Picosecond fluorescence lifetime imaging microscope for imaging of living glioma cells

摘要

In this communication, we report the imaging of living glioma cells using fluorescence lifetime imaging (FLIM)technique. The growing interests in developing novel techniques for diagnosis and minimally invasive therapy of braintumor have led to microscopic studies of subcellular structures and intracellular processes in glioma cells. Fluorescencemicroscopy has been used with a number of exogenous molecular probes specific for certain intracellular structures suchas mitochondria, peripheral benzodiazepine receptor (PBR), and calcium concentration. When probes with overlappingemission spectra being used, separate samples are required to image each probe individually under conventionalfluorescence microscopy. We have developed a wide-field FLIM microscope that uses fluorescence lifetime as anadditional contrast for resolving multiple markers in the same essay. The FLIM microscope consists of a violet diodelaser and a nitrogen-pumped dye laser to provide tunable sub-nanosecond excitation from UV to NIR. The detectionsystem is based on a time-gated ICCD camera with minimum 80 ps gate width. The performance of the system wasevaluated using fluorescence dyes with reported lifetime values. Living rat glioma C6 cells were stained with JC-1 andRhodamine 123. FLIM images were acquired and their lifetimes in living cells were found in good agreements withvalues measured in solutions by a time-domain fluorescence spectrometer. These results indicate that imaging of gliomacells using FLIM can resolve multiple spectrally-overlapping probes and provide quantitative functional informationabout the intracellular environment.