Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse Excitation (original) (raw)
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Fluorescence lifetime images and correlation spectra obtained by multidimensional TCSPC
Proceedings of SPIE, 2005
Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a highrepetition 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 twophoton 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.
Multispectral Fluorescence Lifetime Imaging by TCSPC
2007
We present a fluorescence lifetime imaging technique with simultaneous spectral and temporal resolution. The technique is fully compatible with the commonly used multiphoton microscopes and nondescanned (direct) detection. An image of the back-aperture of the microscope lens is projected on the input of a fiber bundle. The input of the fiber bundle is circular, and the output is flattened to match the input slit of a spectrograph. The spectrum at the output of the spectrograph is projected on a 16-anode PMT module. For each detected photon, the encoding logics of the PMT module deliver a timing pulse and the number of the PMT channel in which the photon was detected. The photons are accumulated by a multidimensional time-correlated single photon counting (TCSPC) process. The recording process builds up a four-dimensional photon distribution over the times of the photons in the excitation pulse period, the wavelengths of the photons, and the coordinates of the scan area. The method delivers a near-ideal counting efficiency and is capable of resolving double-exponential decay functions. We demonstrate the performance of the technique for autofluorescence imaging of tissue.
Fluorescence lifetime imaging by multi-dimensional time correlated single photon counting
Medical Photonics, 2015
Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several features, such as high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals and optical sectioning capability. The combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal or two-photon laser scanning meets these requirements almost ideally. Multi-dimensional 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. Each photon is characterised by its arrival time with respect to the laser pulse and the coordinates of the laser beam in the scanning area. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve. More parameters can be added to the photon distribution, such as the wavelength of the photons, the time from a stimulation of the sample, or the time with respect to an additional modulation of the laser. In this review, the application of the technique will be described for the measurement of molecular environment parameters within a sample, protein interaction experiments by Förster resonance energy transfer (FRET), autofluorescence measurements of cells and tissue, and in-vivo imaging of human skin and the fundus of the human eye.
Picosecond fluorescence lifetime microscopy by TCSPC imaging
Multiphoton Microscopy in the Biomedical Sciences, 2001
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 µs 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.
Fluorescence lifetime imaging by time-correlated single-photon counting
Microscopy Research and Technique, 2003
We present a time-correlated single photon counting (TCPSC) 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 the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.
Microscopy Research and Technique, 2006
Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a highrepetition 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 twophoton 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.
High resolution TCSPC lifetime imaging
SPIE Proceedings, 2003
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 × 1024 pixels. It resolves the components of doubleexponential 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.
IEEE Transactions on Biomedical Engineering, 2013
In time-correlated single photon counting (TCSPC) systems, the maximum signal throughput is limited by the occurrence of pileup and other effects. In many biological applications that exhibit high levels of fluorescence intensity (FI), pileup related distortions yield serious distortions in the fluorescence lifetime (FLT) calculation as well as significant decrease in the signal-to-noise ratio (SNR). Recent developments that allow the use of high-repetition-rate light sources (in the range of 50-100 MHz) in fluorescence lifetime imaging (FLIM) experiments enable minimization of pileup related distortions. However, modern TCSPC configurations that use high-repetition-rate excitation sources for FLIM suffer from dead-time-related distortions that cause unpredictable distortions of the FI signal. In this study, the loss of SNR is described by F-value as it is typically done in FLIM systems. This F-value describes the relation of the relative standard deviation in the estimated FLT to the relative standard deviation in FI measurements. Optimization of the F-value allows minimization of signal distortion, as well as shortening of the acquisition time for certain samples. We applied this method for Fluorescein, Rhodamine B, and Erythrosine fluorescent solutions that have different FLT values (4 ns, 1.67 ns, and 140 ps, respectively). Index Terms-Fluorescence intensity (FI), fluorescence lifetime (FLT), fluorescence lifetime imaging (FLIM), time-correlated single photon counting (TCSPC).
Combined fluorescence and phosphorescence lifetime imaging
APPLIED PHYSICS LETTERS, 2016
We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.