Development of label-free multispectral large-field fluorescence lifetime imaging microscopy

Abstract

Intrinsic fluorophores in biological sample provides structural and biochemical information within tissue. Fluorescence lifetime imaging microscopy (FLIM), which measures the fluorescence intensity and fluorescence lifetime of these endogenous fluorophores with high-resolution images, is becoming increasingly important in the biological science. However, Photobleaching occurring in fluorescence imaging and a narrow field of view of FLIM due to low image acquisition speed has limited versatility of FLIM. This study describes the development of multispectral fluorescence lifetime imaging microscopy using 1MHz pulsed laser and analog mean delay method and proposes a method for reducing photobleaching through utilization of optical property and scanning method that increase light detection efficiency. This study deals with theoretical explanation of the proposed method, the detailed design process of the system and the verification of performance through various sample experiments.