Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera

We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (∼0.2 μm) and temporal (∼50 ps) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in the literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green florescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and fluorescence resonance energy transfer imaging, which are discussed in the context of live cell imaging.

[1]  H.C. Gerritsen,et al.  Combining two-photon excitation with fluorescence lifetime imaging , 1999, IEEE Engineering in Medicine and Biology Magazine.

[2]  Ammasi Periasamy,et al.  TIME-RESOLVED FLUORESCENCE LIFETIME IMAGING MICROSCOPY USING A PICOSECOND PULSED TUNABLE DYE LASER SYSTEM , 1996 .

[3]  S. Mayor,et al.  Fluorescence Methods to Probe Nanometer-Scale Organization of Molecules in Living Cell Membranes , 2001, Journal of Fluorescence.

[4]  Hiro-o Hamaguchi,et al.  Picosecond Raman Spectroscopy Using a Streak Camera , 1993 .

[5]  B. Herman,et al.  Resonance energy transfer microscopy. , 1989, Methods in cell biology.

[6]  P. Bastiaens,et al.  Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. , 1999, Trends in cell biology.

[7]  B R Masters,et al.  Two-photon excitation fluorescence microscopy. , 2000, Annual review of biomedical engineering.

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

[9]  Noriaki Miyanaga,et al.  Development of wide-field, multi-imaging x-ray streak camera technique with increased image-sampling arrays , 2001 .

[10]  B. Herman,et al.  Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. , 1998, Biophysical journal.

[11]  Richard N. Day,et al.  Nanosecond fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell , 2002, Journal of microscopy.

[12]  Paul R. Selvin,et al.  The renaissance of fluorescence resonance energy transfer , 2000, Nature Structural Biology.

[13]  T. Jovin,et al.  Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression. , 2001, Cytometry.

[14]  V. Subramaniam,et al.  One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. , 2000, Biophysical journal.

[15]  H. Gibbs,et al.  Combining a scanning near-field optical microscope with a picosecond streak camera: Statistical analysis of exciton kinetics in GaAs single-quantum wells , 2002 .

[16]  P J Verveer,et al.  Global analysis of fluorescence lifetime imaging microscopy data. , 2000, Biophysical journal.

[17]  Rainer Pepperkok,et al.  Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy , 1999, Current Biology.