TCSPC FLIM is the most direct way to obtain molecular information from live cells and tissues. Data analysis in molecular FLIM applications is usually complicated by the need of multi-exponential decay analysis in presence of low photon budget. Other issues are uncertainty in the instrument response function (IRF) and extremely long data processing times. bh's new generation SPCImage NG software addresses these problems by a combination of time-domain and frequency-domain analysis, image segmentation, MLE fitting, GPU processing, and IRF modelling. We demonstrate the performance of SPCImage NG for a number of advanced FLIM applications.
We describe a metabolic-imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses two-photon excitation by a dual-wavelength femtosecond fibre laser. The two wavelengths of the laser, 780 nm and 880 nm, are multiplexed synchronously with the frames or the lines of the scan. The recording system uses two parallel TCSPC FLIM channels, detecting from 420 to 475 nm and 480 to 600 nm. By using the multiplexing functions of the TCSPC modules, separate images for NAD(P)H and FAD are recorded. A third image is obtained for the SHG of the 880 nm laser wavelength. Data analysis delivers images of the amplitude-weighted lifetime, tm, the component lifetimes, t1 and t2, the amplitudes of the components, a1 and a2, the amplitude ratio, a1/a2, and the fluorescence-lifetime redox ratio (FLIRR), a2nadh/a1fad. We demonstrate the performance of the system for metabolic imaging of mammalian skin.
We report on a fast-acquisition FLIM system comprising a single detector, four parallel TCSPC channels, and a device that distributes the photon pulses 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 hybrid detector is shorter than 25 ps (FWHM). The system is virtually free of pile-up effects and has drastically reduced counting loss. FLIM data can be recorded at acquisition times down to the fastest frame times of the commonly used galvanometer scanners. Fast recording does not compromise the time resolution; the data can be recorded with the TCSPC-typical number of time-channels numbers of up to 1024 or even 4096. Pixel numbers can be increased to 1024 x 1024 or 2048 x 2048 pixels. The system is therefore equally suitable for fast FLIM and precision FLIM applications.
We describe a metabolic-imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses one-photon excitation by ps diode lasers, scanning by galvanometer mirrors, confocal detection, and two parallel TCSPC FLIM recording channels. The two lasers, with wavelengths of 375nm and 405 nm, are multiplexed to alternatingly excite NAD(P)H and FAD. One FLIM channel detects in the emission band of NAD(P)H, the other in the emission band of FAD. The FLIM data are processed by SPCImage data analysis software. For both channels, the data analysis delivers images of the amplitude-weighted lifetime, tm, the component lifetimes, t1 and t2, the amplitudes of the components, a1 and a2, and the amplitude ratio, a1/a2. Moreover, it delivers the fluorescence-lifetime redox ratio (FLIRR), a2nadh/a1fad. We demonstrate the performance of the system at the example of human bladder cells. Normal cells and tumor cells were discriminated by the tm images, the a1 images, and the FLIRR images.
We present a lifetime imaging technique that simultaneously records fluorescence and phosphorescence lifetime images
in laser scanning systems. It is based on modulating a high-frequency pulsed laser by a signal synchronous with the
pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multi-dimensional TCSPC.
Fluorescence is recorded during the on-phase of the laser, phosphorescence during the off-phase. The technique does not
require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using excessively high
pulse power for phosphorescence excitation. Laser modulation is achieved either by electrically modulating picosecond
diode lasers, or be controlling the lasers via the AOM of a standard confocal or multiphoton laser scanning microscope.
The principle of the hybrid PMT is known for about 15 years: Photoelectrons emitted by a photocathode are accelerated
by a strong electrical field, and directly injected into an avalanche diode chip. Until recently, the gain of hybrid PMTs
was too low for picosecond-resolution photon counting. Now devices are available that reach a total gain of a few
100,000, enough to detect single photons at ps resolution. Compared with conventional PMTs, multi-channel PMTs, and
SPADs (single-photon avalanche photodiodes) hybrid PMTs have a number of advantages: With a modern GaAsP
cathode the detection quantum efficiency reaches the efficiency of a SPAD. However, the active area is on the order of
5 mm2, compared to 2.5 10-3 mm2 for a SPAD. A hybrid PMT can therefore be used for non-descanned detection in a
multiphoton microscope. The TCSPC response is clean, without the bumps typical for PMTs, and without the diffusion
tail typical for SPADs. Most important, the hybrid PMT is free of afterpulsing. So far, afterpulsing has been present in
all photon counting detectors. It causes a signal-dependent background in FLIM measurements, and a typical
afterpulsing peak in FCS. With a hybrid PMT, FLIM measurements reach a much higher dynamic range. Clean FCS
data are obtained from a single detector. Compared to cross-correlation of the signals of two detectors an increase in
FCS efficiency by a factor of four is obtained. We demonstrate the performance of the new detector for a number of
applications.
Currently used TCSPC FLIM systems are characterised by high counting efficiency, high time resolution, and multiwavelength
capability. The systems are, however, restricted to count rates on the order of a few MHz. In the majority of
applications, such as FRET or tissue autofluorescence, the photostability of the samples limits the count rate to much
lower values. The limited counting capability of the hardware is therefore no problem. However, if FLIM is used for
samples containing highly photostable fluorophores at high concentrations the available count rates can exceed the
counting capability of a single TCSPC channel. In this paper we describe a TCSPC FLIM system that uses 8 parallel
TCSPC channels to record FLIM data at a peak count rate on the order of 50•106 s-1. By using a polychromator for
spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on
the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live
plants.
The acquisition time of TCSPC FLIM depends on the number of pixels of the image, on the required lifetime accuracy,
and on the count rate available from the sample. For samples with high fluorophore concentrations, such as stained
tissue or plant cells the available count rates may come close to the maximum counting capability of the currently used
TCSPC FLIM techniques. We describe the behaviour of TCSPC at high count rates and estimate the size of counting
loss and pile-up effects. It turns out that systematic lifetime errors are smaller than previously believed. TCSPC FLIM
can therefore be used to record fast sequences of fluorescence lifetime images. Fast sequential FLIM will be
demonstrated for the measurement of chlorophyll transients in living plant tissue.
Time-correlated single photon counting (TCSPC) is based on the detection of single photons of a periodic light signal,
measurement of the detection time of the photons, and the build-up of the photon distribution versus the time in the
signal period. TCSPC achieves a near ideal counting efficiency and transit-time-spread-limited time resolution for a
given detector. We present an advanced TCSPC technique featuring multi-dimensional photon acquisition and a count
rate close to the capability of currently available detectors. The technique is able to acquire photon distributions versus
wavelength, spatial coordinates, and the time on the ps scale, and to record fast changes in the fluorescence lifetime and
fluorescence intensity of a sample. Biomedical applications of advanced TCSPC techniques include time-domain optical
tomography, recording of transient phenomena in biological systems, spectrally resolved fluorescence lifetime imaging,
FRET experiments in living cells, and the investigation of dye-protein complexes by fluorescence correlation
spectroscopy. We demonstrate the potential of the technique for selected applications.
In this study, we present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the
time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated singlephoton-
counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope (LSM 510, Zeiss) and a
Titanium:Sapphire-laser (Mira 900D, Coherent) that is used for pulsed one- and two-photon excitation. Fluorescence
light emitted by the sample is dispersed by a polychromator (250is, Chromex) and recorded by a streak camera (C5680
with M5677 sweep unit, Hamamatsu Photonics) or a 16 channel TCSPC detector head (PML-16, Becker & Hickl)
connected to a TCSPC imaging module (SPC-730/SPC-830, Becker & Hickl).
With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. We
applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages over
fluorescence techniques that are already well established in the field of confocal microscopy, such as spectrally resolved
intensity measurements or single-wavelength fluorescence lifetime measurements.
Time-correlated single photon counting (TCSPC) is based on the detection of single photons of a periodic light signal, measurement of the detection time of the photons, and the build-up of the photon distribution versus the time in the signal period. TCSPC achieves a near ideal counting efficiency and transit-time-spread-limited time resolution for a given detector. The drawback of traditional TCSPC is the low count rate, long acquisition time, and the fact that the technique is one-dimensional, i.e. limited to the recording of the pulse shape of light signals. We present an advanced TCSPC technique featuring multi-dimensional photon acquisition and a count rate close to the capability of currently available detectors. The technique is able to acquire photon distributions versus wavelength, spatial coordinates, and the time on the ps scale, and to record fast changes in the fluorescence lifetime and fluorescence intensity of a sample. Biomedical applications of advanced TCSPC techniques are time-domain optical tomography, recording of transient phenomena in biological systems, spectrally resolved fluorescence lifetime imaging, FRET experiments in living cells, and the investigation of dye-protein complexes by fluorescence correlation spectroscopy. We demonstrate the potential of the technique for selected applications.
We present a multi-dimensional TCSPC technique that simultaneously records the photon distribution over the time in the
fluorescence decay, the wavelength, and the coordinates of a two-dimensional scan or the time since the start of the experiment.
We demonstrate the application of the technique to diffuse optical tomography, single-point autofluorescence measurements
of skin, and multi-spectra autofluorescence lifetime imaging of tissue.
We present an approach which monitors both time- and spectral information of the fluorescence in order to receive the full information content of the light emitted from a sample. Our instrumentation covers a combination of the TCSPC technique extended by a multi-wavelength detection scheme. Based on the spectral properties of the participating chromophores their relative contributions to the fluorescence within each wavelength channel can be derived. This information serves as an additional parameter for the decay curve analysis which allows us to identify emission patterns for both lifetime and wavelength. Potential applications of the multi-wavelength TCSPC technique are FRET experiments, new marker techniques based on environment-dependent lifetime, and the separation of fluorophores in autofluorescence images of tissue.
Advanced time-correlated single-photon counting (TCSPC) devices are able to record several 106 photons per second and deliver an instrument response function down to 25 ps FWHM. Under these conditions the accuracy of fluorescence decay or photon migration times is limited by systematic timing errors rather than by the photon statistics. The experiments described below determined the variation of the instrument response function (IRF) with the count rate and the timing drift for an SPC-140 TCSPC module and a number of commonly used detectors. For count rates from 3×104 to 4×106 s-1 a shift of the first moment of the IRF smaller than 2 ps was obtained. The drift over 16 minutes was within ±0.7 ps.
Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH2 provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.
Changes in cellular metabolism are considered first signs of fundus diseases, e.g. of age-related macular degeneration. Changes in the metabolism can potentially be detected by measuring the autofluorescence of the fundus. The fundus contains a wide variety of fluorophores in different binding and quenching states. The fluorescence signals cannot be clearly discriminated by commonly used steady state imaging techniques, even when these are combined with spectral resolution and excitation wavelength multiplexing. A considerable improvement is obtained by fluorescence lifetime imaging (FLIM). FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Due to its high sensitivity and high time resolution, its capability to resolve multi-exponential decay functions, and its easy combination with fast scanning we use multi-dimensional time-correlated single photon counting for fundus imaging. By analyzing the spectral properties of the expected fluorophores in the fundus, we show that improved discrimination of fluorophores is obtained by FLIM in combination with selected excitation wavelength and emission wavelength. As demonstrated in lifetime histograms of 40° fundus images, several fluorophores are excited at 446 nm, but predominantly lipofuscin at 468 nm excitation. Simultaneous detection of lifetime images in two emission ranges 500 nm to 560 nm and 560 nm to 700 nm improves further the discrimination of fluorophores.
We present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain in conjunction with a laser scanning microscope and a pulsed excitation source. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting (TCSPC) approach. The complete setup consists of a laser scanning microscope (LSM-510, Zeiss), a polychromator (250is, Chromex), a streak camera (C5680 with M5677 sweep unit, Hamamatsu Photonics) or a 16-channel TCSPC detector head (PML-16, Becker and Hickl) connected to a TCSPC imaging module (SPC-730/SPC-830, Becker and Hickl).
With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. The fluorescence emitted by the sample can be recorded in a single measurement. No filters have to be used to separate the contributions of different fluorophores to the overall fluorescence signal. When applied to Forster resonance energy transfer (FRET) measurements, the technique allows to separate the decay components of the donor and acceptor fluorescence. In this way, it is possible to reliably determine FRET efficiencies between acceptor and donor fluorophores in given subcellular structures.
We present a multi-dimensional TCSPC technique that simultaneously records the photon distribution over the time in the fluorescence decay, the wavelength, and the coordinates of a two-dimensional scan. We demonstrate the application of the technique to single-point autofluorescence measurements of skin, to multi-spectral fluorescence lifetime microscopy, and ophthalmic imaging.
We present a multi-dimensional TCSPC technique that combines multi-detector and multiplexing capability, and records fast and virtually unlimited sequences of time-of-flight distributions. The system consists of four fully parallel TCSPC channels. Each channel records simultaneously in up to eight detection channels. Up to four lasers and 32 source positions can be multiplexed. The total count rate is up to 4 x 107 photons per second. Time-of-flight sequences can be recorded with a resolution of 50 to 100 ms per curve. The system is operated within a single personal computer.
We present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain in conjunction with a laser scanning microscope and a pulsed excitation source. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting (TCSPC) approach. When applied to Forster resonance energy transfer (FRET) measurements, these setups are capable to record time-resolved fluorescence decays for the donor and the acceptor simultaneously.
Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterised by its time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multi-dimensional TCSPC makes a fluorescence lifetime technique with multi-wavelength capability, near-ideal counting efficiency, and the capability to resolve multi-exponential decay functions. We show that the same technique and the same hardware can be used to for precision fluorescence decay analysis, fluorescence correlation spectroscopy (FCS), and fluorescence intensity distribution analysis (FIDA and FILDA) in selected spots of a sample.
Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein–protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein–protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.
In this study, we describe a time-correlated single photon counting (TCSPC) technique for multi-wavelength lifetime imaging in laser-scanning microscopes. The technique is based on a four-dimensional histogramming process that records the photon density versus the time in the fluorescence decay, the x-y coordinates of the scanning area and the wavelength. It avoids any time gating or wavelength scanning and, therefore, yields a near-ideal counting efficiency. The decay functions are recorded in a large number of time channels, and the components of a multi-exponential decay can be resolved down to less than 30 ps. A single TCSPC imaging channel works with a high detection efficiency up to a photon count rate of about 5•106s-1. A modified version of the TCSPC fluorescence lifetime imaging (FLIM) technique uses several fully parallel detector and TCSPC channels. It operates at a count rate of more than 107 photons per second and records double-exponential FLIM data within less than 10 seconds.
KEYWORDS: Luminescence, Photons, Sensors, Biomedical optics, Single photon, Signal detection, Imaging spectroscopy, Picosecond phenomena, Imaging systems, Fluorescence resonance energy transfer
Time-correlated single photon counting (TCSPC) is based on the detection of single photons of a periodic light signal, measurement of the detection time of the photons, and the build-up of the photon distribution versus the time in the signal period. TCSPC achieves a near ideal counting efficiency and transit-time-spread-limited time resolution for a given detector. The drawback of traditional TCSPC is the low count rate, long acquisition time, and the fact that the technique is one-dimensional, i.e. limited to the recording of the pulse shape of light signals. We present an advanced TCSPC technique featuring multi-dimensional photon acquisition and a count rate close to the capability of currently available detectors. The technique is able to acquire photon distributions versus wavelength, spatial coordinates, and the time on the ps scale, and to record fast changes in the fluorescence lifetime and fluorescence intensity of a sample. Biomedical applications of advanced TCSPC techniques are time-domain optical tomography, recording of transient phenomena in biological systems, spectrally resolved fluorescence lifetime imaging, FRET experiments in living cells, and the investigation of dye-protein complexes by fluorescence correlation spectroscopy. We demonstrate the potential of the technique for selected applications.
Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging in laser scanning microscopes can be combined with a multi-detector technique that allows to record time-resolved images in several wavelength channels simultaneously. The technique is based on a multi-dimensional histogramming process that records the photon density versus the time within the fluorescence decay function, the x-y coordinates of the scanning area and the detector channel number. It avoids any time gating or wavelength switching and therefore yields a near-ideal counting efficiency. We show an instrument that records dual wavelength lifetime images with up to 512 x 512 pixels, and single wavelength lifetime images with up to 1024 x 1024 pixels. It resolves the components of double-exponential decay functions down to 30 ps, and works at the full scanning speed of a two-photon laser scanning microscope. The performance of the instrument is demonstrated for simultaneous lifetime imaging of the donor and acceptor fluorescence in CFP/YFP FRET systems and for tissue samples stained with several fluorophores.
The Zeiss LSM-510 NLO laser scanning microscope can be combined with a new TCSPC (time-correlated single photon counting) lifetime imaging technique developed by Becker & Hickl, Berlin. This technique is based on a three-dimensional histogramming process that records the photon density over the time within the fluorescence decay function and the x-y coordinates of the scanning area. The histogramming process avoids any time gating and therefore yields a counting efficiency close to one. Upgrading The LSM-510 for TCSPC imaging does not require changes in the microscope hardware or software. A fast detector is attached to the fibre output of the scanning head, and synchronisation of the TCSPC module with scanning is achieved via the user I/O of the scan controller. With an MCP-PMT as a detector, fluorescence decay components down to 30 ps can be resolved. The capability of the instrument is shown for the separation of chromphores by their fluorescence lifetime and for CFP/YFP FRET.
We present a novel time-correlated single photon counting (TCPSC) imaging technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over time, the x-y coordinates of the scanning area and the detector channel number. The histogramming process avoids any time gating or wavelength scanning and therefore yields a near-perfect counting efficiency. Applied to resonance energy transfer (RET) measurements, the setup is capable to record time-resolved fluorescence decays for the donor and the acceptor simultaneously.
An improved Time-Correlated Single Photon Counting (TCSPC) technique features high count rate, low differential nonlinearity and multi-detector capability. The system has four completely parallel TCSPC channels and achieves an effective overall count rate of 20 MHz. By an active routing technique, up to eight detectors can be connected to each of the TCSPC channels. We used the system to record optical mammograms after pulsed laser illumination at different wavelengths and projection angles.
We use a two-photon laser scanning microscope with a new Time-Correlated Single Photon Counting (TCSPC) imaging technique to obtain combined intensity-lifetime images for FRET measurements in living cells. Single photon pulses from a photomultiplier and signals from the scanning head are used to record the three-dimensional photon density over the time- and image coordinates. Double exponential decay analysis delivers the lifetime components of the quenched and the unquenched molecules in all pixels of the image. We use the ratio of the intensity coefficients of the fast and slow decay component to create images that show the size of the FRET effects in different parts of the cell.
A new Time-Correlated Single Photon Counting (TCSPC) imaging technique delivers combined intensity-lifetime images in a two-photon laser scanning microscope. The sample is excited by laser pulses of 150 fs duration and 80 MHz repetition rate. The microscope scans the sample with a pixel dwell time in the +s range. The fluorescence is detected with a fast PMT at the non-descanned port of the laser scanning microscope. The single photon pulses from the PMT and the scan control signals from the scanning head are used to build up a three-dimensional histogram of the photon density over the time within the decay function and the image coordinates x and y. Analysis of the recorded data delivers images containing the intensity as brightness and the lifetime as colour, images within selected time windows or decay curves in selected pixels. The performance of the system is shown for typical applications such as FRET measurements, Ca imaging and discrimination of endogenous fluorophores or different dyes in living cells and tissues.
Simultaneous acquisition of time- and space-information in time-domain single photon counting spectroscopy became feasible by a recent advance in microchannel-plate photomultiplier-tube technology: we present a novel MCP-PMT detector, featuring a space- sensitive delay-line anode. The detector is characterized by temporal and spatial instrument response functions of 75 ps and 100 micrometer FWHM, respectively, at 200 space channels and a dynamic range of 105. By employing a two-dimensional multichannel analyzer with transputer, 70.000 cps through-put or higher is possible. No photons are lost at the exit slit of the monochromator, as in standard, one-channel time-correlated single photon counting spectroscopy, and sensitive biological samples can be studied at reduced excitation energies. We applied the novel detector to study the basic photophysics of DAPI and its interaction with DNA.
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