Fluorescence Lifetime Imaging Microscopy

Alessandro Esposito1, Fred S. Wouters1

1 European Neuroscience Institute‐Goetingen, Goetingen
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.14
DOI:  10.1002/0471143030.cb0414s25
Online Posting Date:  December, 2004
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Fluorescent lifetime imaging microscopy is a powerful tool to enhance the contrast in images of biological samples and to investigate the local environment of a fluorochrome. FLIM allows the detection of protein‐protein interactions and their biochemical state by the quantitative detection of Förster resonance energy transfer (FRET) between molecules in living cells or tissues. The availability of different spectral variants of the visible fluorescent proteins (VFPs) allows the investigation of molecular activities and protein‐protein interactions in living cells by FRET as measured by FLIM.

Keywords: FLIM; FRET; fluorescence microscopy; two‐photon microscopy; protein‐protein interactions

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Operating A Wide‐Field FD‐FLIM Setup
  • Basic Protocol 2: Operating A TD‐FLIM Based on TPLSM
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Operating A Wide‐Field FD‐FLIM Setup

  • Single‐exponential decaying fluorochrome with a well‐defined lifetime (e.g., Rhodamine‐6G)
  • Basic components of a wide‐field FD‐FLIM (see )
  • Positive‐intrinsic‐negative (PIN) diode connected to an oscilloscope (bandwidth up to 100 MHz)
  • Mirror, reflective foil, homogenous flat fluorescent or scattering sample
CAUTION: Protective goggles matching the used laser wavelength and power are recommended when aligning the optical path. Lasers can be harmful and standard laser safety rules should always be followed. Moreover, handle the optics with cotton gloves to avoid dirty or scratched optical surfaces.

Basic Protocol 2: Operating A TD‐FLIM Based on TPLSM

  • Homogenous flat fluorescent sample
  • Basic components of a scanning TD‐FLIM (see and Critical Parameters)
  • Infrared viewer
CAUTION: Wear protective goggles matching near‐infrared radiation when aligning the optical path. Lasers can be harmful and standard laser safety rules should always be followed. Moreover, handle the optics with cotton gloves to avoid dirty or scratched optical surfaces.
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Literature Cited

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Internet Resources
  The microscopes that were described in this unit were built by assembling standard wide‐field or scanning microscopes with detectors purchased from LaVision and Becker&Hickl. LaVision offers MCP‐CCD cameras suitable for wide‐field imaging, while Becker&Hickl sells electronics and detectors for TCSPC, compatible also with FCS and other techniques.
  A complete wide‐field system, except light sources, can be acquired from LaVision BioTec or from its U.S. reseller TauTec.
  Lambert‐Instruments offers a setup with a LED as a light source with the possibility to drive other light sources like lasers.
  A TGSPC system (LIMO) is available from Nikon Instech.
  PicoQuant sells a stage scanning TCSPC microscope and a broad range of lifetime products.
  Jobin‐Yvon sells a frequency‐domain confocal system and a broad range of lifetime products.
  A broad range of lifetime products is available from ISS and Edinburgh Instruments.
  Other manufacturers or resellers for lasers and detectors that are of interest for FLIM are Kentech Instruments, Hamamatsu Photonics K.K., Coherent, Spectra Physics, and Melles‐Griot.
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