Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting (original) (raw)
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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.
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.
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.
Multi-dimensional fluorescence lifetime measurements
Progress in Biomedical Optics and Imaging, 2007
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.
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).
Multi-dimensional fluorescence lifetime measurements
Multiphoton Microscopy in the Biomedical Sciences VIII, 2008
In this study, we present two different approaches that can be used for multi-wavelength fluorescence lifetime measurements in the time domain. One technique is based on a streak-camera system, the other technique is based on the timecorrelated-single-photon-counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope and a Titanium:Sapphire-laser that is used for pulsed one-and two-photon excitation. Fluorescence light emitted by the sample is fed back through the scan head and guided to one of the confocal channels, where it is coupled into an optical fiber and directed to a polychromator. The polychromator disperses the emitted light according to its wavelength and focuses the resulting spectrum on the entrance slit of a streak camera or a 16 channel PMT array, which is connected to a TCSPC imaging module. 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 and pitfalls of fluorescence lifetime measurements.
Journal of Biophotonics, 2019
We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans. K E Y W O R D S fluorescence lifetime imaging (FLIM), lightsheet microscopy, microchannel plate (MCP), SPIM, time-correlated single photon counting (TCSPC)
Fluorescence lifetime microscopy with a time-and space-resolved single-photon counting detector
2006
abstract We have recently developed a wide-field photon-counting detector (the H33D detector) having high-temporal and highspatial resolutions and capable of recording up to 500,000 photons per sec. Its temporal performance has been previously characterized using solutions of fluorescent materials with different lifetimes, and its spatial resolution using sub-diffraction objects (beads and quantum dots).
Multiparametric Time-Correlated Single Photon Counting Luminescence Microscopy
Biokhimiya, 2019
Classic time correlated single photon counting (TCSPC) technique involves detection of single photons of a peri odic optical signal, registration of the photon arrival time in respect to the reference pulse, and construction of photon dis tribution with regard to the detection times. This technique achieves extremely high time resolution and near ideal detec tion efficiency. Modern TCSPC is multi dimensional, i.e., in addition to the photon arrival time relative to the excitation pulse, spatial coordinates within the image area, wavelength, time from the start of the experiment, and many other param eters are determined for each photon. Hence, the multi dimensional TCSPC allows generation of photon distributions over these parameters. This review describes both classic and multi dimensional types of TCSPC microscopy and their applica tion for fluorescence lifetime imaging in different areas of biological studies.