bh FLIM systems record FLIM images of unprecedented temporal and spatial resolution at an
accuracy level close to the theoretical limit given by photon statistics. But bh FLIM systems do more
that that: The bh FLIM technique is based on a new understanding of FLIM in general. FLIM is not
just considered a way to add additional contrast to microscopy images. It is considered and designed
as a molecular imaging technique. bh FLIM exploits the fact that the fluorescence decay function of a
fluorophore is an indicator of its molecular environment, and that multi-exponential decay analysis
delivers molecular information, such as the metabolic state of live cells and tissues, protein
conformation and protein interaction, reaction of cells to drugs and environment parameters, or
mechanisms of cancer development and cancer progression. To reach this target, bh FLIM systems
have features not available in other systems: Compatibility with live-cell imaging, extraordinarily
high time resolution and photon efficiency, capability to split decay functions into several
components, excitation-wavelength multiplexing in combination with parallel-channel detection,
recording of dynamic lifetime effects caused by fast physiological effects, and simultaneous
FLIM/PLIM.

Keywords: FLIM, PLIM, Molecular Imaging, FRET, Protein Structure, Metabolic Imaging, Live Cell Imaging, Physiological Effects

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The bh FLIM Technique: More than Fluorescence-Lifetime Imaging

From Basic FLIM to High-End Molecular Imaging

bh FLIM systems record FLIM images of unprecedented temporal and spatial resolution at an accuracy close to the theoretical limit given by photon statistics [1, 25]. But bh FLIM systems do more that that: The bh FLIM technique is based on a new understanding of FLIM in general [2]. FLIM is not just considered a way to add additional contrast to microscopy images. It is considered and designed as a molecular imaging technique. bh FLIM exploits the fact that the fluorescence decay function of a fluorophore is an indicator of its molecular environment, and that multi-exponential decay analysis delivers molecular information, such as the metabolic state of live cells and tissues, protein conformation and protein interaction, reaction of cells to drugs and molecular environment, or mechanisms of cancer development and cancer progression. To reach this target, bh FLIM systems have features not available by other systems: Compatibility with live-cell imaging, extraordinarily high time resolution and photon efficiency, capability to split decay functions into several components, excitation-wavelength multiplexing in combination with parallel-channel detection, recording of dynamic lifetime effects caused by fast physiological effects, and simultaneous FLIM/PLIM [2]. The most important ones of these features will be described in this brochure.

Precision Megapixel FLIM Images

bh FLIM is characterised by spatial resolution in the megapixel range and temporal resolution in the 10-ps range. An example is shown below.

The Ultimate in FLIM Time Resolution and Timing Stability

The electrical time resolution of the bh SPC-180 NX FLIM modules is 3.5ps fwhm, or about 1.5 ps rms [2]. Timing stability is better than 0.4ps rms. The IRF of multiphoton systems is <19 ps fwhm, or 8.3 ps rms, including detector and laser. No need to record an IRF for a system this fast!

Ultra-High Resolution FLIM

Ultra-short decay times in biological systems are more frequent than commonly believed. They are often considered difficult or impossible to measure. However, lifetimes in the 10-ps range are no problem for bh FLIM systems. The data below were recorded at a time-channel width of 300 femtoseconds, and with an IRF width of 19 ps [9]. The dominating decay component has a lifetime of 7.6 picoseconds.

FLIM Data Analysis by SPCImage NG

Data analysis is an integral part of the bh FLIM systems [3, 26]. Fast GPU processing, MLE fit, multi-exponential analysis, fit with global parameters, combination with phasor plot, automatic IRF synthesis, intelligent binning, batch processing - these are only a few of the SPCImage NG functions. GPU-based analysis runs multi-exponential decay analysis within seconds, MLE yields high fit accuracy even at low photon numbers, automatic IRF generation avoids the need of recording IRFs, batch processing automatically analyses large series of files, and the combination with phasor analysis provides an easy way of image segmentation. Biologically relevant parameters, such or FRET intensities and FRET distances, ion concentrations, membrane potentials, and metabolic ratios are directly available from the decay data. Below: FLIM image displayed in parallel with phasor plot. Second below: Batch processing of a file series. GPU Processing, calculation time <1s per image.

Protein Interaction - Quantitative FRET Results

Precision FLIM-FRET is performed by double-exponential FRET analysis. In contrast to single-exponential techniques, the method delivers correct FRET efficiencies and FRET distances [10] even for incomplete donor-acceptor linking, and without reference measurement of a donor-only sample [11]. Global analysis increases the accuracy of the FRET parameters in case of constant free-donor decay time [27]. Images of the classic FRET efficiency, the FRET efficiency of the interacting donor, the amount of interacting donor, and the donor-acceptor distance are provided directly by SPCImage NG [3]. Please see images below.

Molecular Parameters - Derived from Fluorescence-Decay Data

Molecular parameters, such as local pH, ion concentrations, local viscosity or redox potential are available through precision decay analysis [2]. The results are quantitative, i.e. independent of the laser power, the fluorophore concentration, and the parameters of the optical-system. Examples are shown below.

Membrane Potential

Membrane potentials can be measured by FLIM of voltage sensitive dyes. The lifetime change over the physiological range of membrane potentials is not very large but can well be resolved by bh's TCSPC FLIM systems [12].

Label-Free Multiphoton Imaging of Cells and Tissues

Use high-resolution multiphoton FLIM to record label-free FLIM from deep layers of biological tissue. Benefit from high penetration depth, high image contrast and from the metabolic information contained in the data [2].

Metabolic Imaging by FLIM of NADH

Resolve unbound and bound NADH fractions by double-exponential analysis and MLE fit [3]. Component lifetimes and component amplitudes bear information on the metabolic state of cells and tissues [2]. Please see below. SPCImage NG data analysis resolves even differences in the component lifetimes and the amplitude ratio between different mitochondria.

Metabolic FLIM of NADH and FAD by Laser Multiplexing - Increased Reliability of Tumor Detection

Record Metabolic FLIM by excitation-wavelength multiplexing and simultaneous imaging of NADH and FAD. Benefit from perfect separation of NADH and FAD. Discriminate tumor cells from good cells quantitatively via the amplitudes of the decay components [14, 15]. Below: Human bladder cells, amplitude images, tumor cells marked.

FMN in Cells

Distinguish FMN from FAD by triple-exponential decay analysis [16]. Below: Relative concentration of bound FAD, free FAD, and FMN in human bladder cells. SPCImage NG, global fit.

Metabolic FLIM with Simultaneous pH Imaging

Record metabolic FLIM simultaneously with FLIM of other molecular parameters. Below: NADH FLIM with simultaneous pH imaging via the lifetime of a pH-sensitive probe.

Metabolic FLIM of Macroscopic Objects

Record metabolic FLIM of macroscopic objects [17]. Below: FLIM image of a whole rat brain. Colour indicates the amplitude of the fast decay component, a1, characterising the metabolic state of the tissue [18].

Ultra-Fast Decay Processes in Biological Material

Explore fluorescence-decay processes which have never been seen before [19, 20, 21]. Below: Mushroom spores and pollen grains, fast decay component, amplitude-weighted net lifetime, amplitude of fast decay component. Decay curves shown on the right. Fast component is 10 to 11 ps.

Ultra-Fast Fluorescence Decay in Malignant Melanoma

Use ultra-fast FLIM for melanoma detection. Below: Melanoma sample, decay curves of healthy tissue and tumor tissue. The tumor has a fast decay component of 13 ps [22].

Autofluorescence Imaging of Small Organisms

Study environment effects on small organisms by recording autofluorescence. Benefit from the fact that FLIM parameters are sensitive to the metabolic state.

High-Resolution Z Stacks

Resolve fluorescence dynamics through the entire depth of small organisms. Benefit from high spatial and temporal resolution. Lifetime analysis is performed by bh's fast GPU-based SPCImage NG multi-exponential decay FLIM analysis, results can be displayed as vertical projections of selected ranges of planes or exported to ImageJ / FIJI and stacked to create high resolution 4D representations of the data. Please see [23] for details. Below: Z Stack of a fly, recorded by bh DCS-120 system.

Fast-Acquisition FLIM

For samples that withstand high excitation power bh FLIM systems are able to record FLIM within surprisingly short acquisition time. The figure below shows an autofluorescence lifetime image of a live Enchytraeus albidus taken at 1 second acquisition time.

Express FLIM: Video Sequences from Dynamic Objects

BH FLIM systems are capable of recording video sequences from dynamically changing objects. Frame rates can be 20 fps and more [2]. The data shown below were recorded by bhs Express-FLIM Technique. Enchytraeus albidus, 5 frames per second.

SPCDynamics: See the Heart of Your Mouse Beating

A second way to record fast FLIM sequences is by recording a continuous stream of single-photon data with a fast scanner and extracting subsequent frames from this data stream. The figure below shows a beating mouse heart recorded with a bh/Zeiss FLIM system, displayed by bh SPCDynamics software. Three subsequent frames are shown. The frame rate is 10 fps, a video is available under https://www.becker-hickl.com/see-the-heart-of-your-mouse-beating/.

Triggered Accumulation of Time Series - Recording of Fast Physiological Effects

Use Triggered Accumulation to record precision FLIM data of fast physiological effects. The technique records as fast as the scanner can run [2, 24]. The Signal-to-Noise Ratio does not depend on the image rate! Below, left: Calcium transient in cultured neurons, temporal mosaic imaging, 40 ms per image. Right: Chlorophyll transient, line scanning, 0.5 s per line.

Temporal Mosaic FLIM: Precision Lifetime Analysis of Moving Objects

Record precision decay data from moving objects. Below: Metabolic FLIM on the moving leg of a water flee. bh Temporal Mosaic FLIM with subsequent image segmentation [2, 26]. Precision decay curve shown lower right. DCS-120 confocal FLIM system and SPCImage NG data analysis.

Simultaneous FLIM / PLIM

bh FLIM systems are able to record fluorescence and phosphorescence simultaneously. Use simultaneous FLIM and PLIM to record the metabolic state of cells as a function of the oxygen concentration [28, 29].

FLIM with NIR Dyes

NIR dyes often show large variation in their fluorescence decay with the molecular environment [2]. Explore the use of NIR fluorophores as molecular sensors! Below: Pig skin stained with DTTCC.

Multi-Wavelength FLIM

Explore the unexplored: Simultaneous detection in 16 wavelength channels. A multi-wavelength detector is available for all bh FLIM systems. Please see [1, 2] for details. Below: 16-Wavelength FLIM of plant tissue.

bh FLIM Systems

DCS-120 Confocal FLIM System

The basic bh confocal scanning FLIM system. Fast bh DCS-120 scanner, two ps diode lasers, hybrid detectors, two parallel SPC-180 NX TCSPC / FLIM channels. Simultaneous FLIM/PLIM, 2-laser multiplexing, mosaic FLIM, time-series recording, Z stack recording. Expandable with additional lasers and detectors [4]. Controlled by SPCM software, data analysis by SPCImage NG.

DCS-120 CMI Molecular Imaging Confocal FLIM System

Confocal scanning system for advanced molecular-imaging applications. Fast bh DCS-120 scanner, four ps diode lasers, four hybrid detectors, four parallel SPC-180 NX or SPC-QC-106 TCSPC / FLIM channels. Simultaneous FLIM / PLIM, 4-laser multiplexing, mosaic FLIM, time-series recording, Z stack recording.

DCS-120 MP Multiphoton FLIM System

Basic DCS-120 multiphoton FLIM system. Fast scanning by bh DCS scanner, excitation by fs fibre laser, non-descanned detection, ultra-fast hybrid detectors, two parallel SPC-180 NX TCSPC / FLIM channels. Controlled by SPCM software, data analysis by SPCImage NG [3, 4]. Available also with Ti:Sa laser.

DCS-120 DMP Dual Multiphoton Excitation FLIM System

Dual-Excitation multiphoton system, target application metabolic imaging. Fast scanning by bh DCS scanner, scanner-synchronous laser multiplexing, non-descanned detection. Hybrid detectors, two parallel SPC-180 NX TCSPC / FLIM channels. Controlled by SPCM software, data analysis by SPCImage NG [3, 4].

DCS-120 Super MPC FLIM System

Tuneable multiphoton NDD FLIM by fs NIR laser, tuneable one-photon confocal FLIM by SHG of fs laser. Fast scanning by bh DCS scanner, two fully parallel SPC-180NX recording channels, hybrid detectors, sub-20 ps FWHM time resolution both for multiphoton and confocal operation. Expandable with additional recording channels. Controlled by SPCM software, data analysis by SPCImage NG.

DCS-120 MACRO

Confocal scanning in the image plane of a bh DCS scanner. No microscope or other external optics needed. Image size up to 15 mm, resolution 15 µm. Two ps diode lasers, two hybrid detectors, two parallel SPC-180 NX or SPC-QC-104 TCSPC / FLIM channels. Controlled by SPCM software, data analysis by SPCImage NG [3, 4].

FLIM System for Zeiss LSM 980 Confocals

Confocal scanning with up to four bh ps diode lasers, two hybrid detectors attached to confocal port of scan head, two SPC‑180 NX or SPC-QC-104 TCSPC/FLIM channels [5, 6]. bh SPCM software functions embedded in Zeiss ZEN software. Data analysis by SPCImage NG [4].

FLIM System for Zeiss LSM 980 NLO Multiphoton Microscopes

Multiphoton scanning by Ti:Sa laser, non-descanned detection, hybrid detectors or Zeiss BIG-2 detector, two SPC‑180 NX FLIM channels or two channels of one SPC-QC-104 [5, 6]. Controlled by Zeiss ZEN software. SPCM functions integrated in ZEN via TCP interface. Data analysis by SPCImage NG [3, 4].

FLIM Systems for Other Laser Scanning Microscopes

bh FLIM systems are available for Nikon [8], Leica [7], and a variety of other microscopes. Please see 'The bh TCSPC Handbook' [2], available on www.becker-hickl.com.