TCSPC for FLIM and FRET in - Boston Electronics Corporation

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density is measured best by counting the PMT pulses within subsequent time intervals. Therefore,the application of photon counting techniques is the logical consequence of the high gain and thehigh speed of photomultipliers.The efficiency, i.e. the probability that a particular photon causes a pulse at the output of the PMT,depends on the efficiency of the photocathode. Unfortunately the sensitivity S of a photocathode isusually not given in units of quantum efficiency but in mA of photocurrent per Watt incident power.The quantum efficiency QE ish c S W mQE = S ---- = ---- . 1.24 . 10 6 -----e λ λ AThe efficiency for the commonly usedphotocathodes is shown in fig. 13 (right). TheQE of the conventional bialkali andmultialkali cathodes reaches 20 to 25 %between 400 and 500 nm. The recentlydeveloped GaAsP cathode reaches 45 %. TheGaAs cathode has an improved red sensitivityand is a good replacement for the multialkaliabove 600 nm.Sensitivity1000mA/WGenerally, there is no significant differencebetween the efficiency of similarphotocathodes in different PMTs and fromdifferent manufacturers. The differences are of 1300 400 500 600 700 800 900the same order as the variation betweenWavelengthnmdifferent tube of the same type. Reflectiontype cathodes are a bit more efficient than Fig. 13: Sensitivity of different photocathodes [34]transmission type photocathodes. However,reflection type photocathodes have non-uniform photoelectron transit times to the dynode systemand therefore cannot be used in ultra-fast PMTs. A good overview about the characteristics of PMTsis given in [34] and [79].10010GaAsPbia kaliGaAsmultialkaliQE=0.5QE=0.2QE=0.1Image intensifiersImage intensifiers are vacuum devices consisting ofa photocathode, an acceleration and/ormultiplication system for the photoelectrons, and atwo-dimensional image detection system.First generation systems used a electron-opticalimaging system that accelerates the photoelectronsto an energy of some keV and sends them to afluorescent screen. The image from the screen wasdetected by a traditional camera or later with aCCD camera. First generation devices had arelatively low gain and strong image distortions.-HVPhotocathodeMultichannel PlateFluorescence ScreenCCD CameraFig. 14: Intensified CCD camera11
Second generation image intensifiers use multichannel plates for electron multiplication (fig. 14).One plate gives a typical multiplication factor of 1000 so that a gain of 10 6 can be achieved by twoplates in series.The CCD chip can be placed inside the tube to detect the electrons directly. These EBD CCDs(Electron Bombarded CCDs) give higher gain than a CCD behind a fluorescent screen.Gating of an image intensifier can be accomplished by a grid behind the photocathode. Gainmodulation can also be achieved by modulating the voltage between the cathode and the channelplate or the voltage across the multichannel plate.In general, image intensifiers use the same photocathodes as photomultiplier tubes. Therefore, thedetection efficiency is approximately the same. There can, however, be an appreciable loss ofphotons or signal-to-noise ratio due to gating or modulating.Avalanche photodiodesAvalanche photodiodes (APDs) use a multiplication effectdue to a strong electric field in a semiconductor structure(fig. 15). For use as a linear detector, a gain factor of theorder of 100 can be achieved. However, cooled avalanchephotodiodes can be used to detect single photons if they areoperated close to or slightly above the breakdown voltage.The generated electron-hole pairs initiate an avalanchebreakdown in the diode. Active or passive quenchingcircuits must be used to restore normal operation after eachphoton [36]. Therefore, a single photon avalanchephotodiode (SPAPD) can only be used for photon counting. The advantage of an APD is the highquantum efficiency that can reach 90% in the near infrared. Single photon avalanche photodiode(SPAPD) modules are available from Perkin Elmer [35]. Although a resolution as fast as 20 ps hasbeen reported for especially manufactured SPAPDs [36] the time resolution of these modules is inthe order of 300 to 800 ps and depends on the count rate. This makes them less useful for lifetimemeasurements. SPAPDs are, however, excellently suited for fluorescence correlation measurementswhich do not require sub-ns resolution.200VPhotonQuenching CircuitAvalancheOutputFig. 15: Single Photon Avalanche Photodiode(SPAPD)12
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: ns or less. Due to the high gain an
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 and 28: Fortunately, decay curves are eithe
Page 29 and 30: Time-Correlated Single Photon Count
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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