Four-dimensional multiphoton microscopy with time-correlated single-photon counting.

We report on the implementation of fluorescence-lifetime imaging in multiphoton excitation microscopy that uses PC-compatible modules for time-correlated single-photon counting. Four-dimensional data stacks are produced with each pixel featuring fluorescence-decay curves that consist of as many as 4096 bins. Fluorescence lifetime(s) and their amplitude(s) are extracted by statistical methods at each pixel or in arbitrarily defined regions of interest. When employing an avalanche photodiode the width of the temporal response function is 420 ps. Although this response confines the temporal resolution to values greater than several hundreds of picoseconds, the lifetime precision is determined by the signal-to-noise ratio and can be in the range of tens of picosconds. Lifetime changes are visualized in pulsed-laser-deposited fluorescent layers as well as in cyan fluorescent proteins that transfer energy to yellow fluorescent proteins in live mammalian cells.

[1]  David M. Coleman,et al.  A Two-Dimensional Fluorescence Lifetime Imaging System Using a Gated Image Intensifier , 1991 .

[2]  William H. Press,et al.  Numerical recipes , 1990 .

[3]  Squire,et al.  Multiple frequency fluorescence lifetime imaging microscopy , 2000, Journal of microscopy.

[4]  Joseph R. Lakowicz,et al.  Lifetime‐selective fluorescence imaging using an rf phase‐sensitive camera , 1991 .

[5]  Hans C. Gerritsen,et al.  Fluorescence Lifetime Imaging, a New Tool in Confocal Microscopy , 1995 .

[6]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[7]  Jürgen Wolfrum,et al.  Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC) , 1999 .

[8]  T M Jovin,et al.  Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging. , 1991, Biophysical journal.

[9]  William H. Press,et al.  Numerical recipes in C , 2002 .

[10]  William H. Press,et al.  Numerical Recipes in C, 2nd Edition , 1992 .

[11]  Sytsma,et al.  Time‐gated fluorescence lifetime imaging and microvolume spectroscopy using two‐photon excitation , 1998 .

[12]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[13]  T. Klar,et al.  Ultrafast dynamics microscopy , 2000 .

[14]  H Szmacinski,et al.  Fluorescence lifetime imaging. , 1992, Analytical biochemistry.

[15]  T M Jovin,et al.  Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin. , 1996, The EMBO journal.

[16]  Schrader,et al.  Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two‐photon fluorescence microscopy , 1998, Journal of microscopy.

[17]  Christopher G. Morgan,et al.  Prospects for confocal imaging based on nanosecond fluorescence decay time , 1992 .

[18]  David M. Coleman,et al.  Time-Resolved Fluorescence Microscopy Using Multichannel Photon Counting , 1990 .

[19]  M. Strauba,et al.  Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope , 1998 .

[20]  J. Lakowicz,et al.  Fluorescence lifetime imaging of free and protein-bound NADH. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Stefan W. Hell,et al.  Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope , 1998 .

[22]  S. Hell,et al.  Pulsed laser fluorophore deposition: a method for measuring the axial resolution in two-photon fluorescence microscopy , 1995 .