RESUMO
Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to visualize photophysical characteristics of biological targets. However, conventional FLIM methods have some limitations that restrict obtaining high-precision images in real time. Here, we propose a high-speed time-resolved laser-scanning microscopy by incorporating a novel line-to-pixel referencing method into the previously suggested analog mean-delay (AMD) method. The AMD method dramatically enhances the photon accumulation speed for achieving the certain precision compared to the time-correlated single-photon counting (TCSPC) method while maintaining high photon efficiency. However, its imaging pixel rate can still be restricted by the rearm time of the digitizer when it is triggered by laser pulses. With our line-to-pixel referencing method, the pulse train repeats faster than the trigger rearm time can be utilized by generating a line trigger, which is phase-locked with only the first pulse in each horizontal line composing an image. Our proposed method has been tested with a pulsed laser with 40 MHz repetition rate and a commercial digitizer with a 500 ns trigger rearm time, and a frame rate of 3.73 fps with a pixel rate of 3.91 MHz was accomplished while maintaining the measurement precision under 20 ps.
RESUMO
A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.