Total internal reflectance fluorescence lifetime

Total internal reflection fluorescence lifetime imaging microscopy... 

Total internal reflection fluorescence (TIRF) microscopy, fluorescence in situ hybridization (FISH), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM). 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. 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. 

M. R. Rainbow, S. Arterton, and R. C. Eberhardt, Fluorescence lifetime measurements using total internal reflection fluorimetry Evidence for a conformational change adsorbed to quartz, J. Biomed. Mater. Res. 21, 539-555 (1987). 

Fig. 3. Total internal reflection cell used for the interfacial fluorescence lifetime measurement (left) and the external reflection absorption spectrometry (right).

The excited-state lifetime of the molecule in absence of any radiationless deeay processes is the natural fluorescence lifetime", r . The natural lifetime is a constant for a given molecule and given refraction index of the solvent. Because the absorbed energy can also be dissipated by internal conversion, the effective fluorescence lifetime, is shorter than the natural lifetime, The fluorescence quantum efficiency", i.e. the ratio of the number of emitted photons to absorbed photons, reflects the ratio of the radiative decay rate to the total decay rate. Most dyes of high quantum efficiency, such as laser dyes and fluorescence markers for biological samples, have natural fluorescence decay times of the order of 1 to 10 ns. There are a few exceptions, such as pyrene or coronene, with lifetimes of 400 ns and 200 ns, and rare-earth chelates with lifetimes in the ps range. 

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