Abstract
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 manipulation, 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 conditions 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.
© 2022 Optica Publishing Group
Full Article | PDF ArticleMore Like This
Marijn E. Siemons, Lukas C. Kapitein, and Sjoerd Stallinga
Opt. Express 30(16) 28290-28300 (2022)
Oumeng Zhang and Matthew D. Lew
J. Opt. Soc. Am. A 38(2) 277-287 (2021)
Maxine Xiu, Jeff Field, Randy Bartels, and Ali Pezeshki
J. Opt. Soc. Am. A 40(1) 185-203 (2023)