Abstract
Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation is emerging as a reliable quantitative tool for measuring fluorescence resonance energy transfer (FRET) in living cells. By virtue of being free from spectroscopic artifacts encountered in conventional FRET detection methods, multiphoton FLIM methods offer the advantages of high spatial and temporal resolution, faster data acquisition and data analysis. We compare the FRET results obtained by two different methods namely (i) multiphoton excitation lifetime-based FRET and (ii) single photon excitation intensity-based acceptor photobleaching FRET. Using the same biological samples, we apply these two different methods in understanding the growth hormone receptor dimerization kinetics at the cell surface of human embryonic kidney cells. We conclude that the multiphoton FLIM using the streak-camera approach provides the best ability to monitor FRET in dynamic situations where high temporal and spatial resolution are required with minimal photodamage/phototoxicity.
| Original language | English |
|---|---|
| Pages (from-to) | 63-70 |
| Number of pages | 8 |
| Journal | Proceedings of SPIE - The International Society for Optical Engineering |
| Volume | 5323 |
| DOIs | |
| State | Published - 2004 |
| Event | Progress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV - San Jose, CA, United States Duration: 25 Jan 2004 → 27 Jan 2004 |
Keywords
- Acceptor photobleaching FRET
- FRET
- Multiphoton FLIM
- Streak camera