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Microsecond Decay FLIM: Combined Fluorescence and Phosphorescence Lifetime Imaging

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We present a lifetime imaging technique that simultaneously records fluorescence and phosphorescence lifetime images in laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional TCSPC. Fluorescence is recorded during the on-phase of the laser, phosphorescence during the off-phase. The technique does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using excessively high pulse power for phosphorescence excitation. Laser modulation is achieved either by electrically modulating picosecond diode lasers, or be controlling the lasers via the AOM of a standard confocal or multiphoton laser scanning microscope.

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