Using fluorescent beads to emulate single fluorophores

We study the conditions under which fluorescent beads can be used to emulate single fluorescent molecules in the calibration of optical microscopes. Although beads are widely used due to their brightness and easy manipu-lation, there can be notable differences between the point spread functions (PSFs) they produce and those for single-molecule fluorophores, caused by their different emission patterns and sizes. We study theoretically these differences for various scenarios, e.g., with or without polarization channel splitting, to determine the condi-tions under which the use of beads as a model for single molecules is valid. We also propose methods to model the blurring due to the size difference and compensate for it to produce PSFs that are more similar to those for single molecules.(c) 2022 Optica Publishing Group

Automated summary

This study investigates the conditions for using fluorescent beads to emulate single fluorescent molecules in the calibration of optical microscopes. It examines the differences in point spread functions (PSFs) between beads and single molecules caused by their emission patterns and sizes, and proposes methods to compensate for the size difference and improve the similarity of PSFs.