Chapter 3 Time domain FLIM: Theory, instrumentation, and data analysis

• Published in: Laboratory Techniques in Biochemistry and Molecular Biology Vol. 33; pp. 95 - 132
• Main Authors: Gerritsen, H.C., Agronskaia, A.V., Bader, A.N., Esposito, A.
• Format: Book Chapter
• Language: English
• Published: Elsevier Science & Technology 2009
• ISBN: 9780080549583; 0080549586
• ISSN: 0075-7535; 2589-2789
• DOI: 10.1016/S0075-7535(08)00003-X

This chapter deals with time domain based fluorescence lifetime imaging methods. The chapter introduces the instrumentation and methods of analysis for FLIM in the time domain. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC and time-gating (TG). In TCSPC, the fluorescent molecules are excited with very short light pulses. In TG methods, the fluorescence emission is detected in two or more time-gates each delayed by a different time relative to the excitation pulse. There are many sources of noise that affect the performance of fluorescence imaging systems. Advantages and limitations of time-correlated single photon counting (TCSPC), and time-gated imaging techniques are discussed together, with general issues on photon effciency of detection schemes, data analysis, and practical experimental examples. In the analyses of the FLIM data, it is important to set an appropriate threshold on the number of counted photons above which the data is fitted. An important application of fluorescence lifetime imaging is the imaging of molecular colocalization based on FRET imaging.