Chapter 9 Total internal reflection fluorescence lifetime imaging microscopy

• Published in: Laboratory Techniques in Biochemistry and Molecular Biology Vol. 33; pp. 395 - 412
• Main Author: Gadella, T.W.J.
• Format: Book Chapter
• Language: English
• Published: Elsevier Science & Technology 2009
• ISBN: 9780080549583; 0080549586
• ISSN: 0075-7535; 2589-2789
• DOI: 10.1016/S0075-7535(08)00009-0

The chapter presents the upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. The chapter presents the combination of total internal reflection fluorescence (TIRF) microscopy and fluorescence lifetime imaging Microscope (FLIM). The implementation with of TIRF in a frequency-domain wide-field FLIM system is described. The chapter demonstrates an application of TIRF-FLIM, as TIRF provides the spatial contrast of detecting only molecules that are immediately adjacent to the plasma membrane, and the lifetime contrast reports on the aggregation state of annexin A4.