Megapixel-Size FLIM Images

Images as large as 2048 x 2048 pixels, with 256 time channels per pixel

Diffraction-limited imaging of full field of view of microscope lens

Large number of cells imaged under identical conditions

High-resolution images of large objects

Super-resolution STED images of entire cells

Complex photon distributions:

 Mosaic FLIM, Z Stack FLIM, ultra-fast accumulation of time series

 Physiological effects recorded at millisecond resolution

 Simultaneous FLIM / PLIM

 

 

 

 

 

 

Lateral Mosaic FLIM with Zeiss LSM 710 / 780 / 880 Laser Scanning Microscopes

     - Images larger than the field of view of the microscope lens

     - Pixel numbers up to       2048 x 2048 at 256 time channels per pixel

                                        4096 x 4096 pixels at 64 time channel per pixel

 

 

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Please see: Modular FLIM Systems for Zeiss LSM 710/780/880 Family Laser Scanning Microscopes, user handbook, 7th Edition

 

 

 

 

 

 

Lateral Mosaic FLIM with DCS-120 Confocal and Multiphoton FLIM System

  - Sample stepping by optional motos stage

  - Fully integrated in SPCM data acquisition software

  - Data analysis by standard SPCImage procedures

 

 

Please see: DCS-120 confocal and multiphoton FLIM systems, user handbook, 7th Edition (2017)

 

 

 

FASTAC Fast-Acquisition FLIM System

The bh Fast-Acquition FLIM system uses four parallel TCSPC channels and a device that distributes the photon pulses of a single detector into the four recording channels. The system features an electrical IRF width of less than 7 ps (FWHM), and a time channel width down to 820 fs. The optical time resolution with an HPM-100-06 or -07 hybrid detector is shorter than 25 ps (FWHM). The system is virtually free of pile-up effects. FLIM data can be recorded at acquisition times down to the fastest frame times of the commonly used galvanometer scanners. The data are recorded with the TCSPC-typical number of time-channels of up to 4096, and with pixel numbers from 128 x 128 to 2048 x 2048 pixels. The system is therefore equally suitable for fast FLIM and precision FLIM applications.

                    

 



 

Please see:         1. bh FASTAC Fast Acquisition FLIM System, data sheet, available on www.becker-hickl.com

                         2. Fast-Acquisition TCSPC FLIM System with sub-25 ps IRF Width, available on www.becker-hickl.com

                         3. Fast-Acquisition Multiphoton FLIM with the Zeiss LSM 880 NLO, available on www.becker-hickl.com

                         4. Fast-Acquisition TCSPC FLIM: What are the Options?, available on www.becker-hickl.com

 

 

 

 

 

DCS-120 MACRO Records FLIM and PLIM of cm-Size Objects

 

     - Imaging of objects as large as 12 x 12 mm

     - Resolution down to the cell level

     - Excitation wavelengths from 375 nm to 785 nm

     - Metabolic FLIM of tumors

     - Small-animal imaging

            

 

Please see:   V. I. Shcheslavskiy, M. V. Shirmanova, V. V. Dudenkova, K. A. Lukyanov, A. I. Gavrina, A. V. Shumilova, E. Zagaynova, W. Becker, Fluorescence time-resolved macroimaging. Opt. Lett. 43, No. 13, 3152-5155 (2018)

 

Mosaic FLIM of Macroscopic Objects

     - DCS-120 Macro FLIM System with optional motor stage

     - Beam scanning in primary focal plane of DCS scanner, combined with motor stage

     - Images as large as 4096 x 4096 pixels

     - Scanning of objects in the 10 cm range

     - Scanning of well plates

     - Control of motor stage fully integrated in SPCM data acquisition software

            

Please see:      DCS-120 confocal and multiphoton FLIM systems, user handbook, 7th Edition (2017), available on www.becker-hickl.com

                      W. Becker, The bh TCSPC Handbook, 7th Edition (2017), available on www.becker-hickl.com

 

 

 

 

 

 

Z Stack FLIM by Mosaic Function

     - No stop for data saving between subsequent image planes

     - No plane trigger required

     - Easy implementation in all laser scanning microscopes

     - Easy data analysis over entire data array

     

 

 

 

 

Temporal Mosaic FLIM: Ultra-fast accumulation of time series

Ca2+ Transients in live neurons recorded by temporal mosaic FLIM

 

Please see:     1. Modular FLIM Systems for Zeiss LSM 710/780/880 Family Laser Scanning Microscopes, user handbook, 7th Edition

                      2. bh FLIM systems record calcium transients in live neurons. Application note, www.becker-hickl.com

                      4. Fast-Acquisition TCSPC FLIM: What are the Options? Application note, on www.becker-hickl.com

 

 

 

 

 

DCS-120 MP Multiphoton System

The DCS-120 MP uses multiphoton excitation by a femtosecond titanium-sapphire laser, fast galvanometer scanning, non-descanned detection, hybrid detector technology, and single-photon recording by bh’s multi-dimensional TCSPC process. An AOM is included to control the laser power and to modulate the laser for PLIM acquisition. The system records in two fully parallel NDD channels, runs lateral mosaic scans and Z stacks, accumulates fast FLIM time series, and simultaneously records  FLIM and PLIM. All components, including the laser and the AOM, are controlled by bh’s SPCM 64 bit data acquisition software. Image formats as large as 2048 x 2048 pixels with 256 time channels per pixel are available.

            

 

     

DCS-120 MP system. Left: entire system with laser, AOM, microscope and scanner. Right: Non-descanned detectors attached to back of microscope.

Please see: DCS-120 confocal and multiphoton FLIM systems, user handbook, 7th Edition (2017)

 

 

 

 

 

 

Ultra-fast HPM Detectors Improve NAD(P)H FLIM

In combination with the ultra-fast HPM-100-06 and -07 detectors, bh multiphoton FLIM systems system achieve an instrument response function (IRF) of less than 20 ps FWHM. The fast response greatly improves the accuracy at which fast decay components can be extracted from a multi-exponential decay. Applications are mainly in the field of metabolic FLIM, which requires separation of the decay components bound and unbound NADH. Typical NADH FLIM images of the amplitude-weighted lifetime and of the amplitudes and liftimes of the fast and slow decay component.

 

   

NADH Lifetime image, amplitude-weighted lifetime of double-exponential fit. Right: Decay curve in selected spot, 9x9 pixel area. FLIM data format 512x512 pixels, 1024 time channels. Time-channel width 10ps.

 

 

  

Left to right: Images of the amplitude ratio, a1/a2 (unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay component (t2, bound NADH). FLIM data format 512x512 pixels, 1024 time channels. Time-channel width 10ps.

 

Please see: 1. Ultra-fast HPM detectors improve NADH FLIM. Application note, www.becker-hickl.com

                   2. Modular FLIM Systems for Zeiss LSM 710/780/880 Family Laser Scanning Microscopes, user handbook, 7th Edition

                   3. DCS-120 confocal and multiphoton FLIM systems, user handbook, 7th Edition (2017)

 

 

 

 

 

Simultaneous FLIM / PLIM

Record metabolic effects by FLIM, track O2 concentration by PLIM

High sensitivity due to multi-pulse PLIM excitation

Low concentration of O2 sensor, low generation of reactive oxygen species

DCS-120, DCS-120 MP and DCS-120 Macro systems

Zeiss LSM 710, 780, 880 NLO systems

Leica SP5 and SP8 MP systems

 



Please see:   1. The bh TCSPC Handbook, 7th edition (2017), page 388.

                   2. Becker & Hickl GmbH, Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse  Excitation. Application note, available on www.becker-hickl.com

                   3. S. Kalinina, V. Shcheslavskiy, W. Becker, J. Breymayer, P. Schäfer, A. Rück, Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping. J. Biophotonics 9(8):800-811 (2016)

                   3. V. I. Shcheslavskiy, A. Neubauer, R. Bukowiecki, F. Dinter, W. Becker, Combined fluorescence and phosphorescence lifetime imaging. Appl. Phys. Lett. 108, 091111-1 to -5 (2016)

 

 

Near Infrared FLIM with Ti:Sa Laser and Diode laser

Near Infrared MP FLIM with OPO

Ti:Sa Laser as a 1p excitation source - Diode laser excitation 640, 685, 780 nm - 2p excitation by OPO

- Exploit target-specific lifetimes of NIR dyes

- Obtain clear images from extremely deep tissue layers by 2p excitation with OPO

- Record deep-tissue images with diode-laser based systems

          

Please see:   The bh TCSPC Handbook, 7th edition (2017), page 354

                   W. Becker, V. Shcheslavskiy, Fluorescence Lifetime Imaging with Near-Infrared Dyes. Photonics and Lasers in Medicine 4, 73-83 (2015)

 

 

 

 

 

bh TCSPC Systems Record FLIM/PLIM with Sutter MOM Microscopes

Due to its pulsed excitation source and its high modularity the Sutter MOM microscope 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. FLIM data can be recorded with up to 1024x1024 pixels and 1024 time channels.

                             

Left: Sutter Instrument MOM microscope with two FLIM detectors. Right: Salmon louse (Lepeophtheirus Salmonis), 512 x 512 pixels. Autofluorescence, excitation wavelength 750 nm, detection wavelength 440 to 480 nm.

 

Please see: bh TCSPC Systems Record FLIM with Sutter MOM Microscopes. Application note, www.becker-hickl.com.

 

 

SPCM Software Runs Online-FLIM at 10 Images per Second

Version 9.72 SPCM software of the bh TCSPC/FLIM systems displays fluorescence lifetime images at a rate of 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.  It works for all SPC-150, SPC-150N, SPC-160, and SPC-830 FLIM systems that use fast scanning.

         

FLIM Images recorded and displayed at a rate of 10, 2, and 0.5 images per second (left to right)

 

Please see: SPCM software runs online FLIM at 10 images per second. Application note, available on www.becker-hickl.com.

 

 

 

 

 

 

 

bh - Abberior Combination Records STED FLIM at Megapixel Resolution

The combination of the Abberior STED system with the bh Simple-Tau 150/154 TCSPC FLIM system records FLIM data at a spatial resolution of better than 40 nm. The image format can be as large as 2048 x 2048 pixels with 256 time channels per pixel. 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 from the high data throughput of up to four parallel TCSPC FLIM channels.

 

 

Tubulin fibres in a BPAE cell. Abberior STED system with bh SPC-150 TCSPC-FLIM module. 20nm per pixel, 2048 x 2048 pixels, 256 time channels

 

Please see:  1. The bh TCSPC Handbook, 7th ed. (2017)

                   2. bh - Abberior combination records STED FLIM at megapixel resolution. Application note, www.becker-hickl.com

                   3. Online STED FLIM with bh - Abberior Combination. Application note, www.becker-hickl.com

 

 

 

 

 

Nikon A1 Laser Scanning Microscopes Integrate bh FLIM

FLIM by bh's Multi-Dimensional TCSPC Technique

Integrated in Nikon ‘Elements’ Microscope Software

Excellent Time Resolution, Excellent Spatial Resolution

Megapixel Images

Online-Lifetime Display Function

Precision Data Analysis by bh SPCImage

 



 

       

 

 

 

 

 

 

SPCImage Data Analysis Comes with New User Interface

Since Version 5.4 of February 2016 SPCImage comes with a new user interfac configuration. Taking advantage  of the new wide-screen monitor formats it improves the display of lifetime images of high pixel numbers. Other new features are the implementation of Phasor Analysis, advanced region-of-interest definitions, and two-dimensional lifetime histograms.

     

 

 

SPCImage with New 2-Dimensional Histogram Functions

 



 

 

 

 

 

 

New SPCImage Version Combines Time-Domain FLIM Analysis with Phasor Plot

Version 6.0 SPCImage FLIM analysis software combines time-domain multi-exponential decay analysis with the phasor plot.  Different phasor clusters can be selected, and the corresponding pixels back-annotated in the time-domain FLIM images. Moreover, the decay functions of the pixels within the selected phasor range can be combined into a single decay curve of high photon number. This curve can be analysed at high accuracy, revealing decay components that are not visible by normal pixel-by-pixel analysis.

        

 

      

   

   

Please see:  New SPCImage Version Combines Time-Domain Analysis with Phasor Plot. Application note, www.becker-hickl.com

 

 

 

 

 

 

High Efficiency PML16-GaAsP 16-Channel and MW-FLIM Multi-Spectral FLIM Detectors

Greatly increased efficiency compared to conventional MW FLIM detector. More photons for multi-spectral Megapixel FLIM.

 

      

Left: Megapixel Multi-Spectral FLIM with MW-FLIM GaAsP, 16 wavelengths channels. Right: Each of the channels shown left has 512x512 pixels and 256 time channels per pixel.

 

 

uG             

Fluorescence signal in 16 wavelength channels           PML-16 GaAsP Detector       MW-FLIM Multi-Spectral FLIM Detector

 

Please see: W. Becker, The bh TCSPC Handbook, 7th Edition (2017), page 170

                   PML-16C and PML-16 GaAsP 16-channel detectors, PML-SPEC and MW FLIM Multi-wavelength detectors. User handbook, www.becker-hickl.com

 

 

 

 

 

 

 

Infrared Detection up to 1700 nm with id 230 InGaAs SPAD - IRF width 80 ps FWHM

 

 

                   

Please see: 80 ps FHWM Instrument Response with ID230 InGaAs SPAD and SPC 150 TCSPC Module. Application note, www.becker-hickl.com

 

 

 

 

 

SPC-150NX with SCONTEL NbN Detectors: World Record in TCSPC Time Resolution

Combination of bh SPC-150NX with SCONTEL NbN Detector yields 17.8 ps FWHM, 7.9 ps rms timing jitter

                 

 

Please see: 1. World Record in TCSPC Time Resolution: Combination of bh SPC-150NX with SCONTEL NbN Detector yields 17.8 ps FWHM. Application note, available on www.becker-hickl.com

                   2. V. Shcheslavskiy, P. Morozov, A. Divochiy, Yu. Vakhtomin, K. Smirnov, W. Becker, Ultrafast time measurements by time-correlated single photon counting coupled with superconducting single photon detector, Rev. Sci. Instrum. 053117 (2016)

 

 

 

 

 

 

 

 

 

 

New Ultra-Fast Hybrid Detectors

The new HPM-100-06 and -07 hybrid detectors achieve an IRF width of less than 20 ps. The detectors are available with a C-Mount adapter, with a fibre adapter, and with adapters for the optical ports of the DCS-120 scanning system and the Zeiss LSM 710/780/880 laser scanning microscopes. The HPM-100-06 and -07 detectors are a direct drop-in for HPM-100-40 and -50 detectors.

 

             

 

Please see:     The bh TCSPC handbook, 7th ed. (2017), page 164

 

 

 

 

GaAsP Hybrid detector with Extended Wavelength Range

The HPM-100-42 hybrid detectors cover a wavelength range from 300 to 850 nm. The peak quantum efficiency is about 25%, the IRF width about 140 ps. The detector is especially useful for NIR applications that require low dark count rate. It is direct replacement for the HPM-100-50 detector.

 

     

 

 

 

 

 

 

 

 

Sub-20ps IRF Width from HPM-100-06 and -07 Hybrid Detectors

                   

Left: IRF of HPM-100-06 (bi-alkali cathode). Right: IRF of HPM-100-07 (multi-alkali cathode). The full with at half maximum is 18.9 ps and 19.4 ps, respectively. SPC-150NX TCSPC module, 100 ps/div, 405 fs/channel

Please see:     Sub-20ps IRF Width from Hybrid Detectors and MCP-PMTs. Application note, available on www.becker-hickl.com

                      The bh TCSPC handbook, 7th ed. (2017), page 164

                     

Sub-20ps IRF Width from R3809U MCP PMTs

        

Left: IRF for R3809U MCP PMT, operating voltage -3000 V. CFD threshold -100mV (blue curve) and -400 mV (red curve). Right: Relative efficiency and FWHM of IRF as a function of CFD threshold. 26dB preamplifier, operating voltage ‑3000V.

Please see:     1. Sub-20ps IRF Width from Hybrid Detectors and MCP-PMTs. Application note, available on www.becker-hickl.com

                      2. The bh TCSPC handbook, 7th ed. (2017), page 164

 

 

 

38 ps FWHM Overall System Response with bh ps Diode Laser and HPM‑100-06/07 Hybrid Detectors

- SPC-150N and SPC-150NX TCSPC modules

- 405 nm, 445 nm, and 785 nm BDS-series ps diode lasers

- Excellent time resolution in fluorescence-decay and FLIM applications

                     

Please see: HPM‑100-06/07 data sheet, www.becker-hickl.com

 

 

 

 

 

New PMC-Series Detectors have 120 ps IRF Width

The PMC series of cooled PMT modules got an update. Compared with the older PMC-100 the new PMC-150 series detectors feature improved time resolution and sensitivity. The PMC-150 detectors are a direct replacement for the older PMC-100. The IRF width with the SPC-150N, SPC-150NX and SPC-160 TCSPC modules is about 120 ps for a spot size of 1 mm diameter, and 130 ps for illumination of the entire cathode.

                           

 

Please see:     W. Becker, The bh TCSPC handbook, 7th ed. (2017), page 158. Available on www.becker-hickl.com

 

 

 

 

Fibre-Based Fluorescence-Lifetime Systems for in-vivo Applications

 

Implantable fibre tip, removable from fibre system

Single mode excitation, multi-mode detection

Excitation by BDL or BDS ps diode lasers

Detection by hybrid detectors or SPAD detectors

Multi-wavelength detector option

Simultaneous fluorescence / phosphorescence capability

Detection of NADH, FAD, or exogenous fluorophores

Ca2+ detection in neuronal tissue

pO2 measurement in tissue

 

Please see:   TCSPC fibre-probe system with an exchangeable tip, application note, www.becker-hickl.com

                   W. Becker, The bh TCSPC handbook, 7th ed. (2017), page 526

M. Lukina et al.,  Interrogation of metabolic and oxygen states of tumors with fiber-based luminescence lifetime spectroscopy. Opt. Lett. 42(4) 731-734 (2017)

G. Cui et al., Concurrent activation of strial direct and indirect pathways during action initiation. Nature 494, 238-242 (2013)

 

 

 

 

 

SPC-150NX TCSPC Module: Ultra-Fast Timing for Ultra-Fast Detectors

- Ultra-fast discriminators, 5 GHz input bandwidth

- Extremely low timing jitter

- Electrical response 3.5 ps FWHM, electrical timing jitter 1.5 ps RMS

- Minimum time channel width 405 fs

- IRF width <20ps with hybrid detectors, MCP PMTs, and superconducting NbN detectors

           

 

 

SPC-160 pcie: TCSPC / FLIM Module with PCI Express Interface

Full range of SPC-160 functions

Parallel operation of up to four modules

Excellent resolution

Excellent timing stability

Part of Simple-Tau II systems

               

 

DCC-100 pcie and GVD-120 pcie PCI Express Modules

The DCC-100 detector controller and the GVD-120 Scan Controller are available as PCI express modules. The full range of TCSPC, FLIM, FCS, Single-Molecule detection, and diffuse tomography applications can thus be covered with PCI Express modules. Please see also ‘Simple Tau II Systems’.

                                   

 

Simple-Tau II Systems with Thunderbolt Interface

Compact TCSPC system with Thunderbolt interface

One, two, or three SPC-160pcie TCSPC / FLIM modules

Two SPC-160 pcie modules and one DCC-100 pcie

One SPC-160 pcie, one DCC-100 pcie, one GVD-120 pcie module

 

 

 

 

 

 

BDS Familiy ps Diode Lasers: High Power from Small Lasers

BDS-SM Family: Now with three frequencies and CW mode

                        

Please see: BDS-SM family ps diode lasers. Extended data sheet, www.becker-hickl.com

 

 

BDS-MM Multimode Family: Up to 40 mW average power at 640 nm and 785 nm

                                            

Please see: BDS-MM family ps diode lasers. Extended data sheet, www.becker-hickl.com

 

BDS-SMY series green / yellow picosecond diode lasers

 

The BDS-SMY lasers close the wavelength gap in the spectrum of ps diode lasers in the 520 to 630 nm range. The lasers are based on the QLD series laser modules of QD Laser Inc., Japan. These modules contain an IR laser diode, an amplifier diode, and a frequency doubler. Combined with bh BDS laser series technology, the BDS-SMY lasers provide picosecond light pulses of short pulse width and narrow bandwidth at wavelengths of 532 nm, 561 nm, and 594 nm.

                          

Please see data sheet on www.becker-hickl.com.

 

Laser Combiner / Splitter

 

Combines four lasers into one fibre output

or

Splits one laser into four fibre outputs

Works for single mode and multi-mode fibres

 

 

 

TCSPC and FLIM Literature

 

W. Becker, The bh TCSPC Handbook

7th Edition,  September 2017

 

891 Pages

1155 References, 1043 Figures

 

 

 

Modular FLIM Systems for Zeiss LSM 710/780/880 Family Laser Scanning Microscopes

User handbook, 7th Edition, November 2017

With Appendix for LSM 510 FLIM Systems

 

357 pages

531 References, 458 Figures

 

 

 

DCS-120 Confocal and Multiphoton FLIM Systems

User Handbook, 7th Edition, December 2017

 

391 pages

519 References, 502 Figures

 

 

 

PML-16-C 16 and PML-16 GaAsP 16 Channel TCSPC / FLIM Detectors,

PML-SPEC and MW FLIM Multi-Wavelength Detectors

User Handbook

 

61 pages

75 figures, 99 references

 

 

       

 

 

W. Becker, Ed., Advanced Time-Correlated Single-Photon Counting Applications

Springer, 2015, 624 Pages

 

 

 

 

 

 

Beautiful FLIM

 

The brochure presents a selection of the most beautiful images ever recorded with bh FLIM systems. Every image comes with a description of the technical and scientific background.

 

Please download from www.becker-hickl.com.

For best results print on A3 format paper.

Printed copies are available from bh.

 

 

 

FLIM and TCSPC Posters from bh

 

    

 

FLIM and TCSPC posters are available from bh. Size is A0, or 84 x 120 cm.

For ordering please see poster catalog on www.becker-hickl.com

 

 

 

 

Becker & Hickl GmbH

Nunsdorfer Ring 7-9

12277 Berlin, Germany

Tel. +49  212 800 20                                       Fax +49  30 212 800 213

info@becker-hickl.com       https:/www.becker-hickl.com

 

 

 

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