TCSPC for FLIM and FRET in - Boston Electronics Corporation

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Febr 2003 Printer HP4500PS TCVGBH1.DOCLifetime Imaging Techniques for Optical MicroscopyWolfgang Becker, Axel BergmannBecker & Hickl GmbH, Berlin, becker@becker-hickl.com, bergmann@becker-hickl.com1
ContentsIntroduction.........................................................................................................................................................................3Biological Applications of Lifetime Techniques ...............................................................................................................3Fluorescence Quenching ................................................................................................................................................3Resonance Energy Transfer............................................................................................................................................4Separation of Different Chromophores ..........................................................................................................................5Diffusion in Cells ...........................................................................................................................................................5Microscopy Techniques......................................................................................................................................................6Wide-Field Fluorescence Microscopy............................................................................................................................6Laser Scanning Microscopy ...........................................................................................................................................6Optical Near-Field Microscopy......................................................................................................................................7Light Sources ......................................................................................................................................................................8Titanium-Sapphire Lasers ..............................................................................................................................................8Frequency Doubled Titanium-Sapphire Lasers ..............................................................................................................8Fibre Lasers....................................................................................................................................................................8Pulsed Diode Lasers.......................................................................................................................................................8Modulated CW Lasers....................................................................................................................................................9Mode-locked CW Lasers................................................................................................................................................9Pulse Pickers and Cavity Dumpers.................................................................................................................................9Detectors.............................................................................................................................................................................9Photomultiplier Tubes (PMTs) ......................................................................................................................................9Image intensifiers .........................................................................................................................................................11Avalanche photodiodes ................................................................................................................................................12Cornerstones of Fluorescence Lifetime Imaging...............................................................................................................13Time Resolution...........................................................................................................................................................13Signal-to-Noise Ratio...................................................................................................................................................13Detection efficiency .....................................................................................................................................................13Recording efficiency ....................................................................................................................................................14Sample Saturation ........................................................................................................................................................14Photobleaching.............................................................................................................................................................14Signal Processing Techniques...........................................................................................................................................15Gated Image Intensifiers ..............................................................................................................................................15Modulation Techniques................................................................................................................................................17Single Channel Modulation Techniques..................................................................................................................18Modulated Image Intensifiers..................................................................................................................................20Gated Photon Counters ................................................................................................................................................23Time-Correlated Single Photon Counting (TCSPC) ....................................................................................................26TCSPC Imaging ......................................................................................................................................................27Multi Wavelength TCSPC Imaging.........................................................................................................................29Features of the TCSPC imaging techniques ............................................................................................................32Other TCSPC Techniques ............................................................................................................................................35Application of TCSPC to diffusion in cells.............................................................................................................35TCSPC Wide Field Imaging....................................................................................................................................35Comparison of Signal Processing Techniques ..................................................................................................................36Summary...........................................................................................................................................................................37References.........................................................................................................................................................................382
Page 1 and 2: Boston Electronics Corporation91 Bo
Page 3: FLIM Upgrade Kits for Confocal Micr
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 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
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TCSPC for FLIM and FRET in - Boston Electronics Corporation

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