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Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse Excitation

We present a fluorescence and phosphorescence lifetime imaging (FLIM / PLIM) technique that simultaneously records FLIM and PLIM in confocal or multiphoton laser scanning systems. Different than other techniques, it uses not only one, but multiple laser pulses for every phosphorescence excitation cycle. The sensitivity is thus orders of magnitude higher. Our technique is based on on-off modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multi-dimensional TCSPC. FLIM is obtained by building up a photon distribution over the times of the photons in the laser pulse period and the scan coordinates, PLIM by building up the distribution over the times of the photons in the laser modulation period and the scan coordinates. The technique does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of high pulse energy for phosphorescence excitation.

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