A framework to enhance the signal-to-noise ratio for quantitative fluorescence microscopy.

Abstract

Single-cell fluorescence characterization has gained much attention for studying the dynamics of individual cells in human diseases such as cancer. Despite the abundance of literature on quantitative fluorescence microscopy and its advantages in measuring cell-to-cell variation and spatial variation over other high-throughput instruments, there lacks a concise model that one can follow to maximize the quality of images. Here, we used the signal-to-noise ratio (SNR) model to verify marketed camera parameters and optimize microscope settings to maximize SNR for quantitative single cell fluorescence microscopy (QSFM). We determined the microscope camera's readout noise, dark current, photon shot noise, the clock-induced charge, and validated the additive noise model for each noise source. The dark current and the clock-induced charge were both higher than reported in literature, compromising camera sensitivity. We also reduced excess background noise and improved SNR by 3-fold, by adding secondary emission and excitation filters as well as by introducing wait time in the dark before fluorescence acquisition. Additionally, our work opens new avenues for enhancing superresolution microscopy techniques such as single-molecule localization microscopy (SMLM).