to a sustained count rate of up to 10 MHz per SPC module, independently of the speed of the system computer.
In the software accumulation mode, the photon data are transferred into the system computer photon by photon, and the photon distributions are built up by software. Due to the large memory space available, large photon distributions can be built up, such as FLIM images with 2048x2048 pixels and 256 time channels per pixel. The large memory space is also an advantage when complex photon distributions are built up, for example for spatial or temporal mosaic FLIM, fast FLIM time series, or multi-wavelength FLIM. The software accumulation mode is also used to record FCS, combined FLIM/PLIM, or photon counting histograms. Please see section 'Multi-Dimensional TCSPC' for an overview.
In the software accumulation modes, parameter-tagged single-photon data can be stored instead or in parallel with the build-up of photon distributions. Such data are used especially for single-molecule spectroscopy. The transit of single molecules through a laser focus can be identified in the data, spectroscopic information be derived from the photon data of the individual molecules, and the properties of the molecules be statistically analysed.
bh TCSPC systems have a high degree of modularity. By combining TCSPC modules, various experiment control modules, detectors, bh picosecond and CW diode lasers, titanium-sapphire lasers, super-continuum lasers, motorised sample stages, optical scanners, microscopes, and other optical components time-resolved optical recording systems for a wide range of experiment tasks are feasible. bh deliver complete one-photon and multiphoton FLIM laser scanning microscopes, FLIM systems for laser scanning microscopes of a wide variety of manufacturers, scanning systems for macroscopic FLIM systems, fibre-based systems for metabolic measurements in animals, and sub-systems for diffuse near-infrared imaging (NIRS and fNIRS).
All functions of the SPC modules and of peripheral devices are controlled by 64-bit SPCM ‘Multi SPC Software’. The software provides functions such as measurement control, online calculation and display of data, set-up of measurement parameters, 2‑dimensional and 3‑dimensional display of measurement results, mathematical operations, selection of subsets from multi-dimensional data sets, loading and saving of results and system parameters, control of the measurement in the selected operation mode. The SPCM software includes also the control of the bh DCS‑120 confocal scanning FLIM system, resulting in a seamless interaction of TCSPC operation and scanning. The 64-bit SPCM Software runs under Windows 7 and Windows 10. It is able to control up to four TCSPC modules simultaneously.
The SPCM software has a direct link to the SPCImage Fluorescence decay and FLIM data analysis. SPCImage calculates fluorescence decay parameters and lifetime images by various decay models. It runs analysis of single fluorescence decay curves, analysis of single- and multi-wavelength FLIM data, batch-processing of FLIM time series an FLIM Z stacks, and analysis of PLIM data.
Due to their superior time resolution all bh TCSPC devices are an excellent choice for the traditional fluorescence lifetime experiments. However, their true strength are advanced applications, such as multi-spectral fluorescence lifetime spectroscopy, single- and multi-wavelength FLIM, simultaneous FLIM and PLIM of cell metabolism and oxygen partial pressure, fast sequential recording of time-of-flight distributions in diffuse optical tomography, measurement of transient fluorescence lifetime effects in biological systems, combined fluorescence correlation (FCS) and lifetime spectroscopy, and spectroscopy of single molecules, quantum dots and semiconductor nano-structures by FCS, FIDA, MFD, and BIFL techniques.
The SPC‑130 modules are optimised for small size and low price. The SPC-130 targets on basic fluorescence lifetime applications, and on applications requiring a large number of parallel TCSPC channels.
Fig. 5: SPC-130 board
The boards have all standard photon distribution modes, the continuous-flow mode, and the FIFO (time-tag) mode implemented. However, the boards have no imaging modes, and very limited routing capability. The SPC‑130 board can thus be used for traditional fluorescence lifetime experiments, diffuse optical tomography, stopped flow experiments, single molecule detection and combined FCS/lifetime experiments. The SPC-130 has especially overvoltage-hardened signal inputs. They are reliable and rugged, and can be used even under harsh conditions. When ordered in large quantities for OEM applications, the bh SPC‑130 modules offer unprecedented price-performance ratio.
The SPC‑130 EM module is an improved version of the SPC-130. The SPC-130 EM has the same set of functions as the SPC‑130, but it got the faster bus interface, the large memory, and the full routing capability of the SPC‑150. This is an advantage especially for high-throughput applications, such as dynamic brain imaging in diffuse optical tomography, or fluorescence correlation and BIFL measurements in single-molecule spectroscopy.
Fig. 6: SPC-130 EM board
The SPC‑150 modules have a large internal memory and a fast bus interface, combined with a large set of functions and operating modes.
Fig. 7: SPC-150 board
The SPC‑150 board can be used for traditional fluorescence lifetime experiments, diffuse optical tomography, stopped flow experiments, single molecule spectroscopy, FCS and FCCS recording, FLIM, Mosaic FLIM, FLITS, and combined FLIM / PLIM. One or more SPC-150 modules are used the bh DCS-120 confocal FLIM system. They are also the basis of the standard FLIM systems for the Zeiss LSM 880, 780, 710 and 510 microscopes and various other confocal and multiphoton laser scanning microscopes.
The SPC‑150 N differ from the SPC‑150 in that they have ultra-fast discriminators in the detector- and synchronisation inputs. This allows the modules to obtain superior time resolution with detectors that both have low transit time spread and an extremely fast single-photon response. They are recommended especially for superconducting detectors, MCP PMTs, and fast hybrid detectors with bi-alkali or multi-alkali cathodes.
Fig. 8: SPC-150 N board
The SPC‑150 NX is a resolution-enhanced version of the SPC-150N. The minimum time-channel width is 405 fs in comparison to 810 fs for the SPC-150 and SPC-150N. The SPC-150NX is recommended especially for applications that require ultimate time resolution from superconducting detectors, MCP PMTs, and fast hybrid detectors. The SPC-150NX achieves sub-20 ps (full-width-half maximum) IRF width with these detectors.
Fig. 9: SPC-150 NX board
The SPC‑160 modules have a large internal memory and a fast bus interface, combined with the largest set of functions and operating modes of all bh TCSPC modules. The SPC‑160 modules have the fast discriminators of the SPC‑150 N. Moreover, they have an additional parallel photon counting channel in the FIFO Imaging (FLIM) mode. The 160 modules are thus able to record intensity images up to a detector peak count rate of about 100 MHz. Intensity information from the parallel counter channel and lifetime information from the TCSPC imaging channel are combined to obtain FLIM images that have a linear intensity scale up to extremely high count rates.
Fig. 10: SPC-160 board
As the SPC‑150 and SPC‑150 N, the SPC‑160 modules can be used for traditional fluorescence lifetime experiments, diffuse optical tomography, stopped flow experiments, single molecule spectroscopy, FCS and FCCS recording, FLIM, multi-wavelength FLIM, Mosaic FLIM, and simultaneous FLIM / PLIM.
The SPC-160pcie is a PCI-Express-bus version of the SPC-160. The range of functions and operation modes is the same as for the SPC-160. Together with the GVD-120pcie scan controller and the DCC-120pcie detector controller a complete set of PCI-Express cards for FLIM systems is available.
Fig. 11: SPC-160pcie (left), shown together with DCC-120pcie (middle) and GVD-120pcie (right)
All SPC modules are available as two-channel, three-channel, and four-channel packages. The devices are used in applications where several optical signals have to be recorded simultaneously, and maximum throughput is required. Typical examples are bh FLIM systems, which normally have two parallel SPC channels. Three- and four-channel systems are commonly used in near-infrared diffuse optical imaging applications.
Fig. 12: SPC-134 (left) and SPC-154 (right) four-channel TCSPC packages
There is a number of SPC modules that are still in use in large quantities but which are not recommended for new systems.
The SPC-830 module was the first solution to the complete range from the traditional fluorescence lifetime and anisotropy experiments, photon correlation experiments, confocal and two-photon fluorescence lifetime microscopy, single molecule lifetime, anisotropy and fluorescence correlation, cross-correlation, down to burst-integrated lifetime experiments. The SPC-830 module is still an active product. In special applications (such as ultra-fast scanning) it even has advantages over the SPC-150 or SPC-160 modules because it has an extremely large on-board memory.
Fig. 13: SPC-830 module
The SPC-830 combines the functions of the older SPC-630 and the SPC-730 modules. It has a memory four times as large as the SPC‑730 and an extremely fast bus interface. It aims at high-end applications that require both high-resolution lifetime imaging, single molecule detection and fluorescence correlation spectroscopy (FCS). As the SPC-730, the SPC-830 modules can be coupled to almost any scanning device. The modules can be synchronised by the frame/line synchronisation pulses or by X/Y signals from free running scanners such as confocal or two-photon laser scanning microscopes or ultra-fast video-compatible ophthalmic scanners. Furthermore, the SPC-830 modules can actively control a scanning device by sending appropriate synchronisation pulses. All scanning applications that require the Scan Sync In mode benefit from the large memory of the SPC-830. For single-wavelength detection the maximum scanning area is up to 2048 x 2048 pixels for lifetime images and up to 4096 x 4096 pixels for steady state images. Multi-spectral lifetime data with 16 wavelength channels can be recorded with up to 512 x 512 pixels. Due to the fast bus interface, even large images can be read out and displayed in intervals of less than one second.
The SPC-830 modules can be operated in the parameter-tag (‘FIFO’) mode and used for single molecule detection and combined FCS / lifetime experiments. In the parameter-tag mode the SPC‑830 benefits from its large memory and fast bus interface. The FIFO buffers up to 8×106 photons, which is often enough to buffer a complete FCS, FIDA, or BIFL measurement. Moreover, the readout rate is on the order of 4×106 photons/s. A sustained count rate close to the maximum useful count rate of the SPC‑830 does therefore not overload the FIFO. Another advantage of the SPC‑830 is that the macro-time clock can be synchronised with the laser repetition rate. FCS artefacts due to aliasing of the time-tag clock with the laser repetition rate are thus avoided.
From May 2007 the SPC‑830 got the ‘FIFO Imaging’ mode of the SPC-150 and SPC-160 modules implemented. It facilitates the recording of large images, especially in combination with the bh multi-wavelength FLIM detector. It also makes the SPC‑830 capable of simultaneously recording fluorescence and phosphorescence decay data.
The SPC-600/630 modules were the first TCSPC devices that had both the photon distribution modes and the single-photon parameter-tag mode implemented.
Fig. 14: SPC-630 module
Thus, the modules can be used for the complete range of applications from the traditional fluorescence lifetime and anisotropy decay experiments, photon correlation experiments, and single-molecule spectroscopy by FCS, FIDA and BIFL techniques. The SPC‑600/630 modules have a dual memory structure for simultaneous measurement and data readout. A ‘Continuous Flow’ mode is available to record extremely fast and virtually infinite sequences of waveforms. Thus, the SPC-600/630 modules can be used to record transient phenomena in the absorption, scattering and fluorescence parameters of a sample.
Introduced in 1997, the SPC‑600/630 modules are still used in large quantities. However, due to the limitations of the FPGA technique of the late 90s the SPC‑600/630 are inferior to newer designs in terms of bus transfer rate and functionality. In particular, the SPC-600/630 have no imaging modes implemented. The SPC-6 series modules are still supported but are no longer manufactured. Please use the more advanced SPC‑830, SPC‑130, SPC‑130 EM, or SPC‑150 modules instead.
The SPC-700/730 modules were the first devices designed for TCSPC imaging with laser scanning microscopes. They combine the features of the earlier SPC-500/530, SPC-505/535 and the SPC-506/536 modules.
Fig. 15: SPC-730 module
The disadvantage is the (for modern standards) small device memory and the lack of a FIFO imaging mode. Images recorded with the SPC-730 therefore do not reach pixel numbers higher than 256 x 256. The manufacturing of the SPC-730 boards has been discontinued. Nevertheless, the boards are still supported by bh SPCM device software, and the data can be processed by SPCImage data analysis software.
A comparison of the bh SPC board versions is given in Table 1 and Table 2, page 27.
The Simple-Tau systems are compact table-top systems based on a lap-top computer and a bus-extension box. The box contains the TCSPC and experiment control electronics.
There is a wide variety of Simple-Tau systems with different TCSPC boards, different numbers of TCSPC boards, and different experiment control modules. The ‘small’ Simple Tau systems (see Fig. 16) have space for four PCI modules, and come in the following configurations:
Type TCSPC module(s) Detector control Scanner Control
Simple-Tau 830 1 x SPC‑830 DCC-100 GVD-120
Simple-Tau 130 1 x SPC‑130 DCC-100
Simple-Tau 132 2 x SPC-130 DCC-100
Simple-Tau 134 4 x SPC-130 - -
Simple-Tau 150 1 x SPC-150 DCC-100 GVD-120
Simple-Tau 152 2 x SPC-150 DCC-100 GVD-120
Simple-Tau 154 4 x SPC-150 - -
Simple-Tau 160 1 x SPC-150 DCC-100 GVD-120
Simple-Tau 162 2 x SPC-150 DCC-100 GVD-120
Simple-Tau 164 4 x SPC-150 -
The DCC-120 detector controller and the GVD-120 scan controller are optional.
For applications requiring more than four PCI modules a ‘big’ Simple-Tau version is available, see Fig. 16. The electronics boxes of these systems provide space for 8 cards. Thus, there is space for additional DCC-100 detector controllers and for a GVD-120 scan controller card even in four-channel TCSPC systems.
Fig. 16: Simple-Tau systems. Left: Standard version. Right: Simple-Tau BIG version with space for 8 cards.
The small size of the Simple-Tau systems is an advantage especially when a FLIM system has to fit into a crammed laboratory environment. The disadvantage of the small size is that also the screen area is small. Since 2012 the Simple-Tau systems are therefore available with large screens and external keyboards. The ‘Simple-Tau 152 LS’ with a 27” screen and 1920 x 1080 pixels resolution is shown in Fig. 17.
Fig. 17: Simple-Tau 152 LS system
The Simple-Tau II systems contain one or two SPC-160 pcie TCSPC modules, a DCC-100 pcie detector controller, and (for scanning applications) a GVD-120 pcie scan controller. The modules come in an extension box which is coupled to a laptop computer via a Thunderbolt interface. The systems offer the full range of functions of the SPC-160 pcie modules. An advantage of the Simple-Tau II systems is small size, low weight, and a high degree of portability.
Fig. 18: Simple-Tau II system
The Power-Tau TCSPC systems are used in applications where maximum computation power, maximum memory space, and maximum data throughput rate are required. Up to six TCSPC or measurement control cards are run in a high-performance PC. The cards communicate with the CPU directly via the computer bus, thus avoiding any bottleneck in the data transfer by interface or data transmission lines. The Power-Tau systems are especially used for multi-module systems, such as the SPC‑154, SPC‑154N, or SPC-164. Typical applications are high-throughput FLIM, clinical FLIM, and diffuse optical imaging.
Fig. 19: Power-Tau system, shown with optical scanner, ps diode laser, and several detectors
The DPC-230  especially targets at a new type of fluorescence correlation spectroscopy that records correlation over a time range of 10 orders of magnitude . Thus, singlet decay rates, rotational relaxation rates, rate constants of conformational changes, triplet transition and decay rates, and diffusion times of free fluorophores and fluorophores bound to proteins, lipids or nanoparticles can be obtained within a single measurement. Another application may be diffuse optical correlation experiments which require autocorrelation in a large number of detection channels.
The DPC-230 photon correlator card (Fig. 20) records absolute photon times in up to 16 parallel detection channels. The time resolution of the recording is 165 ps per time channel. Depending on how the photons are correlated, fluorescence correlation (FCS), fluorescence cross correlation (FCCS), or photon counting histograms are obtained. A fluorescence correlation curve from 160 ps to 1 ms is shown in Fig. 20, right.
Fig. 20: DPC‑230 16 channel correlator (left) and fluorescence cross-correlation function of two detector channels recorded at ps resolution (right)
Moreover, the DPC-230 can be used for recording luminescence decay curves and fluorescence lifetime images from the sub-nanosecond to the millisecond range, for LIDAR experiments, time-of flight mass spectroscopy, or any other experiments based on acquiring the temporal distribution of detection events in a large number of detector channels. Fluorescence decay curves of a ruthenium dye and a fluorescence lifetime image of a convallaria sample are shown in Fig. 21. Details of the DPC‑230 are described in a separate handbook .
Fig. 21: Fluorescence Lifetime applications of DPC‑230. Left: Luminescence decay of a ruthenium dye for different oxygen concentration. Right: Lifetime image of a convallaria sample, bh DCS‑120 scanning system, data analysis by SPCImage
For photon counting at medium time resolution and for steady-state applications a number of multichannel scalers are available from bh [68, 69]. Multichannel scaling differs from TCSPC in that it is able to record several photons per signal period. Moreover, count rates on the order of several 100×106 s-1 can be recorded. The PMS-400, MSA-300, and MSA-1000 multichannel scalers are shown in Fig. 22.
Fig. 22: Photon counters based on multichannel scaling. Left to right: PMS-300 (250 ns per channel), MSA-300 (5 ns per channel), MSA-1000 (1 ns per channel)
The minimum time-channel width of these devices is 250 ns, 5ns, and 1 ns, respectively. In optical spectroscopy, the multichannel scalers are excellent instruments for recording phosphorescence decay curves of organic dyes, luminescence decay curves of rare-earth chelates, and luminescence decay curves of inorganic fluorophores. Other applications are time-of-flight mass spectrometry and LIDAR.
For the MSA‑1000 and MSA‑300 photon multichannel scalers compact table-top systems are available, see Fig. 23. These Simple-Tau MSA systems contain one or two MSA‑1000 modules or one or two MSA‑300 modules along with a DCC‑100 detector controller card and a PMC‑100-1 detector modules. Simple-Tau-MSA systems with other detectors are available on request.
Fig. 23: Simple-Tau MSA system
In the ‘Single’ mode the intensity versus time (usually a fluorescence decay curve) is measured, see Fig. 24, left. The ‘Single’ mode is equivalent of the traditional TCSPC procedure used in classic TCSPC devices. However, the bh modules are able to record at much higher pulse repetition rates and count rates. Thus, results can be obtained within acquisition time intervals far less than one second. More importantly, the bh TCSPC modules can record the signals of several detectors or detector channels simultaneously. With the bh PML‑Spec and MW‑FLIM detectors a signal is recorded in 16 wavelength intervals, see Fig. 24, middle and right. The same principle can be used to record signals excited by several multiplexed lasers.
Fig. 24: ‘Single’ mode. Left: Fluorescence decay curve detected by one detector. Middle and Right: 16 fluorescence decay curves detected simultaneously at different wavelength (Curves 1 - 8 and 9 -16, PML‑SPEC detector)
The memory of the TCSPC modules can be divided into several ‘measurement pages’. The pages hold data of different measurements with the same system parameters. That means, several measurements can be performed one after another, the data be recorded into different pages, and stored together in a single data file. The ‘Single’ mode has a ‘page stepping’, a ‘cycle’ and an ‘autosave’ function. Using the page stepping function, several measurements can be performed automatically and the data be stored into subsequent pages. The recorded data set can be saved into a single file at the end of the procedure. The entire procedure can be repeated by the cycle function and subsequent data sets be saved in subsequent data files.
In the 'Oscilloscope' mode a fast repetitive measurement is performed and the results are displayed in short intervals. The measurement and display update rate can be on the order of 50 measurements per second and faster. Thus, the TCSPC device works like an oscilloscope. However, compared with a measurement with fast photodiode and an oscilloscope the sensitivity and the time resolution are orders of magnitude higher. As described for the ‘Single’ mode, the signals of several detectors or several multiplexed lasers can be measured and displayed simultaneously. Fig. 25 gives an impression of the performance of the bh SPC modules in the oscilloscope mode.
Fig. 25: Oscilloscope mode. Measurement and display update rate 0.1 s. Three subsequent curves were overlaid to reproduce the visual appearance.
The ‘f(t,x,y)’ mode is used for measurements with one-dimensional or two-dimensional detector arrays or for other multi-dimensional recording tasks. The principle of data recording is the same as in the ‘Single’ mode. However, the results are interpreted as a multi-dimensional photon distribution over the time in the excitation pulse sequence, the coordinates of the elements of a detector array, or one or two other variables encoded in the digital ‘routing’ input signals of the SPC module. A number of different display modes are available to display these multi-dimensional results. Two examples are shown in Fig. 26.
Fig. 26: F(t,x,y) mode: Decay curves recorded for 16 wavelengths simultaneously
In the ‘f(t,T)’ mode the measurement is repeated in specified time intervals, and the results are written into subsequent memory pages of the SPC module. The result is a sequence of waveforms (usually fluorescence decay curves). With several detectors or a PML‑SPEC multi-wavelength detector every step of the time-series delivers decay curves in several wavelength intervals. An example is shown in Fig. 27.
Fig. 27: Time-series of decay curves recorded in the f(t,T) mode. Fluorescence of live plant after start of exposure, time series starts from the back. Simultaneous recording in four wavelength intervals.
In the ‘f(t,EXT)’ mode an external parameter is assigned to the steps of the series. The parameter may be the wavelength selected by a monochromator, the sample temperature varied via an external controller, a voltage or electrical field strength, or any other parameter. The results represent the change of the waveform as a function of the external parameter.
Fig. 28: Series of decay curves recorded during a wavelength scan of a monochromator
The ‘fi(ext)’ and ‘fi(T)’ modes record a time-controlled sequence of decay curves. However, instead of storing the complete decay curves, gated intensities in selectable time windows are stored. Up to eight independent time windows can be defined on the measured waveforms. The intensities within these windows are displayed as functions of time or of an externally variable parameter. The mode is also called spectrum scan mode because it was originally introduced to record time-resolved spectra.
Fig. 29: fi(t,T) mode: Time resolved spectra in 8 time-windows, recorded during a wavelength scan of a monochromator
The ‘Continuous Flow’ mode records an unlimited sequence of decay curves or other optical waveforms. The memory of the SPC module is split into two banks. Each bank provides space for a large number of recordings. The continuous flow modes writes the waveforms (either from a single detector or from an array of detectors) in subsequent memory blocks of the current memory bank. When one bank is full the banks are swapped, and the recording is continued in the other bank. In the meantime the results from the first bank are read and written to the hard disc of the computer. Thus, a virtually infinite sequence of waveforms can be measured. Unlike f(t,T), the Continuous Flow mode is strictly hardware controlled and thus provides an extremely fast and accurate recording sequence. The Continuous Flow mode was originally developed for single molecule detection or DNA analysis in a gel electrophoresis setup. Now the mode is mainly used for functional brain imaging by optical tomography techniques, for stopped-flow measurements, measurements of the Kautski effect in living plants, and other applications which require a large number of decay curves to be recorded in short and exactly defined time intervals without time gaps between subsequent recordings. An example is shown in Fig. 30.
Fig. 30: Part of a Continuous-Flow sequence
The Continuous Flow mode is available for the SPC-130, SPC-130EM, SPC‑150, SPC-150N, SPC-150NX, SPC-160, and SPC-160pcie modules, and the corresponding multi-module packages.
The Scan Sync Out mode was originally designed to record images with piezo scan stages. The SPC module controls the scan by sending synchronisation pulses to the scan controller and, pixel by pixel, builds up a photon distribution over the coordinates of the scan, the times of the photons in the laser pulse sequence, and a detector number or the wavelength of the photons. Since piezo scanner have widely been replaced with galvanometer scanners the Scan Sync Out mode has come out of use for imaging applications. It is now mainly used to accumulate ultra-fast triggered time series. An example is shown in Fig. 31.
Fig. 31: Photochemical quenching transient of chlorophyll in living plant. Scan Sync out mode, accumulated triggered time series, 100µs per curve.
In the parameter-tag (or ‘FIFO’) mode the hardware of the SPC module does not build up photon distributions but records the full information about each individual photon. Each photon delivers three pieces of information: the time in the signal period, or ‘micro time, the number of the detector channel where the photon was detected, and the time from the start of the experiment, or ‘macro time’. Additional information can be fed in via additional ‘routing bits’ and by encoding external events into four ‘marker’ bits. These data are put into in a first-in-first-out (FIFO) buffer. During the measurement the FIFO is continuously read. The photon data are processed online or stored in the computer memory or on the hard disc.
The FIFO mode is mainly used for single molecule experiments. The results are used to calculate fluorescence correlation (FCS) curves, fluorescence decay curves, and photon counting histograms (PCH), and intensity (MCS) traces over specified time intervals within the measurement interval or as a function of time. These data can be calculated both off-line or online in the incoming data stream, see Fig. 32.
Fig. 32: Decay function, FCS curve, and MCS trace recorded and displayed online in the FIFO mode.
Moreover, FIFO data are used to obtain Burst Integrated Fluorescence Lifetime (BIFL) and Multiparameter Fluorescence Detection (MFD) data. The data delivered are compatible with the MFD analysis software of the University Düsseldorf .
The FIFO mode is also the basis of FLIM recording with large pixel numbers, for recording optical waveforms on the microsecond and millisecond time scale, and for simultaneous FLIM / PLIM.
The FIFO mode is available for all active bh SPC modules. It is also available in the SPC-830, with the restriction that the ‘marker’ inputs are available only in modules manufactured later than May 2007.
With the SPCM software version 9.0 of April 2010 ‘Triggered Accumulation MCS’ acquisition has been added to the FIFO mode. The mode builds up the distribution of the photons versus their macro times after an excitation pulse. The time of the excitation pulse is inserted in the photon data stream via one of the ‘marker’ inputs of the SPC-150 or SPC-830 modules. The Triggered Accumulation MCS mode is used to record luminescence decay curves in the microsecond or millisecond range. The mode can be combined with fluorescence decay recording and with scanning, see Fig. 45.
Fig. 33: Microsecond luminescence decay curves recorded in the Triggered Accumulation MCS mode. Left: Single PMC‑100 detector. Right: PML-SPEC multi-wavelength detector, 8 of 16 channels displayed
The ‘Scan Sync In’ mode was implemented for recording fluorescence lifetime images in conjunction with fast scanning devices. The mode not only works with piezo scan stages but also with fast galvanometer or even fast polygon and resonance scanners. In the ‘Scan Sync In’ mode the data acquisition is controlled by receiving synchronisation pulses from a free-running scanner. The SPC module builds up a photon distribution over the coordinates of the scan, the times of the photons in the laser pulse period, and a detector number or the wavelength of the photons. The recording process works at any combination of photon rate and scan rate. In particular, it is not required that a sufficient number of photons is recorded within the dwell time of the scanner in a single pixel. For fast scanning, the data are accumulated over a large number of frames, therefore the number of photons depends on the total acquisition time, not on the pixel dwell time.
The scan sync in mode is used in laser scanning microscopes to acquire images with pixel dwell time down to 100 ns, and to obtain preview images at high count rate. Different than in the FIFO Imaging mode, high count rates do not saturate the data transfer from the TCSPC module into the computer. An example of an image recorded in the scan sync in mode is shown in Fig. 34.
Fig. 34: Fluorescence lifetime image recorded in the Scan Sync In mode
Both the scan sync in and the scan sync out can be used in combination with multi-wavelength detection. An example is shown in Fig. 35. Please note that the recording process does not use any time gating or wavelength scanning. Thus, multi-wavelength lifetime images are obtained with near-ideal efficiency and minimum sample exposure.
Fig. 35: Multi-wavelength fluorescence lifetime image recorded in the Scan Sync In mode
The SPC‑150, SPC-150N, SPC-160, SPC-160pcie modules and the corresponding multi-module packages are able to combine Scan Sync In imaging with the Continuous Flow (memory swapping) technique. The memory is split in two banks. The banks are swapped periodically, and during the image acquisition in one bank the data from the other bank are read and written on the hard disc. Because the data readout and the data acquisition are performed in different memories photons can be recorded at a sustained rate up to the saturated count rate of the SPC module. Fig. 36 shows a time series of images recorded at a rate of 2 frames per second.
Fig. 36: Continuous flow imaging sequence, recorded at rate of 2 imager per second. Lifetime images of chloroplasts in a leaf.
The FIFO Imaging mode combines parameter-tag recording with fast scanning and imaging. It is implemented in the SPC‑150, SPC-150N, SPC-150NX, SPC-160, and SPC-160pcie modules, in the corresponding multi-module packages, and in SPC-830 modules manufactured later than May 2007. An example of a FIFO-mode image is shown in Fig. 37.
Fig. 37: Image recorded in the FIFO imaging mode. BPAE cells, 2048 x2048 pixels, 256 time channels. Decay data in selected spots of the images are shown on the right.
In contrast to the Scan Sync In mode, the FIFO Imaging mode does not build up a photon distribution in the memory in the SPC module. Instead, it transfers parameter-tag data of the individual photons and of the scan clock pulses into the computer. The lifetime images are built up from these data in the memory of the computer. Thus, the size of the images, i.e. the number of pixels and time channels, is not limited by the size of the on-board memory of the TCSPC module. FIFO imaging can therefore record images with enormous pixel numbers. The image shown in Fig. 37 has 2048 x 2048 pixels and 256 time channels per pixel. The instrument software can be adapted to a wide variety of scan procedures and scan clock definitions, including bi-directional scanning. Several such images can be recorded simultaneously by a parallel-channel FLIM system.
In addition to online calculation of intensity images and lifetime images, the time-tag data themselves can be stored for off-line analysis, e.g. to obtain correlation data or sequences of images in individual frames of the scan.
The FIFO imaging mode can be used in combination with multi-wavelength detection. As for the Scan Sync In mode, the data in all wavelength intervals are recorded simultaneously, i.e. without any wavelength scanning or time gating. An example is shown in Fig. 38.
Fig. 38: Multi-wavelength FLIM of a convallaria sample.
Z stack FLIM acquires FLIM images in a large number of subsequent focal planes of a confocal or multiphoton laser scanning microscope. FLIM Z stacks can be obtained by controlling the microscope from the SPCM software , or by running a Z sequence in the microscope and running a triggered series of FLIM acquisitions in the TCSPC system [61, 86]. Please see also page 368 of this handbook. An example is shown in Fig. 39.
Fig. 39: Z stack recording, part of a water flee, autofluorescence. Images 256x256 pixels, 256 time channels.15 steps in Z, step width 4 µm. bh DCS-120 confocal scanning FLIM system with HPM‑100-40 hybrid detector.
Several lasers or laser wavelengths can be multiplexed on a pixel-by-pixel, line-by-line, or frame-by-frame basis, and separate images be recorded within the same scan. The high multiplexing rate avoids artefacts by photobleaching or dynamic effects in the sample. In the bh DCS-120 scanning system excitation wavelength multiplexing is integrated in the scanner control . An example of a wavelength-multiplexed recording is shown in Fig. 40. Please see also page 351 of this handbook.
Fig. 40: Excitation wavelength multiplexing, 405 nm and 473 nm. Detection wavelength 432 nm to 510 nm and 510 nm to 550 nm. Mouse kidney section, stained with Alexa 488 WGA, Alexa 568 phalloidin, and DAPI.
Mosaic FLIM records an array of FLIM images. The individual elements of the array can represent a spatial array of images obtained by a sample-stepping device, images in different wavelength channels of a multi-wavelength detector, images in different depth of a sample, or images at different times after a stimulation of a sample. The entire mosaic is recorded into one single, large photon distribution. The advantage of Mosaic FLIM is that there are no readout times between the recordings of the mosaic elements, and that the entire data set can be analysed in a single data analysis run. This makes it possible to perform a fit with global parameters, please see data analysis chapter of this book. A lateral mosaic is shown in Fig. 41 left, a Z stack recorded by mosaic FLIM in Fig. 41, right.
Because there is no readout time between the recording of the individual mosaic elements the procedure can be used to record fast time series. An example of ‘Temporal Mosaic FLIM’ is shown in Fig. 42. The figure shows the change of the fluorescence lifetime of the chloroplasts in a moss leaf for different time after the start of illumination.
Fig. 41: Examples of Mosaic FLIM. Left: Spatial mosaic, convallaria sample, 16 elements of 512 x 512 pixels each, 256 time channels, entire mosaic is 2048 x 2048 pixels x 256 time channels. Right: Z Stack recorded by Mosaic FLIM. Pig skin, stained with cyanine dye, each element 512x512 pixels x 256 time channels
Fig. 42: Temporal mosaic, lifetime change of chlorophyll in a leaf, 64 elements of 128 x 128 pixels, time per element 1 second. Time is running upper left to lower right. Decay curves for two identical spots at different time after the start of the illumination are shown right.
An important feature of temporal mosaic FLIM is that the sample can be stimulated periodically and the data be accumulated. This allows extremely fast image sequences to be recorded without the need of trading lifetime accuracy against the speed of the sequence. An example is shown in Fig. 43. The figure shows the change in the calcium concentration in a culture of live neurons after stimulation with an electrical pulse. Please see 111 and page 372 for details.
Fig. 43: Ca2+ transient in cultured neurons, incubated with Oregon Green. Electrical stimulation, stimulation period 3s, data accumulated over 100 stimulation periods. Scan time per mosaic element 38 ms.
FLITS records transient effects in the fluorescence lifetime of a sample along a one-dimensional scan. The maximum resolution at which lifetime changes can be recorded is given by the line scan time. With periodic stimulation and triggered accumulation transient lifetime effects can be resolved at a resolution of about one millisecond [101, 136].
Fig. 44: FLITS of chloroplasts in a grass blade, change of fluorescence lifetime after start of illumination. Experiment time runs bottom up. Left: Non-photochemical transient, transient resolution 60 ms. Right: Photochemical transient. Triggered accumulation, transient resolution 1 ms.
With the SPCM software version 9.0 of April 2010 phosphorescence lifetime imaging (PLIM) in the microsecond and millisecond range has been introduced [94, 135]. PLIM is performed in the FIFO Imaging mode. However, instead of the micro time of the photons it uses the macro time to build up the photon distribution. Minimum time-channel width is 25 ns. An example is shown in Fig. 45.
Fig. 45: Luminescence lifetime image of an inorganic fluorophore. Left to right: Intensity image, phosphorescence decay functions of pixels along a horizontal stripe of the image, lifetime image, red to blue corresponds to a range of 10 to 15 ms.
Phosphorescence lifetime imaging can be run simultaneously with FLIM. The technique is based on on/off modulation of a high-frequency pulsed laser. It is performed in the FIFO Imaging mode. For each photon both the time in the laser pulse period and the time in the modulation period are determined. PLIM is obtained from the times of the photons in the modulation period, FLIM from the times of the photons in the laser pulse period. To avoid aliasing of the laser modulation with the pixel frequency the modulation period is synchronised with the pixels of the scan. An example is shown in Fig. 46. Please see page 113 or page 388 for details.
Fig. 46: Simultaneous FLIM and PLIM of yeast cells. Autofluorescence of the cells (left) and phosphorescence of a ruthenium compound (right)
The Wide-Field FLIM mode requires three SPC-150N, SPC-160 or SPC-160pcie modules with special discriminators and a position-sensitive detector with delay-line anode [105, 145]. The first module measures the times of the photons in the laser pulse period. The second and third module measure the time differences of the pulses at the outputs of the X and the Y delay lines of the detector. The detection events, i.e. the times, t, and the time differences, tx and ty, are transferred into the computer photon by photon. From these data the software builds up a photon distribution over the coordinates x, y, and the times, t. This is the usual photon distribution of FLIM: It is an array of pixels, each of which contains a fluorescence decay curve consisting of photon numbers in consecutive time channels. This is the normal photon distribution of TCSPC FLIM. The data can thus be analysed by SPCImage FLIM data analysis software. For parameter setup and other technical details please see page 410 and page 678 of this book.
Fig. 47: Wide-Field FLIM, data analysis by SPCImage. Invitrogen BPAE cells, 512x512 pixels, 1024 time channels per pixel.
General Features SPC-830 SPC-130 SPC-130 EM SPC-150 SPC‑150N SPC‑160
TCSPC Channels / Module 1 1 1 1 1
Time Channels /Curve 1 to 4096 3) 1 to 4096 3) 1 to 4096 3) 1 to 4096 3) 1 to 4096 3) 1 to 4096 3)
Time-Channel Width, min. 815 fs 815 fs 815 fs 815 fs 815 fs 815 fs
Time Resol. el., FWHM / rms 6 ps / 2.5 ps 7 ps / 3 ps 7 ps / 3 ps 7 ps / 3 ps 7 ps / 3 ps 7 ps / 3 ps
IRF w. MCP PMT, FWHM / rms 25 ps / 10 ps 25 ps / 10 ps 25 ps / 10 ps 25 ps / 10 ps 25 ps / 10 ps 25 ps / 10 ps
Discriminator Bandwidth 1 GHz 1 GHz 1 GHz 1 GHz 4 GHz 4 GHz
No. of Curves in internal Memory, 4096 time channels 4) 4096 32 1024 1024 1024 1024
No. of Curves in internal Memory, 256 time channels 4) 65,536 512 16,384 16,384 16,384 16,384
Largest image, Scan Sync mode, 256 time channels 4) 256x256 - - 128x128 128x128 128x128
Largest image, FIFO Img. mode, 256 time channels 4) 512x512 - - 512x512 512x512 512x512
Largest image, FIFO Img. mode, 256 time channels 4, 9) 2048x2048 - - 2048x2048 2048x2048 2048x2048
Memory structure single or FIFO dual or FIFO dual or FIFO dual or FIFO dual or FIFO dual or FIFO
Memory size, photon distribution modes 32 Mbyte 2 x 256 kByte 2 x 8 MByte 2 x 8 Mbyte 2 x 8 Mbyte 2 x 8 Mbyte
FIFO size in Time-tag Mode, photons 8×106 128×103 2×106 2×106 2×106 2×106
Dead Time 125ns 100 ns 100 ns 100 ns 100 ns 80 ns
Saturated Count Rate 1) 8 MHz 10 MHz 10 MHz 10 MHz 10 MHz 12.5 MHz
Useful Count Rate 2) 4 MHz 5 MHz 5 MHz 5 MHz 5 MHz 6,25 MHz
No of routing channels, Photon distribution / FIFO mode 16,384 / 16 128 / 16 6) 128 / 16 128 / 16 128 / 16 128 / 16
Operation Modes SPC-830 SPC-130 SPC-130 EM SPC-150 SPC‑150N SPC‑160
‘Single’ Mode X X X X X X
Oscilloscope Mode X X X X X X
3D mode, f(t,x,y) X X 6) X X X X
Sequence, f(t,T) X X X X X X
Sequence, f(t,ext) X X X X X X
Spectrum, fi(T) X X X X X X
Spectrum, fi(ext) X X X X X X
Unlimited Sequence, Continuous Flow X X X X X
FIF0 (Parameter-Tag) Mode X X X X X X
FIFO, MCS w. triggered accumulation X 7) X X X X
FIFO Imaging X 7) X X X
Imaging, Scan Sync In X X X X
Imaging, Scan Sync In with Cont. Flow X X X
Imaging, Scan Sync Out X X X X
Imaging, Parallel Intensity Channel X
Sequence, by Scan Sync Out X X X X
Control Functions SPC-830 SPC-130 SPC-130 EM SPC-150 SPC-150N SPC-160
Routing, Multi-Detector Operation X x 6) X X X X
Multiplexing X X X X X X
Page stepping X X X X X X
Accumulate X X X X X X
Autosave X X X X X X
Experiment Trigger X X X X X X
Ext. event markers in FIFO mode X 7) X X X
Applications SPC-830 SPC-130 SPC-130 EM SPC-150 SPC‑150N SPC‑160
Fluorescence Decay X X X X X X
Phosphorescence Decay X 7) X X X
Multi-wavelength Fluorescence Decay X X X X X X
Dynamic Fluorescence. Lifetime Phenomena X X X X X X
Fluorescence Spectra X X X X X X
Single-Molecule Spectroscopy, FCS / FCCS, FIDA, BIFL X X X X X X
Opt. Tomography X X X X X X
FLIM, Laser Scanning Microscopy X X X X
Multi-Wavelength FLIM X X X X
Sequential FLIM X X X X
Megapixel FLIM X 9) X 9) X 9) X 9)
Mosaic FLIM X 9) X 9) X 9) X 9)
Triggered Accumulation of FLIM sequences X 9) X 9) X 9) X 9)
FLITS X X X X
PLIM, combined with FLIM X X X X
Parallel-Channel FLIM and PLIM See SPC-154, SPC‑154N, SPC‑164
1) The saturated count rate is the reciprocal dead time. It is the theoretical maximum for infinite detector count rate.
2) The maximum useful count rate is the recorded count rate for 50% counting loss.
3) Number of time channels is 1, 4, 16, 64, 256, 1024, or 4096.
4) Max. number of curves or pixels depends on selected number of time channels and routing channels. Please see ‘System Parameters’.
5) The SPC-134, SPC-134EM, SPC‑154, SPC‑154N, and SPC-164 devices are packages of four parallel SPC-130, SPC‑130EM, SPC‑150 SPC‑150N, or SPC‑160 TCSPC modules.
6) Limited routing capability. 6) Limited routing capability. Multi-detector operation requires 7m delay cable in detector line.
7) Modules manufactured later than May 2007. Modules later than May 2005 can be upgraded
8) For each module of the four-channel package
9) 64 bit SPCM operating software
Table 1: Comparison of bh TCSPC modules
General Features SPC-134 SPC-134 EM SPC-154 SPC-154N SPC‑164
TCSPC Channels / Module 4 4 4 4 4
Time Channels /Curve 1 to 4096 3) 1 to 4096 3) 1 to 4096 3) 1 to 4096 3) 1 to 4096 3)
Time-Channel Width, min. 815 fs 815 fs 815 fs 815 fs 815 fs
Time Resol. el., FWHM / rms 7 ps / 3 ps 7 ps / 3 ps 7 ps / 3 ps 7 ps / 3 ps 7 ps / 3 ps
IRF w. MCP PMT, FWHM / rms 25 ps / 10 ps 25 ps / 10 ps 25 ps / 10 ps 25 ps / 10 ps 25 ps / 10 ps
Discriminator Bandwidth 1 GHz 1 GHz 1 GHz 4 GHz 4 GHz
No. of Curves in internal Memory, 4096 time channels4) 32 8) 1024 8) 1024 8) 1024 8) 1024 8)
No. of Curves in internal Memory, 256 time channels4) 512 8) 16,384 8) 16,384 8) 16,384 8) 16,384 8)
Largest image, Scan Sync mode, example f. 256 time channels4) - - 128 x 128 128 x 128 128 x 128
Largest image, FIFO Img. mode, example f. 256 time channels4) - - 512x512 512x512 512x512
Largest image, FIFO Img. mode, 256 time channels 4, 9) - - 2048x2048 2048x2048 2048x2048
Memory structure (each module) dual or FIFO dual or FIFO dual or FIFO dual or FIFO dual or FIFO
Memory size, photon distribution modes 2 x 256 kByte 8) 2 x 8 Mbyte 8) 2 x 8 Mbyte 8) 2 x 8 Mbyte 8) 2 x 8 Mbyte 8)
FIFO size in Time-tag Mode, photons 128×103 8) 2×106 8) 2×106 8) 2×106 8) 2×106 8)
Dead Time 100 ns 100 ns 100 ns 100 ns 80 ns
Saturated Count Rate 1) 40 MHz total 40 MHz total 40 MHz total 40 MHz total 50 MHz
Useful Count Rate 2) 20 MHz total 20 MHz total 20 MHz total 20 MHz total 25 MHz
No of routing channels, Photon distribution / FIFO mode 128 / 16 8) 6) 128 / 16 8) 128 / 16 8) 128 / 16 8) 128 / 16 8)
Operation Modes SPC-134 SPC-134 EM SPC-154 SPC-154N SPC‑164
‘Single’ Mode X X X X X
Oscilloscope Mode X X X X X
3D mode, f(t,x,y) X 6) X X X X
Sequence, f(t,T) X X X X X
Sequence, f(t,ext) X X X X X
Spectrum, fi(T) X X X X X
Spectrum, fi(ext) X X X X X
Unlimited Sequence, Continuous Flow X X X X X
FIF0 (Parameter-Tag) Mode X X X X X
FIFO, MCS w. triggered accumulation X X X X
FIFO Imaging X X X
Imaging, Scan Sync In X X X
Imaging, Scan Sync In with Cont. Flow X X X
Imaging, Scan Sync Out X X X
Imaging, Parallel Intensity Channel X
Sequence, by Scan Sync Out X X X
Control Functions SPC-134 SPC-134 EM SPC-154 SPC-154N SPC‑164
Routing, Multi-Detector Operation x 6) X X X X
Multiplexing X X X X X
Page stepping X X X X X
Accumulate X X X X X
Autosave X X X X X
Experiment Trigger X X X X X
Ext. event markers in FIFO mode X X X
Applications SPC-134 SPC-134 EM SPC-154
Fluorescence Decay X X X X X
Phosphorescence Decay X X X
Multi-wavelength Fluorescence Decay X X X X X
Dynamic Fluorescence Lifetime Phenomena X X X X X
Fluorescence Spectra X X X X X
Single Molecule Spectroscopy, FCS / FCCS, FIDA, BIFL X X X X X
Opt. Tomography X X X X X
FLIM, Laser Scanning Microscopy X X X
Multi-Wavelength FLIM X X X
Sequential FLIM X X X
Megapixel FLIM X 9) X 9) X 9)
Mosaic FLIM X 9) X 9) X 9)
Triggered Accumulation of FLIM sequences X 9) X 9) X 9)
FLITS X X X
PLIM, combined with FLIM X X X
Parallel-Channel FLIM and PLIM X X X
2) The maximum useful count rate is the recorded count rate for 50% counting loss.
3) Number of time channels is 1, 4, 16, 64, 256, 1024, or 4096.
4) Max. number of curves or pixels depends on selected number of time channels and routing channels. Please see ‘System Parameters’.
5) The SPC-134, SPC-134EM, SPC‑154, SPC‑154N, and SPC-164 devices are packages of four parallel SPC-130, SPC‑130EM, SPC‑150 SPC‑150N, or SPC‑160 TCSPC modules.
6) Limited routing capability. Multi-detector operation requires 7m delay cable in detector line.
7) Modules manufactured later than May 2007. Modules later than May 2005 can be upgraded
8) For each module of the four-channel package
9) 64 bit SPCM operating software
Table 2: Comparison of bh TCSPC multi-module systems
The DCS-120 [57, 60] is a complete confocal laser scanning microscope system for fluorescence lifetime imaging. The basic system uses picosecond diode laser excitation, fast galvanometer-mirror scanning, confocal detection, bh’s multi-dimensional TCSPC technique, and 64‑bit SPCM data acquisition software. The DCS-120 scan head has two input channels for lasers, two confocal detection paths, internal beamsplitters and filters, individually selectable pinholes, and two outputs to direct-coupled detectors.
The DCS‑120 system uses two parallel SPC‑150, SPC-150N, or SPC-160 TCSPC FLIM channels. The scanner and the lasers are controlled by a GVD-120 scan controller card. The user interface for scanner and laser control is integrated in the SPCM software of the bh TCSPC systems. The data analysis part is based on bh’s SPCImage FLIM data processing software [60, 61, 65], see page 741 of this handbook.
Complete DCS‑120 laser scanning microscopes are available with a Nikon TE 2000 U microscope, a Zeiss Axio Observer, a Zeiss Axio Examiner, or an Olympus IX microscope. Alternatively, the DCS scanner and the DCS electronics can be attached to an existing research-grade microscope. The Zeiss Axio Observer and the Zeiss Axio Examiner versions are shown in Fig. 48 and Fig. 49.
There is a number of other DCS‑120 versions: The DCS‑120 MP uses multiphoton excitation and non-descanned detection. It is an excellent instrument for deep-tissue imaging. The DCS‑120 WB uses a wideband beamsplitter. It allows the user to freely swap lasers at the inputs of the scanner, and to use tuneable excitation sources. The DCS‑120 MACRO system records images of macroscopic objects directly in the image plane of the scanner. For technical details please see DCS-120 handbook , DCS-120 Overview Brochure , and page 228 of this handbook. Essential features of the DCS‑120 system are summarised in the following paragraphs. A DCS-120 MP and a DCS-120 MACRO system are shown in Fig. 50.
Fig. 48: DCS‑120 Confocal Scanning FLIM system. Version with Zeiss Axio Observer
Fig. 49: DCS-120 system with Zeis Axio Examiner
Fig. 50: Left: DCS‑120 MP multiphoton FLIM system. Right: DCS‑120 MACRO version for imaging macroscopic objects.
The DCS-120 scan head contains the complete beam deflection and confocal detection optics. The principle is shown in Fig. Fig. 51. The laser beams are deflected by fast-moving galvanometer mirrors, and sent down the microscope beam path. The axis of the galvanometer mirrors is projected into the plane of the microscope lens. With the motion of the galvanometer mirrors the laser focus thus scans over the focal plane in the sample. The emission light is collected back through the microscope lens. The emission beam is descanned by the galvanometer mirrors, separated from the excitation beam, split into two channels of different wavelength or different polarisation and focused into pinholes in a plane conjugate with the focal plane in the sample. Out-of-focus light is not focused into the pinholes and thus suppressed. Please see page 228 of this handbook or  for details of the optical system. The confocal detection principle efficiently suppresses out-of-focus light and laterally scattered light. It thus avoids contamination of the recorded decay functions in the individual pixels by decay components from other sample planes or other pixels.
Fig. 51: Optical diagram of the DCS-120 scan head. Simplified, please see page 228 for details
The DCS-120 uses fast beam scanning by galvanometer mirrors. A complete frame is scanned within a time from 100 ms to a few seconds, with pixel dwell times down to one microsecond. Compared with sample scanning, beam scanning is not only much faster, it avoids also induction of cell motion by exerting dynamic forces on the sample. Moreover, live cell imaging requires a fast preview function for fluorescence images for sample positioning and focusing. This can only be provided if the beam is scanned at a high frame rate. With its fast scanner and its multi-dimensional TCSPC process the DCS system achieves surprisingly short acquisition times, see Fig. 52. Please see also 'Time-Series FLIM', page 371.
Fig. 52: FLIM images recorded within 5 seconds acquisition time. 256 x 256 pixels (left) and 512 x 512 pixels (right), both with 256 time channels.
With 64 bit SPCM software pixel numbers can be increased to 2048 x 2048 pixels, with a temporal resolution of 256 time channels. Two such images are recorded simultaneously in different wavelength channels. Fig. 53 and Fig. 54 (facing page) show an example.
Fig. 53: BPAE sample (Invitrogen) scanned with 2048 x 2048 pixels. Green channel, 485 to 560 nm
The large pixel numbers available in SPCM 64 bit allow the full field of view of even the best microscope lenses to be scanned with an oversampling factor of two or more. In other words, extremely large image areas can be scanned without compromising spatial resolution.
Large pixel numbers are especially important for tissue imaging. They are also useful in cases when a large number of cells have to be investigated and the FLIM results to be compared. Megapixel FLIM records images of many cells simultaneously, and under identical environment conditions. Moreover, the data are analysed in a single analysis run, with identical IRFs and fit parameters. The results are therefore exactly comparable for all cells in the image area.
Fig. 54: BPAE sample (Invitrogen), scanned with 2048 x 2048 pixels. Red channel, 560 to 650 nm
With the bh multispectral FLIM detectors the DCS‑120 records FLIM simultaneously in 16 wavelength channels [119, 130]. The images are recorded by a multi-dimensional TCSPC process which uses the wavelength of the photons as a coordinate of the photon distribution. There is no time gating, no wavelength scanning and, consequently, no loss of photons in this process. The system thus reaches near-ideal recording efficiency. Moreover, dynamic effects in the sample or photobleaching do not cause distortions in the spectra or decay functions. Multi-wavelength FLIM got an additional push from the new 64-bit SPCM software, and from the introduction of a highly efficient GaAsP multi-wavelength detector. 64-bit software works with enormously large photon distributions, and the GaAsP detector delivers the efficiency to fill them with photons. As a result, images in 16 wavelength channels can be recorded at a resolution of 512x512 pixels and 256 time channels. An example is shown in Fig. 491.
Fig. 55: Multi-wavelength FLIM with the bh MW-FLIM GaAsP 16-channel detector. 16 images with 512 x 512 pixels and 256 time channels were recorded simultaneously. Wavelength from upper left to lower right, 490 nm to 690 nm, 12.5 nm per image. DCS‑120 confocal scanner, Zeiss Axio Observer microscope, x20 NA=0.5 air lens.
Fig. 492 demonstrates the true spatial resolution of the data. Images from two wavelength channels, 502 nm and 565 nm, were selected form the data shown Fig. 491, and displayed at larger scale and with individually adjusted lifetime ranges. With 512x512 pixels and 256 time channels, the spatial and temporal resolution of the individual images is comparable with what previously could be reached for FLIM at a single wavelength. Decay curves for selected pixels of the images are shown in Fig. 493.
Fig. 56: Two images from the array shown in Fig. 491, displayed in larger scale and with individually adjusted lifetime range. Wavelength channels 502 nm (left) and 565 nm (right). The images have 512 x 512 pixels and 256 time channels.
Fig. 57: Decay curves at selected pixel position in the images shown above. Blue dots: Photon numbers in the time channels. Red curve: Fit with a double-exponential model.
The control of the DCS‑120 scanner is fully integrated in the SPCM data acquisition software, see Fig. 58. The scanner control panel allows the user to select the image format, scan rate, and scan area, and to control the lasers. Changes in the scan parameters can be made at any time, even without stopping the scan. The DCS‑120 has automatic scan speed control. It automatically selects the fastest possible scan rate available for the scan parameters used.
Fig. 58: Data acquisition software with integrated scanner and detector control
The DCS-120 has a fast preview function that scans the sample at high speed, and displays fluorescence images in intervals of one second or less. With the preview function it is easy to bring the sample into focus, shift it in the desired position, and select the region to be scanned. The scanner control is fully integrated in the SPCM data acquisition software. The zoom factor and the position of the scan area can be adjusted via the scanner control panel or via the cursors of the display window. The scanner can be operated in the raster-scan mode, in the line mode, or in the single-point excitation mode. Changes in the scan parameters are executed online, without stopping the scan.
Fig. 59: Preview function with interactive scanner control
Frequently used instrument configurations are stored in a ‘Predefined Setup’ panel. Changing between the different configurations and user interfaces is just a matter of a single mouse click, see Fig. 60.
Fig. 60: Changing between different instrument configurations: The DCS-120 system switches from a FLIM configuration into an FCS configuration by a simple mouse click
Starting from Version 9.72 SPCM software the DCS-120 system is able to display lifetime images online, both during the accumulation of FLIM data and for the individual steps of a fast image sequence, please see 'Fast Online FLIM', page 379. Lifetime images can be displayed at images rates as fast as 10 images per second. The calculation of the lifetime images is based on the first moment of the decay data in the pixels of the images. The first-moment technique combines short calculation times with near-ideal photon efficiency. Importantly, it does not require to reduce the time resolution (time channels per pixel) to obtain high image rates. Even if the fast online lifetime function is used during the FLIM acquisition the data can later be processed by precision SPCImage multi-exponential data analysis.
Fig. 61: 256x256-pixel images obtained by the online FLLIM display function. Acquisition time 0.2s, 0.5s, and 2s.
The two ps-diode lasers of the DCS‑120 system can be multiplexed on a pixel-by-pixel, line-by-line, or frame-by-frame basis. Laser multiplexing helps discriminate the signals of several fluorophores, or allows one to excite two fluorophores that cannot efficiently be excited at the same wavelength. The capability of fast multiplexing avoids artefacts by photobleaching or dynamic effects in the sample. An example is shown in Fig. 62.
Fig. 62: Excitation wavelength multiplexing, 405nm and 473 nm.
Time-series FLIM is available for all system versions, and all detectors. Time series as fast as 2 images per second can be obtained . A time series taken at a moss leaf is shown in Fig. 63.
Fig. 63: Time-series FLIM, 2 images per second. Chloroplasts in a leaf, the fluorescence lifetime of the chlorophyll decreases with the time of exposure.
Exceptionally fast time series of FLIM images can be recorded by the Mosaic FLIM function of the SPCM software. Temporal Mosaic FLIM records a large number of subsequent FLIM images into a single data array. The advantage of Mosaic FLIM over a traditional record-and-save procedure is that there are no readout times between the recordings of the mosaic elements, and that the data can be accumulated. This allows extremely fast image sequences to be recorded without the need of trading lifetime accuracy against the speed of the sequence. With the DCS-120 scanner and temporal mosaic FLIM, time series at a speed of 100 ms per image can be recorded. An example is shown in Fig. 526, page 373.
In combination with the Zeiss Axio Observer Z1 microscope the DCS‑120 system records Z-stacks of FLIM images. The sample is continuously scanned. For each plane, a FLIM image is acquired for a specified ‘collection time’. Then the data are saved in a file, the microscope is commanded to step to the next plane, and the next image is acquired. The procedure continues for a specified number of Z planes. A Z stack of autofluorescence images taken at a water flee is shown in Fig. 64.
Fig. 64: Z stack recording, part of a water flee, autofluorescence. 15 steps in Z, step width 4 um.
The DCS-120 WB wideband version can be used with tuneable excitation. Images obtained with a Toptica Ichrome laser  are shown in Fig. 65.
Fig. 65: Tuneable excitation with DCS-120 WB and Toptica Ichrome laser. Left to right: Excitation 488 nm emission 525±15 nm, excitation 488 nm emission 620±30 nm, and excitation 580 nm emission 620±30 nm.
A two-photon FLIM image of a convallaria sample recorded by the DCS-120MP system is shown in Fig. 66.
Fig. 66: Two-photon FLIM image of a convallaria sample. 2048 x 2048 pixels x 256 time channels
The signals are detected by non-descanned HPM-100 detectors connected to the lamp port of the microscope. Multiphoton excitation penetrates deep into biological tissue. Moreover, excitation occurs only in the focus of the laser. Multiphoton NDD FLIM images can therefore be detected from deep layers within biological tissue, see Fig. 67.
Fig. 67: Pig skin, autofluorescence, image in different depth in the sample. Amplitude-weighted lifetime of triple-exponential decay model. Excitation 805 nm, 512x512 pixels, 256 time channels. Zeiss Axio Observer Z1, Water C apochromate NA=1.2, non-descanned detection, HPM‑100-40 hybrid detector.
The DCS‑120 WB version is able to record lifetime images with near-infrared (NIR) fluorophores, see page 354 of this book. NIR fluorophores often display large variations of their fluorescence lifetimes depending on the binding target. Moreover, because both the excitation and the emission wavelengths are in the near infrared a high penetration depth is obtained. NIR FLIM is therefore a promising technique to obtain metabolic information from biological tissue. An image of a pig skin sample incubated with 3,3’-diethylthiatricarbocyanine is shown in Fig. 68.
Fig. 68: Near-Infrared FLIM. Pig skin sample stained with 3,3’-diethylthiatricarbocyanine, detection wavelength from 780 nm to 900 nm.
The DCS MACRO version of the DCS system scans objects directly in the focal plane of the scanner. Objects up to a size of 12 mm can be imaged at high resolution. Fig. 69 shows a leaf with a fungus infections.
Fig. 69: Leaf with a fungus infection. ps diode laser excitation, 405nm, scan format 512 x 512 pixels. Right: Decay functions of healthy and infected areas.
The image area of the DCS MACRO can be extended by a motorised sample stage. Mosaic FLIM is used to record images of objects with dimensions in the 10-cm range, see Fig. 70.
Fig. 70: Mosaic FLIM image of a $20 bill
The DCS-120 is able to simultaneously record fluorescence and phosphorescence lifetime images. The technique is based on modulating the ps diode laser synchronously with the pixel clock of the scanner. Fluorescence is recorded during the on time, phosphorescence during the off time of the laser. Please see [94, 135] and page 388 of this handbook.
Fig. 71: FLIM (autofluorescence, left) and PLIM (2,2’-bipyridyl) dichlororuthenium (II) hexahydrate, right) yeast cells. Excitation 473 nm, 256x256 pixels.
FLITS records transient effects in the fluorescence lifetime of a sample along a one-dimensional scan. The maximum resolution at which lifetime changes can be recorded is given by the line scan time. With repetitive stimulation and triggered accumulation transient lifetime effects can be resolved at a resolution of about one millisecond [101, 136, 60]. Please see also page 374 of this handbook.
Fig. 72: FLITS of chloroplasts in a grass blade, change of fluorescence lifetime after start of illumination. Left: Non-photochemical transient, transient resolution 60 ms. Right: Photochemical transient. Triggered accumulation, transient resolution 1 ms.
Due to the superior performance of the HPM‑100-40 hybrid detectors the DCS‑120 system delivers highly efficient FCS. There is no afterpulsing peak in autocorrelation data . Thus, accurate diffusion times and molecular-brightness parameters are obtained from a single detector. Compared to cross-correlation of split signals, correlation of single-detector signals yields a four-fold increase in correlation efficiency. The result is a substantial improvement in the signal-to-noise ratio of FCS recordings. Gated FCS is possible by hardware gating via the TAC limits of the TCSPC modules, FCCS by cross-correlating the signals of the two DCS channels.
Fig. 73: Left: FCS curve recorded by a single HPM-100 detector. The data are free of an afterpulsing peak. Right: Dual-colour FCS, autocorrelation blue and red, cross-correlation green. Online fit with FCS procedures of SPCM software.
FLIM systems for the Zeiss LSM 510 laser scanning microscopes were introduced by bh in 2000. The LSM 510 was in use for more than a decade, and a number LSM 510 FLIM systems are in use until today. The LSM 510 as well as the bh FLIM systems got several upgrades during this time. As a result, a wide variety of bh FLIM systems and of FLIM system configurations have been (and probably still are) in use for the LSM 510. There are systems based on a single SPC-730 module, systems with a single SPC-830, and systems with one or two SPC-150 modules. The detectors were PMC-100 PMT modules, H7422P‑40 modules, R3809U MCP PMTs, and, later, HPM-100-40 hybrid detectors. Some LSM 510 FLIM systems used ps diode lasers and confocal detection. Due to limitations in the coupling of lasers into the scan head and limited availability of confocal outputs from the scan head these systems are not the most frequent ones. The majority of LSM 510 FLIM systems are two-photon systems based on the LSM 510 NLO . The FLIM detectors are attached to the outputs of the Zeiss ‘NDD Switch Box’, see Fig. 74.
Fig. 74: NDD FLIM detectors at the LSM 510. Left: One FLIM detector at the 0° output of the NDD switch box. Right: NDD switch box with two R3809U MCP PMTs.
FLIM detectors for the LSM 510 NLO come with shutters to avoid damage by excessive light intensities from the microscope lamp. The shutters and the detectors are controlled via the DCC-100 detector controller card. Detectors for the LSM 510 NLO with their shutter assemblies are shown in Fig. 75. The dove-tail adapters insert directly into the detector ports the Zeiss NDD box.
Fig. 75: NDD FLIM detector modules for the LSM 510 NLO. Left to right: R3809U MCP PMT, PMC‑100, H7422P‑40, HPM‑100‑40
The LSM 510 FLIM systems are also available with multi-wavelength detection. The fluorescence light is projected at the input of a fibre bundle. The fibre bundle transfers this light into the input slit of a polychromator, the dispersed light is projected on the input of a PML‑16 multichannel detector. Please see pages 346 and 347 for details. An example of a multi-wavelength FLIM recording is shown in Fig. 76.
Fig. 76: Multi-wavelength FLIM images taken with bh FLIM system and LSM 510 NLO
If you have an LSM 510 NLO FLIM system with an SPC-830 or with SPC‑150 FLIM modules there is actually no reason to stop using the system. You may rather consider pushing the performance by upgrading the system with new SPCM and SPCImage software and a 64 bit computer with 64 bit windows. You may then gain many of the advanced features of the LSM 710 / 780 / 880 FLIM systems, please see next section.
The FLIM systems for the LSM 710, 780, 880 family microscopes use advanced features of bh’s multi-dimensional TCSPC FLIM technique. The systems have two or more parallel TCSPC channels and highly efficient GaAsP hybrid detectors. By combining extremely high efficiency with large active area, high counting speed, high time-resolution, and low counting background, these detectors have resulted in a breakthrough in FLIM recording. Using bh’s ‘Megapixel’ technology and 64 bit operating software, FLIM data are recorded at unprecedented pixel numbers, high dynamic range, short acquisition time, and minimum exposure of the sample. New operation modes of the bh TCSPC modules have resulted in new FLIM functions, like time-series FLIM, Z stack FLIM, Mosaic FLIM, combined fluorescence and phosphorescence lifetime imaging (FLIM / PLIM), and fluorescence lifetime-transient scanning (FLITS).
The LSM 710/780/880 family (LSM 710, LSM 710 NLO, LSM 7 MP, LSM 780, LSM 780 NLO, LSM 880, LSM 880 NLO) microscopes are available with Ti:Sapphire lasers and integrated ps-diode lasers. A tuneable (‘Intune’) ps laser is integrated in some of the microscopes but, unfortunately, is no longer available for new systems. All these versions can be used with FLIM. Both inverted and upright versions are in use. Typical optical FLIM configurations are shown in Fig. 77 and Fig. 78.
Fig. 77: LSM 710 family FLIM systems, inverted microscopes. Left: Multiphoton-excitation FLIM with non-descanned detection. Right: One-photon FLIM with confocal detection.
Fig. 78: LSM 710 family FLIM systems, upright microscopes. Left: Multiphoton-excitation FLIM with non-descanned detection. Right: One-photon excitation FLIM with confocal detection.
Detectors with adapters for the NDD ports and the confocal ports of the LSM 710 family microscopes are shown in Fig. 79.
Fig. 79: Detectors for the LSM 710 family. Left to right: PMZ‑100 with NDD adapter, PMZ‑100 detectors at Zeiss NDD T adapter, HPM‑100 with NDD adapter, bh beamsplitter assembly with two HPM‑100 detectors with BIG port adapter.
In 2014 Zeiss has introduced a new ‘BIG-2’ detector. The BIG‑2 has outputs to the bh FLIM system and can be used for FLIM and FCS recording. The bh FLIM systems work also with the new LSM 880 and LSM 880 NLO. NDD detectors are connected as shown above. Confocal detectors are connected to a beam switch that is inserted between the san head and the Airy Scan detector, see Fig. 80.
Fig. 80: LSM 880, confocal FLIM detectors. Left to right: BIG‑2 detector, one HPM‑100‑40 HPM‑100 hybrid detector, two HPM‑100‑40 detectors
The TCSPC systems for the LSM 710 / 780 / 880 family come as compact ‘Simple-Tau’ TCSPC systems. Since 2012 the FLIM systems are available with large screens an with an option to operate them from the same keyboard and mouse that operates the Zeiss Zen software. The ‘Simple-Tau 152 LS’ with a 27” screen and 1920 x 1080 pixels resolution is shown in Fig. 17.
Fig. 81: Simple-Tau 152 LS system
Details of the FLIM systems for the Zeiss LSM 710/780/880 and LSM 510 families are described in a separate 240-page handbook  and a 47-page overview brochure . An overview on the functions of the LSM 710/780/880 family FLIM systems is given below.
FLIM data acquisition is performed by two parallel SPC‑150, SPC-150N, or SPC-160 TCSPC channels and controlled by bh 64-bit SPCM software. Based on parameter-tagged photon data delivered by the TCSPC modules, the SPCM software builds up the photon distributions for the two detection channels or, if multi-spectral FLIM is used, for the 16 spectral cannels of the bh MW FLIM detector. During the FLIM acquisition the SPCM software displays intermediate results in predefined intervals, usually every few seconds. The SPCM software is also able to record FCS and FCCS, or to deliver parameter-tagged photon data for single-molecule spectroscopy. Moreover, it records combined fluorescence/phosphorescence lifetime images (FLIM/PLIM) and Fluorescence Lifetime-Transient Scanning (FLITS) data. The acquisition can be stopped after a defined acquisition time, or by a user command when the desired signal-to-noise ratio has been reached. According to the different operation modes the SPCM software comes up in different user-interface configurations as shown in Fig. 82.
Fig. 82: SPCM software panel configured for LSM 710 FLIM. Left: FLIM in two detector channels. Right: Multi-wavelength FLIM, images in 8 of the 16 wavelength channels shown.
The FLIM systems have a fast preview functions for sample positioning, focusing, laser power adjustment, and region-of-interest selection. The preview function displays images in intervals on the order of 1 second and less, see Fig. 83.
Fig. 83: SPCM software in fast preview mode. 1 second per image.
Any change in the microscope parameters, such as sample position, scan area, the zoom factor, depth of focal plane, pinhole size or laser power, becomes immediately visible in the preview images, see Fig. 84.
Fig. 84: When the scan area definition in the Zeiss ZEN software is changed the result becomes visible in the images immediately.
Frequently used operation modes and user interface configuration can be selected from a panel of predefined setups. Switching between Preview modes, FLIM acquisition, different pixel and time-channel numbers, time-series recording, Z-stack recording, FCS, or any other conceivable recording procedure is a matter of a single mouse click, see Fig. 85.
Fig. 85: Switching the instrument configuration via the ‘Predefined Setup’ panel
The bh FLIM systems are perfectly compatible with the fast beam scanning used in the Zeiss microscopes. Frame times can be from about 50 ms to a few seconds, with pixel dwell times down to one microsecond. The multi-dimensional TCSPC process used in the bh FLIM systems delivers identical results for different scan rates, provided the total acquisition time is the same. FLIM can be acquired at short acquisition time. Fig. 86 shows lifetime images of a BPAE cell recorded within 5 seconds acquisition time.
Fig. 86: FLIM acquired within 5 seconds of acquisition time. Left 485 to 560 nm, right 560 to 650 nm. BPAE cell stained with Alexa 488 and Mito Tracker Red.
Starting from Version 9.72 SPCM software the FLIM systems for the Zeiss LSM 710 / 780 / 880 family are able to display lifetime images online, both during the accumulation of FLIM data and for the individual steps of a fast image sequence, see page 379 of this book. An example is shown in Fig. 87. The calculation of the lifetime images is based on the first moment of the decay data in the pixels of the images. The first-moment technique combines short calculation times with near-ideal photon efficiency. It does not require to reduce the time resolution (time channels per pixel) to obtain high image rates. Even if the fast online lifetime function is used during the FLIM acquisition the data can later be processed by precision SPCImage multi-exponential data analysis.
Fig. 87: Intensity image (left) and online lifetime image (right) calculated by SPCM software
With Version 9.73 SPCM Software, the bh TCSPC / FLIM systems are able to record FLIM with bidirectional scanning. As usual, data recording is synchronised with the scanning by frame clock, line clock, and pixel clock pulses from the scanner. Each first line clock pulse indicates the beginning of a forward scan, each second one the beginning of a backward scan. The recording procedure automatically reverses the data from the backward scan and compensates for the line shift caused by the dynamic behaviour of the scanner. The FLIM data structure is the same as for unidirectional scanning. Thus, standard online intensity and lifetime display functions of the SPCM software are available, and data can be analysed by SPCImage as usual.
Fig. 88: FLIM of Convallaria sample (left, 512x512 pixels) and BPAE Cell sample (right, 1024x1024 pixels), recorded with bidirectional scanning. Images created by online-lifetime function of SPCM software.
Pixel numbers of FLIM data can be increased up to 2048 x 2048. Fig. 89 shows a FLIM image of a BPAE sample recorded at a resolution of 1024 x 1024 pixels. The image on the left shows the entire area of the scan. The image on the right have been selected from the full scan by the zoom function of the SPCImage data analysis software.
Fig. 89: Left: Image recorded with 1024 x 1024 pixels. Right: Digital zoom into the data of Fig. 89, showing the two cells on the upper left. Effective resolution 256x256 pixels. LSM 710 Intune system, excitation 535 nm, emission from 550 nm up. BPAE cell stained with Alexa 488 and Mito Tracker Red.
Standard bh FLIM systems record in two wavelength intervals simultaneously. The signals are detected by separate detectors and processed by separate TCSPC modules. There is no intensity or lifetime crosstalk. Even if one channel overloads the other channel is still able to produce correct data.
Fig. 90: Dual-channel detection. BPAE cells stained with Alexa 488 phalloidin and Mito Tracker Red. Left: 460 nm to 550 nm. Right: 550 nm to 650 nm.
The bh Multiphoton FLIM systems use the non-descanned detection (NDD) path of the LSM 710/780/880 NLO or LSM 7 MP microscopes. With non-descanned detection, fluorescence photons scattered on the way out of the sample are detected far more efficiently than in a confocal system. The result is that clear images are obtained from deep tissue layers. An example is shown in Fig. 91.
Fig. 91: Two-photon FLIM of pig skin. LSM 710 NLO, excitation 800nm, HPM‑100‑40, NDD. Left: Wavelength channel <480nm, colour shows percentage of SHG in the recorded signal. Right: Wavelength channel >480nm, colour shows amplitude-weighted mean lifetime.
The LSM 710 confocal microscopes are available with fully integrated FLIM lasers based on bh / Lasos BDL‑SMC picosecond diode lasers. Together with the superior efficiency of the bh hybrid detectors and of the Zeiss LSM 710 / LSM 780 scan head FLIM is performed at excellent sensitivity, see Fig. 92.
Fig. 92: Confocal FLIM with diode-laser excitation. Left: Plant tissue, autofluorescence. Right: HEK cell, interacting proteins, FRET from GFP into RFP.
Tuneable-excitation FLIM uses the ‘Intune’ laser of the Zeiss LSM 710 / 780 systems. With the Intune laser FLIM images of the same sample can be obtained for different excitation wavelength, see Fig. 93.
Fig. 93: Confocal
FLIM with tuneable ‘Intune’ laser. BPAE cells stained with Alexa 488 phalloidin
The bh multispectral FLIM system detects simultaneously in 16 wavelength intervals . By using bh’s multi-dimensional TCSPC process it avoids any time gating or wavelength scanning. The systems thus reach near-ideal recording efficiency. Dynamic effects in the sample or photobleaching do not cause distortions of the spectra or decay functions. An example for confocal detection and one-photon (diode-laser) excitation is shown in Fig. 94.
Fig. 94: Confocal multispectral FLIM. Part of a water flee, excitation by 405 nm ps diode laser, LSM 710 confocal port, bh MW FLIM detector
bh’s MW FLIM is the world’s first simultaneously detecting multiphoton multispectral NDD FLIM system. It uses an optical interface that connects to the NDD ports of the LSM 710/780/880 NLO microscopes . A typical result is shown in Fig. 95.
Fig. 95: Multiphoton Multispectral NDD FLIM. LSM 710 NLO, bh MW FLIM detector
For many years, Z-stack FLIM had been prevented by photobleaching and photodamage caused by the high excitation dose. With the LSM 710 family microscopes and the new GaAsP hybrid detectors photobleaching is no longer a problem. The FLIM system automatically acquires FLIM in consecutive Z planes and saves the data into a sequence of files. Z‑stack FLIM is particularly interesting in combination with the deep-tissue imaging capability of multiphoton NDD FLIM. It can, however, be used also in combination with diode-laser FLIM and Intune FLIM. An example is shown in Fig. 96.
Fig. 96: Z-stack of FLIM images. Pig skin, autofluorescence, 5 µm to 60µm below the surface. LSM 710 Multiphoton NDD FLIM, GaAsP hybrid detector
Time-series FLIM by the traditional record-and-save procedure is available for all system versions, and all detectors. Time-series FLIM can be recorded at a rate fast as 1 images per second, see Fig. 97.
Fig. 97: Moving amoeba. Autofluorescence, acquisition time 0.5 s, image rate 1 image per second.
Mosaic FLIM records an array of FLIM images. The ‘elements’ of the mosaic may represent images for different lateral offset obtained by a sample-stepping device, images in different wavelength channels of a multi-wavelength detector, images in different depth of a sample, or images at different times after a stimulation of a sample. The entire mosaic is recorded into one single, large photon distribution. Mosaic FLIM can thus be used to increase the field of view of a high-NA microscope lens, to record multi-wavelength FLIM data, to record FLIM Z-stacks, or to record fast FLIM time series. An advantage is that no time for readout operations has to by reserved. This significantly simplifies the parameter setup for Z stack recording, and allows extremely fast time series to be recorded. A time series can even be accumulated, thus avoiding the need of trading lifetime accuracy against speed of the sequence. Examples of a spatial and a temporal mosaic are shown in Fig. 98. A Z stack recorded by mosaic FLIM is shown in Fig. 100.
Fig. 98: Mosaic FLIM with the Zeiss LSM 710 / 780 / 880 microscopes. Left: Spatial mosaic, convallaria sample, 16 elements of 512 x 512 pixels, 256 time channels, entire mosaic is 2048 x 2048 x 256. Right: Temporal mosaic, Ca2+ transient in cultured neurons, electrical stimulation, 64 elements of 64 x 64 pixels, time per element 38 ms. 100 stimulation periods accumulated. Time runs from upper left to lower right, the stimulation was applied during the 4th element.
Near-infrared FLIM can be performed by using the Ti:Sa laser of a multiphoton (NLO) microscope as a one-photon excitation source [90, 143] or by two-photon excitation with an LSM 780 OPO system . Please see page 354 for details. Images are shown in Fig. 99. Because near-infrared light penetrates thick layers of tissue clear images from deep sample layers are recorded. This makes NIR FLIM especially attractive in combination with Z stack recording. An example is shown in Fig. 100.
Fig. 99: Left: Pig skin sample stained with DTTCC. Zeiss LSM 710 NLO, one-photon excitation with Ti:Sa laser. Right: Pig skin stained with methylene blue. OPO system, two-photon excitation at 1200 nm
Fig. 100: Z stack recorded at a pig skin sample stained with Indocyanin Green. OPO excitation, Z-stack recorded by Mosaic FLIM, 16 planes from 0 to 60 µm from top of sample, each plane 512x512 pixels, 256 time channels.
In combination with the LSM 710 / 780 / 880 family multiphoton microscopes the bh FLIM systems are able to simultaneously record fluorescence and phosphorescence lifetime images. The technique is based on modulating the excitation laser synchronously with the pixel clock of the scanner. Photon times are determined both with reference to the laser pulses and the laser modulation pulse [94, 135]. Please see page 388 for details. A typical result is shown in Fig. 101.
Fig. 101: Yeast cells stained with a Ruthenium dye. Left: FLIM image and fluorescence decay curve in selected spot. Right: PLIM image and phosphorescence decay curve in selected spot.
FLITS records transient effects in the fluorescence lifetime of a sample along a one-dimensional scan. The maximum resolution at which lifetime changes can be recorded is given by the line scan time. With repetitive stimulation and triggered accumulation transient lifetime effects can be resolved at a resolution of about one millisecond [61, 101, 136]. Please see page 374. FLITS of the Ca2+ transients in cultured neurons on electrical stimulation is shown in Fig. 102.
Fig. 102: FLITS of Ca2+ transient in cultured neurons. Left: FLIM image, location of one-dimensional FLITS scan indicated. Right: FLITS image, FLITS time scale 0 to 512 ms, resolution 2 ms, 200 stimulation periods accumulated.
The GaAsP hybrid detectors of the bh FLIM systems deliver highly efficient FCS. Because the detectors are free of afterpulsing diffusion times are obtained from a single detector, without the loss in correlation events that occurs when the signals from two detectors are cross-correlated. FCS is be obtained with the diode-laser systems, the Intune system, with the CW lasers of the LSM, and with the Ti:Sa laser of the multiphoton systems.
Fig. 103: FCS with GaAsP hybrid detectors. Left to right: Confocal FCS with ps diode laser, Confocal FCS with Intune laser, Two-photon NDD FCS
SPCImage FLIM data analysis is integrated in the TCSPC software package delivered with the Systems. For single-molecule burst-analysis bh ‘Burst Analysis’ software is available. Please see Fig. 149, page 81 and Fig. 152, page 83.
The Leica SP2, SP5 and SP8 MP multiphoton microscopes use a Ti:Sapphire laser for excitation. The Ti:Sapphire laser is also used for FLIM excitation. Thus, no changes or additions on the excitation side of the microscope are required [63, 77, 79].
FLIM detectors can be used both in a descanned and a non-descanned optical path. Non descanned detection is, of course, superior in detecting photons from deep sample layers. It requires the ‘RLD port adapter’ of Leica to be installed at the microscope. Detector/shutter assemblies for NDD FLIM with adapters to the Leica RLD port are available from bh.
The whole range of detectors can be used, i.e. the PMC‑100 for standard applications and fast-acquisition FLIM, the R3809U or HPM-100-07 for high time resolution, the HPM-100-40 hybrid detector for highest efficiency. Also the bh MW-FLIM assembly for multi-spectral FLIM can be used. The R3809U, the PMC-100, and the HPM-100 detectors with the Leica RLD adapters are shown in Fig. 104.
Fig. 104: NDD FLIM detectors with adapters to the Leica RLD port. Left to second right: R38909U with shutter, PMC‑100, HPM‑100-40. Right: PMC‑100 detector attached to Leica RLD port.
Dual-detector systems can be assembled by using external beam splitters available from bh (see Fig. 136).
FLIM detectors for multiphoton microscopes can also be attached to the ‘X1’ port of the Leica SP2 or SP5 scan head. All bh FLIM detectors can be attached directly to this port.
Recently, Leica have introduced hybrid detectors that deliver the single-photon pulses of the detected signals to external photon counters. The pulses are directly compatible with the inputs of the bh TCSPC FLIM modules. A non-descanned FLIM detector is shown in Fig. 105, left, a FLIM image recorded from a BPAE sample in Fig. 105, right.
Fig. 105: Left: Leica dual-channel hybrid detector with FLIM outputs, attached to the non-descanned (RLD) port of an SP8 multiphoton microscope. Right: FLIM image recorded from a BPAE sample, 512x512 pixels, 256 time channels. bh Simple-Tau 152 TCSPC FLIM system
A frequent concern is that the sinusoidal scan used in the Leica SP 5 and SP8 microscopes may cause a nonlinearity in the x coordinate of the FLIM images. This is, however not the case. The microscopes compensate for the nonlinearity of the scan by a variable pixel clock period. Since the bh FLIM systems use the pixel clock to synchronise the acquisition with the scan the x coordinate of the image remains a linear function of the x coordinate of the sample [80, 142].
One-photon FLIM systems for the FV 1000 use one of the fibre inputs of the scan head to connect a bh BDL-SMC or -SMN picosecond diode laser [64, 71, 72]. The FLIM detectors are connected to a confocal output from the FV 1000 scan head. A wide variety of detectors can be used. PMTs are coupled directly, i.e. without any optical fibre, to the FV 1000. The fibre adapter is removed (or not installed altogether), and an a direct coupling optics attached instead. The FLIM beam pass contains a long-pass or bandpass filter and, in case of the R3809U detector, an electronically controlled shutter. Details are shown in Fig. 106.
Fig. 106: Coupling an R3809U MCP PMT to the FV 1000 scan head
Multi-wavelength FLIM is possible by using the PML-SPEC spectral detection assembly , see page 170 and page 347 of this handbook. The PML‑SPEC assembly can be connected both via the fibre output from the FV 1000 scan head or via a fibre bundle attached to the FLIM adapter for PMTs.
Non-descanned FLIM systems are available for the inverted (IX) versions of the FV 300 and FV 1000 multiphoton microscopes. Once the Olympus NDD port is installed, the PMC‑100 detector, the R3809, the HPM‑100-40, or the MW FLIM detector can be used as shown in Fig. 107. A lifetime image obtained by a bh standard FLIM system attached to an FV 300 multiphoton is shown in Fig. 108. Please see [77, 81] for details.
Fig. 107: FLIM detectors
at the non-descanned port of an
Fig. 108: Left: Simple-Tau 150 system for Olympus FV 300 or FV 1000 MP. Right: FLIM of heart tissue sample, fluorescence from endogenous fluorophores and SHG from the collagen in the tissue.
The bh FLIM systems are also available for the Nikon C1, A1 and A2 laser scanning microscopes. One-photon FLIM systems use one or several BDL‑SMN ps diode lasers for excitation. The lasers are connected to a fibre input port of the scan head. In confocal systems bh HPM‑100‑40 hybrid detectors are connected to the fibre output of the scanner, see Fig. 109, left. In multiphoton systems the detectors are preferably connected to an optional C-Mount NDD port at the Nikon non-descanned detector module, see Fig. 109, right. Multi-wavelength detectors are either coupled to the confocal port via a single optical fibre or to the NDD port via a fibre bundle.
Fig. 109: Left: Confocal A1 system. Two hybrid detectors (lower left) connected to the confocal fibre port of the A2. Right: A2 multiphoton system. Hybrid detector (left) connected to the NDD detector module.
The Sutter Instrument MOM microscope  is a modular platform for imaging deeply within live samples. It uses multi-photon excitation by a titanium-sapphire laser in combination with non-descanned detection. Due to its pulsed excitation source and its high modularity the MOM system can easily be combined with the bh TCSPC FLIM systems . Up to four FLIM detectors can be attached to the system. The signals are processed in up to four entirely parallel TCSPC FLIM channels. A setup with two detectors is shown in Fig. 110. Due to the parallel system architecture, high photon count rates and short acquisition times can be achieved. FLIM data can be recorded with up to 1024x1024 pixels and 1024 time channels.
Fig. 110: Left: Two FLIM detectors attached to Sutter MOM microscope. Right: Salmon louse (Lepeophtheirus Salmonis), 512 x 512 pixels. Autofluorescence, excitation wavelength 750 nm, detection wavelength 440 to 480 nm. Amplitude-weighted lifetime of double-exponential decay.
The combination of the bh TCSPC-FLIM system with the Sutter MOM microscope is an efficient and flexible solution to fluorescence lifetime imaging of live cells and live tissues. The system can be used for all the typical FLIM applications, such as metabolic imaging by recording the fluorescence of NADH and FAD, protein interaction experiments by FLIM-FRET techniques, and ion concentration measurements with environment-sensitive fluorescent dyes. With an additional DDG‑120 pulse generator card it can also record simultaneous FLIM / PLIM. For technical details and parameters setup please see .
The combination of the Abberior STED system  with the bh Simple-Tau TCSPC FLIM system records FLIM data at a spatial resolution of better than 40 nm [99, 100]. The FLIM system can either be integrated in the Abberior microscope software or operated independently via bh SPCM software. The image format can be as large as 2048 x 2048 pixels with 256 time channels per pixel, or 4096 x 4096 pixels with 64 time channels. An image area of 40 x 40 micrometers can thus be covered with <20 nm pixel size, fully satisfying the Nyquist criterion. The system especially benefits from Windows 64 bit technology used both in the Abberior and in the bh data acquisition software, and the high data throughput of up to four parallel TCSPC FLIM channels. The system uses fast galvanometer scanning, and achieves peak count rates in excess of 5 MHz per FLIM channel, resulting in unprecedented signal-to-noise ratio and short acquisition time. The system can thus be used with the online-FLIM display function of the SPCM software. Technical details are described in  and . Please see also page 403 of this book. To our knowledge, the Abberior systems achieves the best spatial resolution and the best sensitivity of all STED systems currently available. This is probably a result of dynamic wavefront correction used in the Abberior optics. A typical image is shown in Fig. 111.
Fig. 111: FLIM image recorded with the Abberior - bh combination. Tubulin fibres in a BPAE cell, recorded with 2048 x 2048 pixels and 256 time channels per pixel.
The PZ‑FLIM-110 Piezo Scanning FLIM system uses bh’s multi-dimensional TCSPC technique in combination with a piezo scanner. The scanner is controlled via a bh GVD-120 scanner control card, the FLIM data are recorded by an SPC-150 or SPC-160 TCSPC / FLIM module. Data acquisition is controlled by 64 bit bh SPCM TCSPC software. The system is able to run X-Y scans, X-Z (vertical) scans, and to record simultaneously FLIM and PLIM data. Maximum FLIM data formats are 512 x 512 pixels, 4096 time channels, or 2048 x 2048 pixels, 256 time channels.
Fig. 112: Left: Photo of the bh PZ-FLIM-110 piezo scanning system. Right: Lifetime image of a convallaria sample. 512 x 512 pixels, 1024 time channels per pixel.
Sample scanning yields high efficiency and good optical resolution. Problems do, however, arise from the slow scan speed of the piezo stage. Under typical imaging conditions, the acquisition time is determined by the speed of the scanner, not by the time needed to acquire the desired number of photons. Scan times for 512 x 512 pixel images are on the order of 100 seconds, scan times for 2048 x 2048 pixel images are at least 6 minutes. These scan times may be acceptable for high quality FLIM but not for imaging dynamic effects in a life sample. Moreover, the slow scan rate makes focusing into a defined image plane within a sample difficult. If these restrictions are taken into account the PZ‑FLIM-110 is a cost-efficient alternative to a DCS‑120 galvanometer scanner system. Please see  and page 401 of this book for technical details.
The WF-FLIM-01 wide-field FLIM system uses a Photek FGN 392‑1000 position-sensitive detector. This detector is an MCP PMT with a delay-line structure coupled to a resistive anode [105, 145]. The photoelectrons emitted by the photocathode are accelerated towards the microchannel plate. In the microchannel plate the electrons are multiplied by a factor of 106 or more. The electron clouds of the individual photoelectrons are injected in a delay line structure at the output of the tube. The X position of a photon is proportional to the delay between X1 and X2, the Y position of a photon is proportional to the delay between Y1 and Y2. The time of the photon is derived from a signal from the low-side of the microchannel plate, t.
The TCSPC system consists of three synchronised SPC‑150N modules. The first module measures the times of the photons in the laser pulse period. The second and third module measure the relative times of the pulses at the outputs of the X and the Y delay lines. The detection events, i.e. the times, t, and the positions, x and y, are transferred into the computer photon by photon. From these data the software builds up a photon distribution over the coordinates, x, y, and the times, t. This is the usual photon distribution of FLIM: An array of pixels each of which contains a fluorescence decay curve consisting of photon numbers in consecutive time channels.
The optical system consists of an inverted microscope, a bh BDS-SM family picosecond diode laser, and the Photek FGN 392-1000 detector. The excitation light is delivered into the microscope by a single-mode fibre. The microscope beam-splitter cube reflects the laser towards the microscope lens. Fluorescence excited in the sample passes the dichroic mirror of the beamsplitter cube and an emission filter. It is directed out of the microscope via one of the side ports. An achromatic negative lens a few cm in front of the image plane magnifies the image to match the active area of the detector. A shutter protects the detector against excessive light intensities. Details are described in  and . Please see also page 410 of this book.
The data obtained with the system feature good time resolution, and reasonably good spatial resolution. A typical result is shown in Fig. 113.
Fig. 113: Wide-field FLIM of an Invitrogen BPAE cell sample. 512x512 pixels, 1024 time channels per pixel. Data analysis by SPCImage FLIM analysis software
Compared to scanning systems, the system does, however, suffer from the general problems of wide-field imaging: Missing suppression of out-of-focus fluorescence and lateral scattering, and contamination by fluorescence and scattering in the optics . These effects restrict the use of the system to thin samples with low internal scattering. Possible applications are prism-type TIRF and light-sheet microscopy which are inherently wide-field . Another application may be combined FLIM/PLIM with phosphorescence markers of millisecond lifetimes. Such long lifetimes require extremely slow scanning but do not pose problems to wide-field FLIM. Wide-field FLIM may also by useful for recording fast physiological processes in cells.
BH deliver a number of picosecond diode lasers with wavelengths from the NUV to the NIR [70, 72, 71]. All lasers feature simple +12V power supply, high repetition rate, short pulse width, and an extremely low electrical noise level. The complete driver electronics is integrated in the laser module. All bh diode laser modules are directly compatible with the bh TCSPC modules.
bh BDL series lasers are manufactured in cooperation of bh and Lasos GmbH,
The corrected beams of the BDL-SMC lasers can be efficiently coupled into single-mode fibres. The coupling efficiency is 50 to 70 %. Both a version with Point-Source compatible couplers  and a version with a pig-tail fibre is available. A BDL-SMC laser, the beam profile, and typical pulse shapes are shown in Fig. 114, lasers with fibre coupling are shown in Fig. 115.
Fig. 114: BDL-SMC laser with beam-profile corrector (left), beam profile (middle) and typical pulse shapes for different average power at a repetition rate 50 MHz (right)
Since 2013, the BDL-SMC lasers have been more and more replaced with the more advanced BDL‑SMN version. Please see section below.
The BDL-SMN lasers  are advanced successors of the BDL-SMC lasers. As the BDL-SMC lasers, they are manufactured in cooperation of bh and LASOS. The SMN lasers have the same mechanical dimensions, the same beam correction optics, and the same control features as the SMC-Lasers. However, there are several advanced features.
The optical power is stabilised by an internal regulation loop also in the pulsed mode. The result is low optical noise and excellent power stability both in the CW and in the pulsed mode, and a strictly linear power control characteristic. The SMN lasers use improved driver electronics, resulting in improved pulsed shape at high pulse power. The SMN lasers can thus be run at substantially higher power than the SMC lasers. For some laser wavelengths, an average power of 8 mW (at 80 MHz) can be reached. The pulse width at medium power is typically 50 to 80 ps. It usually stays below 200 ps up to 4 to 8 mW. Please see Fig. 117.
Fig. 116: BDL-SMN laser, pulse shapes for four typical laser wavelengths. 80 MHz, power measured in free beam.
The lasers have a synchronisation output the bh TCSPC modules, and a trigger input for synchronisation with other BDL-SMN lasers or other pulsed excitation sources. As the SMC lasers, the SMN version is available with a free-beam output and with various single-mode fibre couplers, see Fig. 116.
Fig. 117: BDL-SMN lasers
The BDL‑Series lasers are part of the bh FLIM systems for laser scanning microscopes [61, 63, 64]. The are also used in the bh DCS‑120 Confocal Scanning FLIM System . There are separate manuals for the BDL-SMC and the BDL‑SMN lasers, please see  and .
The BDS-SM lasers  are OEM Modules with a size of only 40 mm x 40 mm x 110 mm. The lasers contain the entire driver electronics. They are operated from a simple +12V power supply.
The BDS ps diode lasers are available both with free-beam and single-mode fibre output. The pulse width is on the order of 50 to 90 ps, the pulse repetition rate can be switched between 50 MHz and 20 MHz. Recently, a third repetition rate has been added. All the typical diode laser wavelengths from 375 nm to 785 nm are available. The BDS lasers use the same driver principle as the BDL-SMC lasers. Thus, high optical power at good pulse shape is available, see Fig. 118. The output power is stabilised by an internal regulation loop, and fast on-off switching is implemented. The lasers have a synchronisation output to the bh TCSPC modules and a trigger input for synchronisation with other pulsed lasers.
Fig. 118: BDS-SM laser, pulse shape for different output power. 473 nm and 640 nm, power measured at fibre output.
Fig. 119: BDS-SM series laser with pig-tail single-mode fibre, Qioptiq Kineflex adapter, Lasos Precision Connector, and free-beam output through C-Mount adapter
Over several years, improvement in laser diode performance has resulted in a constant decrease in the pulse width of the BDS-SM lasers. Currently, the 375 nm, 405 nm, and 445 nm versions achieve pulse widths of less than 40 ps. These pulse widths are achieved in an intensity range where the lasers deliver clean pulse shapes, without much afterpulsing or large shoulders. Examples are shown in Fig. 120.
Fig. 120: Pulse shapes of a 405 nm BDS-SM laser. SPC-150N with HPM-100-07 detector. The recorded FWHMs are below 40 ps, corrected for the detector IRF of 19 ps the laser pulse width is about 35 ps.
The BDS-MM lasers  are multi-mode versions of the BDS-SM lasers. Depending on the wavelength version, the CW equivalent power at 50 MHZ repetition rate can be as high as 20 to 50 mW. In most cases, the pulse shape remains free of tails and afterpulses up to more than 10 mW. A few compromises had to be made, however: Due to limitations in power dissipation the MM lasers have no CW mode, and the light is difficult to focus into optical fibres. If possible, the BDS-MM lasers should be used with free-beam optics or, if fibre coupling is unavoidable, with multi-mode fibres with core diameters of 300 µm or larger.
Fig. 121: Left: BDS-MM laser, with 300 µm fibre. Right: Pulse shape for a 640-nm version. Indicated power is for 50 MHz repetition rate, and measured in free beam.
There is a number of older bh lasers which are no longer manufactured. The BHL-600 modules have wavelengths from 635 nm to 1300 nm . They were optimised for short pulse width. Due to their short pulse width down to 50 ps they can be used for fluorescence excitation from 635 nm to 780 nm and for testing purposes.
The BHLP-700 modules are available for wavelengths from 680 nm to 980 nm . They are optimised to deliver an output power of up to 10 mW (CW equivalent) at 50 MHz repetition rate. As the BDL lasers, the BHLP modules have a fast multiplexing input.
Fig. 122: Red and NIR ps diode lasers. BHLP-700 (left) and BHL-600 (right)
A laser beam combiner is available for up to four lasers of different wavelength. The beams from four single-mode fibres are combined into a single collimated output beam or a single-mode fibre. The beam combiner is permanently aligned.
Fig. 123: Beam combiner for four lasers
An AOM (Acousto-Optical Modulator) for the Ti:Sa lasers of multiphoton laser scanning microscopes is available from bh, see Fig. 124, left. The AOM is part of the DCS-120 MP system but can also be used for other applications that require control or modulation of a femtosecond laser. The control of the Ti:Sa laser and the control of the AOM are integrated in the same SPCM software panel, see Fig. 124, right. This way, the driver frequency of the AOM is automatically adjusted to the wavelength of the laser. Please see page 738 for details.
Fig. 124: Left: AOM for Ti:Sa lasers. Right: Control panel in the SPCM data acquisition software.
The DCC-100 card (see Fig. 138, page 76) is able to control the output power of one or two bh ps diode lasers. For systems with one laser and one detector both can be controlled by a single DCC-100 card. For details, please see page 188 and page 734 of this handbook.
For on-off modulation and multiplexing of the BDL and the BDS series lasers the DDG‑210 digital delay generator is available. The DDG‑210 provides on/off signals for up to four lasers and the corresponding routing signal for the TCSPC module.
Fig. 125: DDG-210 digital delay generator
On/off modulation is used to record fluorescence signals, time-of-flight distributions, and FLIM images for several excitation wavelengths simultaneously. It is also used for simultaneous fluorescence and phosphorescence decay detection and simultaneous FLIM/PLIM. Please see page 388 and page 737 of this handbook.
The frequency of the multiplexing sequence and the duty cycle of the individual lasers within the sequence are fully programmable. The sequence can also be triggered, such as to synchronise laser on/off operation with the pixel clock of a scanner.
bh ps diode lasers can also be controlled by the GVD-120 scan controller card. The GVD-120 controls both axis of a galvanometer scanner, and two lasers. It provides for intensity control, beam blanking in the flyback phases of the scanner, laser multiplexing within one pixel, or between subsequent lines and subsequent frames. Pixel-synchronous on-off modulation for FLIM/PLIM is provided by multiplexing function within the individual pixels. A photo of the GVD-120 is shown in Fig. 143, page 78. For a description of the GVD functions please see  and page 729 of this handbook.
The bh TCSPC modules can be used with almost any photon counting detector. For reasonable operation it is only required that the detector delivers a reasonably fast and stable single-electron response. This section gives a brief description of the detectors typically used with the bh photon counters. For a detailed description and discussion of the parameters of various detectors please see section 'Detectors for TCSPC'.
In earlier TCSPC applications often the bh PMH-100 detector head was used, see Fig. 126, left. This device contains a fast, small PMT, the high voltage generator and a preamplifier in a 32x38x92mm housing. The PMH‑100 was powered directly from an SPC module or a DCC-100 card. By now, the PMH‑100 has almost entirely been replaced with the PMC‑100 detector.
The PMC-100, shown in Fig. 126, middle, is a cooled version of the PMH‑100. The PMC‑100 is fully controlled via the bh DCC‑100 detector controller, see Fig. 138. The DCC‑100 provides for power supply, gain control, and overload shutdown. The PMC detector comes in different cathode versions. The PMC‑100-0 has a bialkali cathode with good efficiency in the wavelength range from 330 to 600 nm. The PMC‑100-1 has a bialkali cathode. This cathode can be used from about 330 to 800 nm. UV-sensitive versions are available as well. The PMC‑100-20 has a ‘high efficiency extended-red’ multialkali cathode. This cathode works up to almost 900 nm and red and near-infrared range yields a noticeably higher sensitivity than the conventional multialkali cathode.
Since 2016, there is an improved version of the PMC-100 detectors. The new PMC-150 detectors are functionally identical with the PMC-100 but contain PMTs with higher cathode efficiency and better time resolution. Please see page 159 of this book.
The PMZ-100 (Fig. 126, right) was originally designed as a FLIM detector for the NDD ports Zeiss LSM 710 NLO microscopes. It has its active area very close to the front face, and an adapter to the LSM 710 NDD ports. In the meantime, it has almost entirely been replaced with HPM-100 hybrid detectors. Currently, the PMZ-100 modules are used as FLIM detectors for the Sutter MOM microscopes where the HPM-100 detectors are to large and heavy. Also the PMZ-100 has been upgraded by new PMTs, and thus got improved sensitivity and time resolution.
All these detectors have an exceptionally stable timing response function at high count rates, see .
Fig. 126: Left to right: PMH-100, PMC‑100 and PMZ‑100 PMT modules
There is a number of other PMT modules that easily integrate into the bh TCSPC system. Please see chapter ‘Detectors for TCSPC’ of this handbook.
For several decades, fastest available single-photon detectors were MCP PMTs of the Hamamatsu R3809U family. An detector-shutter assembly with an R3809U is shown in Fig. 127. The R3809U needs a high-voltage power supply that delivers an operating voltage of -3000 V. A power supply module is available from bh, see Fig. 127, right. The module is controlled via the bh DCC‑100 detector controller.
Fig. 127: R3809U MCP PMT (left) and bh HVM‑100 power supply module (right)
The R3809U is commonly believed to deliver about 25 to 30 ps (FWHM) instrument response width in TCSPC applications. Recent experiments with the bh SPC‑150NX modules have shown that the detectors are even faster: IRF measurements with a 100 fs fibre lasers showed than an IRF width of <20 ps can be achieved . Please see page 153 of this book.
Hybrid detectors are currently the most promising light sensors for TCSPC. The detectors are extremely sensitive, have a clean TCSPC response function, and are free of afterpulsing background [75, 133]. Recently, it turned out that some hybrid detectors (with bi-alkali and multialkali cathodes) yield sub-20 ps FWHM instrument response width . Please see section ‘Description of Selected Detectors’ of this handbook. Bare hybrid detector tubes are difficult to use because the output pulse amplitude is low, and a cathode voltage of -8000 V is needed. The detectors can only be reasonably used if the tube, the preamplifier, and the high voltage generators are integrated in a perfectly shielded housing. bh deliver several versions of hybrid detector modules: The HPM-100-40 with a GaAsP cathode, the HPM-100‑50 with a GaAs cathode, and the HPM-100-06 and HPM-100-07 with bi-alkali and multialkali cathodes. The HPM-100 modules are available with different diameter of the active area, with C-Mount adapters, with fibre adapters, and with adapters to the Zeiss LSM 710/780/880 family laser scanning microscopes. Cooled versions are available as well, see Fig. 128. All HPM-100 detectors are controlled by the DCC-100 detector controller. Please see page 161 for details.
Fig. 128: bh PMH‑100 hybrid detector modules
For multichannel measurements, e.g. spectrally resolved lifetime measurements, the bh PML‑16‑C detector head is available . The PML‑16‑C is shown in Fig. 129, left. It contains a 16-channel multi-anode PMT and the routing electronics that connects all PMT channels to a single TCSPC channel. The PML‑16‑C has an internal high-voltage generator, i.e. no external high-voltage power supply is required. The PML‑16‑C is fully controlled via the DCC‑100 detector controller, which provides for power supply, gain control, and overload shutdown. The PML‑16‑C detector is the basis of the PML‑SPEC multi-wavelength detection modules and the MW‑FLIM multi-spectral FLIM detection assembly.
PML-Spec consists of a bh PML-16‑C sixteen-channel PMT module  and a polychromator. With a bh TCSPC module 16
wavelength channels are recorded simultaneously. Typical applications of the
PML-Spec are single-point autofluorescence measurements of biological tissue
and multi-spectral time-resolved laser scanning microscopy. The MW-FLIM system,
shown in Fig. 129, right, is a spectral detection module optimised for
direct (non-descanned) detection in multi-photon laser scanning microscopes.
The multi-spectral detection modules can be used in all bh FLIM systems,
including the DCS‑120 confocal scanning FLIM system, and the FLIM systems
for the Zeiss, Leica,
Fig. 129: Left: PML-16-C multi-channel PMT module. Right: MW-FLIM 16-channel spectral detection unit for laser scanning systems
Since 2014 a PML version with a GaAsP cathode (PML-16 GaAsP) is available. The PML-16 GaAsP and the MW-FLIM GaAsP multi-wavelength FLIM detectors based on it have about five times the efficiency of the PML and MW FLIM versions with bi-alkali and multi-alkali cathodes. The IRF width is between 200 and 250 ps. An instrument response function and a fluorescence signal recorded by the MW FLIM GaAsP are shown in Fig. 130.
Fig. 130: Instrument response function (FWHM 212 ps, left) and fluorescence signal recorded with MW-FLIM GaAsP detector (right)
The PML‑16‑C, the PML‑SPEC, and the MW‑FLIM system are described in a separate handbook .
For measurements that require both high sensitivity and high time-resolution single-photon Avalanche diodes (SPADs) can be used. A SPAD from id Quantique is shown in Fig. 131, a SPAD from Micro Photon Devices in Fig. 132. The IRFs for a 20 µm version of idQuantique and for a 100 µm version of MPD are shown on the right. The id-100 is connected to the SPC module via an electrical attenuator and an A-PPI pulse inverter, see Fig. 146, the PDM-100 is coupled directly via its fast timing (Lemo connector) output.
Fig. 131: id100 SPAD. Left: C-mount adapter. Middle: Fibre-coupled version. Right: IRF measured with a 20 ps diode laser at 785 nm. Corrected for the pulse width of 20 ps, the IRF width is 40 ps, FWHM.
Fig. 132: Left: SPAD module from Micro Photon Devices (from www.micro-photon-devices.com). Right: Instrument response function of a PDM PD-100-CBT-FC measured with SPC-150NX TCSPC module and 100 fs fibre laser. The IRF width is 35.6 ps.
The bh SPC modules also work with other SPADs, such as the SPCM-AQR modules of Perkin Elmer / Excelitas or the COUNT modules of Laser Components. These modules have large area and high NIR sensitivity bot do not reach the time resolution of the idQ or MPD modules.
The bh SPAD-8 module contains 8 parallel SPAD channels. It contains an id150-1x8, a generator for the SPAD operating voltage, and the routing electronics for multi-dimensional TCSPC. The device is controlled via the bh DCC‑100 detector controller and connects directly to the bh SPC modules. Please see page 177 for details.
Fig. 133: Left: SPAD-8 eight-channel SPAD module. Right: Instrument response function.
InGaAs SPADs work in the spectral range from 900 nm to 1700 nm. For TCSPC operation, the devices must work in a continuous (none-gated) mode. This is the case for the id Quantique id 220 , and id 230 detectors . The ID230 delivers an instrument response width of 80 ps FWHM with the bh SPC-150 TCSPC module. This is the fastest TCSPC response reported for any commercially available InGaAs SPADs. The detector we tested had less than 300 dark counts when operated with a dead time of 40 µs. Optical signals as weak as 800 photons per second count rate could be detected at high signal-to-background ratio . For details please see  or pages 178 and 181 of this handbook.
Fig. 134: Left: id 230 InGaAs SPAD detector. Right: Instrument response function with SPC‑150
NbN detectors compatible with the bh SPC modules are available from SCONTEL,
Fig. 135: Left: SCONTEL TCORPS-UF-10 detector. Right: Instrument response function with SPC-150 NX
Photon counting detectors, especially MCP-PMTs, can easily be damaged or destroyed by overload. Even when an MCP or PMT is switched off the cathode performance is impaired temporarily if the cathode is exposed to a high light intensity . Especially in microscopy applications the microscope lamp - usually a mercury, xenon or halogen lamp - is a potential source of detector damage. A simple operator error can destroy one or several detectors. BH deliver detector/shutter/beamsplitter assemblies for one, two, and four detectors. The assemblies contain a field lens for efficient light transfer in the non-descanned beam path of two-photon laser scanning microscopes. The assemblies can also be used with fibre adapters at the inputs. The can be used with a wide variety of detectors, including the R3809U, the H7422, the PMH-100, the PMC-100, and the PML‑16C. Although primarily developed for laser scanning microscopes, the detector assemblies can be used for any application in which detector overload cannot be reliably excluded. Please see .
Fig. 136: Detector assemblies. Left to right: One R3809U MCP with shutter, two R3809U with shutter and beamsplitter, the PMH-100 with shutter and beamsplitter, four PMH-100 with three beamsplitters
A beamsplitter assembly for the BIG port of the Zeiss LSM 710 family microscopes is shown in Fig. 137, left. It uses standard microscopy beamsplitter cubes (Zeiss type) that can be configures with different dichroic mirrors and filters. The same assembly is available with a C‑mount thread input, see Fig. 137, right.
Fig. 137: Left: Beamsplitter assembly for BIG port of Zeiss LSM 710 family microscopes. Right: Beamsplitter with C-Mount thread input. Both shown with HPM‑100 hybrid detectors.
The bh DCC-100 and DCC-100 pcie modules  are used to control detectors in conjunction with bh photon counters. they provides power supply, gain control, and overload shutdown for two detectors. For cooled PMT modules the DCC also provides the power supply to the thermoelectric coolers. It can thus be used to operate a wide variety of detectors, including the bh PMC-100, the bh PML‑16C, the Hamamatsu H7422, H5783, H6773 or the Hamamatsu R3809U. The DCC‑100 and DCC-100 pcie provide also high-current digital outputs for controlling shutters or electromagnetic actuators. Due to its versatility the DCC modules are part of almost any bh TCSPC system. The DCC‑100 is a PCI module, the DCC-100 pcie a PCI Express module for IBM compatible computers, see Fig. 138. The modules work under Windows 7 and Windows 10.
Fig. 138: DCC-100 and DCC-100 pcie detector controllers. The DCC modules provide power supply, cooler current, gain control, shutter control and overload shutdown for two detectors. The control panel is shown on the right.
For TCSPC experiments with most PMTs and MCPs preamplifiers are recommended. For safe operation of MCPs and PMTs the HFAC-26 (26 dB, 1.6 GHz) and the HFAH amplifiers (26 dB, 1.6 GHz or 40dB, 500 MHz) with current sensing are available. These amplifiers indicate overload conditions in the detector by a LED and by a TTL signal. In combination with the DCC‑100 detector controller overload shutdown for the detector is provided. For multidetector measurements the HFAM-26 with eight amplifier channels is available. Other amplifiers are the ACA-2 and ACA-4 devices with gains from 10 dB to 40 dB and a bandwidth up to 2 GHz.
Fig. 139: HFAH series and HFAC series preamplifiers
The HRT-41 and HRT-81 routers are used to connect up to four (or eight) individual PMTs or MCPs to one bh SPC module. For SPAD modules with TTL and low-level TTL outputs the HRT-82 router is available. With the HRT devices, all detector channels work simultaneously and the detected photons are ‘routed’ into individual memory blocks. Please individual manual  or data sheets. In 2013 the HRT-41 got an upgrade. The internal discriminator threshold was made adjustable to better adapt the router to detectors with different single photon response, see Fig. 140, right. Connection diagrams are given in chapter ‘System Connection’, page 207 and ‘Cable Diagrams of FLIM Systems’, page 222.
Fig. 140: HRT-81 (left) and HRT-41 (middle and right) routers. The routers connect several detectors to a single TCSPC module
Hybrid detectors, such as the bh HPM-100 modules, deliver output pulses the amplitude of which depends on the number of simultaneously detected photons. This feature can by used to identify detection events as single-, double-, or triple-photon events and route then into different memory blocks of the SPC device. The principle is described in . Please see also page 597 of this handbook.
To generate the synchronisation signal for SPC modules from a laser pulse sequence fast photodiode modules are available. The bh PHD-400 and the bh PDM-400 use fast PIN photodiodes. The modules use single +12V power supply which is provided by the SPC card, the DCC-100 detector controller, or by a wall-mounted AC-DC adapter.
Fig. 141: PHD‑400 photodiode module
A new development became necessary for Ti:Sa lasers with wide tuning range. Over the tuning range, the output power of the laser changes by at least one order of magnitude. Simultaneously, the efficiency of the photodiode changes almost by the same ratio. That means the optical power needed to generate a reasonable synchronisation signal by a simple photodiode must be adjusted depending on the wavelength. This inconveniency is avoided by the APS-100 Sync module. The APS-100 delivers an almost constant output signal over an intensity range of about 1:100. Gain regulation in the APS-100 is achieved by keeping the average photodiode current constant. That means, a constant pulse frequency in the range of 70 MHz to 90 MHz is required for the module to work properly. The APS-100 contains a beamsplitter that directs a few percent of the laser power into the photodiode, see Fig. 142, left. The laser beam enters from the back and leaves the beamsplitter to the front. The output signal for an 80 MHz laser pulse sequence is shown in Fig. 142, right.
Fig. 142: APS-100 Synchronisation module. Right: Output pulses for an 80 MHz laser pulse sequence.