Fluorescence lifetime imaging by asynchronous pump-probe microscopy.
We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE.
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Department of Physics, University of Illinois at Urbana-Champaign 61801, USA. chen@lfd.physics.uiuc.edu
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French
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Gratton
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D000818:Animals; D004912:Erythrocytes / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology; D005456:Fluorescent Dyes; D006801:Humans; D007840:Latex; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D008863:Microspheres; D017785:Photons; D013997:Time Factors
D005456:Fluorescent Dyes; D007840:Latex