Precision-enhanced two photon fluorescence lifetime imaging microscopy with controlled repetition rate

Abstract

Fluorescence lifetime that provides information on the fluorophore and local environment is one of the important parameters in the field of medical and life sciences. Two-photon fluorescence lifetime imaging (TP-FLIM) microscopy with a high wavelength pulsed laser stimulates fluorophore by two-photon to obtain fluorescence. TPFLIM is being studied in diagnosing various diseases like atherosclerosis and cancer due to its high penetration depth, safety, high resolution, and strong against photobleaching. Most studies use a high laser repetition rateabout ~80MHz, however, it can affect the fluorescence, penetration depth, and photodamage/bleaching. This study suggests a method to find the proper pulse repetition rate of TP-FLIM microscopy with AMD method by finding out the relationship of repetition rate and system performance. The method is represented by accuracy, precision, phototoxicity, and signal to noise ratio with a controlled repetition rate with simulation and experiment. Finally, the high-quality of tissue images can be obtained by optimal system.