Experimental investigation of noise characterization in real-time, multiphoton fluorescence lifetime imaging microscopy

Multiphoton Fluorescence Lifetime Imaging Microscopy (MP-FLIM) is an important tool for biological studies, enabling the detection of cellular microenvironment dynamics in deeper resolution. However, imaging quality of FLIM is often limited under low-photon efficiency conditions. To enhance fluorescence lifetime estimation through inverse modeling techniques such as deconvolution or denoising, we developed a physics-based mathematical model for noise characterization in FLIM. In this work, we primarily focus on validating our model’s performance by analyzing single-lifetime fluorophores and stained samples. By extracting statistical indicators and examining their relationship with excitation power, we assess the model’s performance under real FLIM experimental conditions. The results confirm that our model accurately characterizes the noise distribution by analyzing statistical properties across different power levels.