TCSPC for FLIM and FRET in - Boston Electronics Corporation

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part of the signal can probably not be used because it is convoluted with the system response.Assuming a loss of a factor of two in the number of counted photons the +method ends up at F = 1.5to 2.1 which is much better than for the image intensifiers and noticeably better than sine-wavemodulation techniques.Acquisition times for biological samples stained with highly fluorescent dyes are in the range 10 to100 seconds [58,11]. Generally the acquisition times for comparable lifetime accuracy should beexpected in the same range as for TCSPC imaging (see below) unless count rates exceeding 5 MHzare available. As for TCSPC imaging, the acquisition time can be reduced by decreasing the numberof pixels of the scan.Multi-gate photon counting has its merits in applications that require to record lifetimes in the rangeof a few ns at high count rate and with short acquisition times. Typical applications are lifetimeimaging with marker dyes for Ca++, Na+, O-- or other parameters within cells and tissue [11b].Probing of DNA and RNA by the lifetime change of SYTO13 is described in [11, 11a].25
Time-Correlated Single Photon Counting (TCSPC)Time-Correlated Single Photon Counting is based on the detection of single photons of a periodicallight signal, the measurement of the detection times of the individual photons and the reconstructionof the waveform from the individual time measurements [62, 63]. The principle is shown in fig. 29.The fluorescence is excited by a pulsed laser with arepetition rate of typically 80 MHz. The fluorescencephotons are detected by a fast PMT, MCP or a singlephoton avalanche photodiode. For precisionmeasurements these detectors allow count rates of theorder of a few MHz, i.e. much less than the laserrepetition rate. The TCSPC method makes use of thefact that the detection of several photons in one laserperiod is small enough to be neglected. When a photonis detected the time of the corresponding detector pulsein the laser pulse sequence is measured. The measuredtimes are used to address a histogram memory in whichthe photons are accumulated. After acquiring a largenumber of photons the photon density versus timebuilds up in the memory.Detector Signal:Period 1Period 2Period 3Period 4Period 5Period 6Period 7Period 8Period 9Period 10Period NResult aftercollecting manyPhotonsOptical WaveformTimeThe method may look circumstantial at first glance, butit has some striking benefits - a near-ideal countingefficiency and an ultra-high time resolution.memory locationFig. 29: Time-correlated single photon countingAs long as the count rate is not so high that the conversion time of the time measurement andhistogramming procedure becomes noticeable there is no loss of photons. The F value describingthe ratio of the ideal SNR to the actually obtained SNR is < 1.1 [38,39].The resolution is limited by the transit time spread in the detector, by the timing accuracy of thediscriminator that receives the detector pulses and by the accuracy of the time measurement. Withan MCP-PMT the width of the instrument response function is of the order of 25 to 30 ps. Thewidth of the time channels of the histogram can be made less than 1 ps [63]. Therefore, theinstrument response function is sufficiently sampled to determine lifetimes down to a few ps.The TCSPC technique is often believed to be extremely slow and unable to reach short acquisitiontimes. This ill reputation comes from older NIM systems used in conjunction with low repetitionrate light sources which are indeed not applicable for time-resolved imaging.However, high count rate techniques exist [64] since the late 80s. An early - probably the first -implementation of TCSPC in a microscope is described in [65].State-of-the-art TCSPC devices use a new conversion principle and achieve count rates of several10 6 photons per seconds [63]. A system with four parallel TCSPC channels and 20 MHz usefulcount rate is described in [66]. The system was used in a slow-scan setup for optical tomographyand can be combined with a multi-detector technique [63,66]. Although a combination with ascanning microscope has not become known yet the system is basically able to record images in upto 32 detector channels with slow scan rates at count rates comparable to multi-gate photoncounting [57].26
Page 1 and 2: Boston Electronics Corporation91 Bo
Page 3 and 4: FLIM Upgrade Kits for Confocal Micr
Page 5 and 6: ContentsIntroduction...............
Page 7 and 8: concentration, fig. 2.Fluorescence
Page 9 and 10: Microscopy TechniquesWide-Field Flu
Page 11 and 12: fluorescence lifetime of the sample
Page 13 and 14: ns or less. Due to the high gain an
Page 15 and 16: Second generation image intensifier
Page 17 and 18: Appreciable loss of photons can occ
Page 19 and 20: Standard gated image intensifier de
Page 21 and 22: If a frequency between 100 MHz and
Page 23 and 24: fraction of unmodulated light is de
Page 25 and 26: intensifiers. The dielectric losses
Page 27: Fortunately, decay curves are eithe
Page 31 and 32: Zeissb&hSPC-730 TCSPC Imaging Modul
Page 33 and 34: R5900L16DiscriminatorsEncoderDetect
Page 35 and 36: Features of the TCSPC imaging techn
Page 37 and 38: containing CFP and YFP measured by
Page 39 and 40: CathodeCathodeMultichannelPlateMult
Page 41 and 42: FCS / lifetime data in selected spo
Page 43 and 44: [44]G. J. Brakenhoff, M. Müller, R
Page 45 and 46: Time-Correlated Single Photon Count
Page 47 and 48: The TCSPC Microscopy SolutionSPC-83
Page 49 and 50: The TCSPC Imaging PackageSPC-144Fou
Page 51 and 52: The TCSPC Imaging PackageSPC-154Fou
Page 53 and 54: Multi-Spectral FLIM SystemMS-FLIMFl
Page 55 and 56: Lifetime Upgrade Kit for Zeiss LSM-
Page 57 and 58: FLIM Upgrade Kits for Olympus FV100
Page 59 and 60: DCS-120Confocal Scanning SystemSyst

TCSPC for FLIM and FRET in - Boston Electronics Corporation

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