Using fluorescent beads to emulate single fluorophores

Luis A Alemán-Castaneda (1) , Sherry Yi-Ting Feng (2) , Rodrigo Gutiérrez-Cuevas (3) , Isael Herrera (1) , Thomas Brown (2) , Sophie Brasselet (1) , Miguel Alonso (1)

1 FRESNEL - Institut FRESNEL
2 University of Rochester [USA]
3 IL - Institut Langevin - Ondes et Images (UMR7587)

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.

Domaines: Optique / photonique

Luis A Alemán-Castaneda, Sherry Yi-Ting Feng, Rodrigo Gutiérrez-Cuevas, Isael Herrera, Thomas Brown, et al.. Using fluorescent beads to emulate single fluorophores. Journal of the Optical Society of America. A Optics, Image Science, and Vision, 2022, 39 (12), pp.C167-C178. ⟨10.1364/JOSAA.474837⟩. ⟨hal-03887458⟩