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Fluorescence Lifetime Imaging Microscopy

The CFS has developed fluorescence lifetime imaging microscopy (FLIM). In the FLIM method, image contrast in the fluorescence microscope is derived from the fluorescence lifetime at each point in the image. This method provides chemical imaging of intracellular ions. We now know that the lifetimes of many fluorophores are altered by the presence of analytes such as Ca2+, Mg2+, Cl , pH or K+, to name a few. Fluorescence lifetimes are mostly independent of the local probe concentration and photobleaching, Importantly, FLIM does not require wavelength-ratiometric probes. Consequently, FLIM allows quantitative Ca2+ imaging using visible-wavelength illumination. A new FLIM instrument is under construction and will be available to external users of the CFS.

References:

  1. Sodium Green as a Potential Probe for Intracellular Sodium Imaging Based on Fluorescence Lifetime. Szmacinski, H. and Lakowicz, J.R. (1997). Analytical Biochemistry, 250:131-138.  
  2. Lifetime-based pH Sensors: Indicators for Acidic Environments, Lin, H.-J., Szmacinski, H., and Lakowicz, J. R. (1999). Anal. Biochem. 269:162-167.  
  3. Release Rates of the Anticancer Drug Topotecan Measured By Fluorescence Lifetime Imaging Microscopy (FLIM), Nowaczyk, K., Herman, P., Demir, A.S., Tanyeli, C., Burke, T.G., Lakowicz, J.R., and Johnson, M.L. (1999). Biophysical Journal, 76: A361
  4. Fluorescence Lifetime Imaging of Nuclear DNA: Effect of Fluorescence Resonance Energy Transfer, S. Murata, P. Herman, H.-J., Lin, and J. R. Lakowicz (2000). Cytometry 41:178-185.
  5. Texture Analysis of Fluorescence Lifetime Images of Nuclear DNA with Effect of Fluorescence Resonance Energy Transfer, S. Murata, P. Herman and J. R. Lakowicz (2001). Cytometry 43:94-100.
  6. Frequency-Domain Fluorescence Microscopy with the LED as a Light Source, P. Herman, B. P. Maliwal, H.-J. Lin, and J. R. Lakowicz (2001). J. Microscopy, 203(2): 176-181.
FLIM
Ionophore and weak base treatments perturbed
the cytosolic pH of CHO cells.

Book Chapters

  1. Imaging Applications of Time-Resolved Fluorescence Spectroscopy (1996). Lakowicz, J.R. and Szmacinski, H. In: Fluorescence Imaging Spectroscopy and Microscopy. Vol. 137. (X.F. Wang and B. Herman, Eds.), John Wiley & Sons, Inc. Publishers. pp. 273-311.
  2. Recent Developments in Fluorescence Spectroscopy: Fluorescence Lifetime Imaging Microscopy, Long Lifetime Metal-Ligand Probes, Multi-Photon Excitation and Light Quenching (1997). Lakowicz, J.R. In: Applications of Optical Engineering to the Study of Cellular Pathology (E. Kohen, Ed.), Research Signpost, India, pp. 33-46.
  3. Emerging Applications of Fluorescence Spectroscopy to Cellular Imaging: Lifetime Imaging, Metal-Ligand Probes, Multi-Photon Excitation and Light Quenching (1996). Lakowicz, J. R. In: Scanning Microscopy Supplement Vol. 10. Scanning Microscopy International. pp. 213-224.