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.
