Fluorescence saturation imaging microscopy: molecular fingerprinting with a standard confocal microscope

Author:

Yakimov Boris12,Rovnyagina Natalia1,Hasan Afraa3,Zhang Juntao4,Wang Haibo4,Fadeev Victor,Urusova Liliya5,Khoroshilov Evgeny6,Sharkov Andrey6,Mokrysheva Nataliya5,Shirshin Evgeny5

Affiliation:

1. Sechenov First Moscow State Medical University

2. Vorohobov’s City Clinical Hospital

3. HSE University

4. Wuhan Polytechnic University

5. Endocrinology Research Center

6. P.N. Lebedev Physical Institute, Russian Academy of Sciences

Abstract

Molecular specificity in fluorescence imaging of cells and tissues can be increased by measuring parameters other than intensity. For instance, fluorescence lifetime imaging became a widespread modality for biomedical optics. Previously, we suggested using the fluorescence saturation effect at pulsed laser excitation to map the absorption cross-section as an additional molecular contrast in two-photon microscopy [Opt. Lett. 47(17), 4455 (2022).10.1364/OL.465605]. Here, it is shown that, somewhat counterintuitive, fluorescence saturation can be observed under cw excitation in a standard confocal microscopy setup. Mapping the fluorescence saturation parameter allows obtaining additional information about the fluorophores in the system, as demonstrated by the example of peptide hydrogel, stained cells and unstained thyroid gland. The suggested technique does not require additional equipment and can be implemented on confocal systems as is.

Funder

Moscow government

Russian Science Foundation

Moscow Center for Innovative Technologies in Healthcare

Publisher

Optica Publishing Group

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