Abstract

We use a two-photon laser scanning microscope with a new Time-Correlated Single Photon Counting (TCSPC) imaging technique to obtain combined intensity-lifetime images for FRET measurements in living cells. Single photon pulses from a photomultiplier and signals from the scanning head are used to record the three-dimensional photon density over the time- and image coordinates. Double exponential decay analysis delivers the lifetime components of the quenched and the unquenched molecules in all pixels of the image. We use the ratio of the intensity coefficients of the fast and slow decay component to create images that show the size of the FRET effects in different parts of the cell.

© 2001 OSA/SPIE