Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells

摘要: A new method of fluorescence microscopy for cell imaging has been developed that takes advantage the spatial variations lifetimes in single cells as a source image contrast, and thus it is named "fluorescence lifetime (flimscopy)". Since time-resolved measurements are sensitive to molecular dynamics interactions, flimscopy allows information be visualized cells. In measurements, several (nanosecond) images sample obtained at various delay times after pulsed laser excitation microscope's entire field view. Lifetimes calculated pixel-by-pixel from these images, then displayed pseudocolor format (flimscopy image). The total data acquisition time needed obtain with diffraction-limited resolution (approximately 250 nm) decreased just approximately 30 s 300 fluorescent molecules/micron2. This was achieved by developing high-frequency (400 kHz) nanosecond-gating (9 ns full width half height)-signal accumulation system. technique extent resonance energy transfer living cells, free errors due path length, light scattering, number fluorophores necessitate complex corrections steady-state microfluorometry ratio microscopy. Flimscopy applied here observe fusion individual endosomes Results revealed occurrence extensive between primary endocytic vesicles and/or sorting endosomes, thereby raising possibility biogenesis involves multiple fusions vesicles.