Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy

• DOI: 10.1038/s41377-022-01064-4
• Publication Date: 2023-01-26T11:02:52Z
• Authors: Júlia Ferrer Ortas; Pierre Mahou; Sophie Escot; Chiara Stringari; Nicolas B. David; Laure Bally-Cuif; Nicolas Dray; Michel Négrerie; Willy Supatto; Emmanuel Beaurepaire
• Keywords: Temporal resolution

Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain tissue physiology. Current microscopy methods are limited either in sensitivity or spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) third-harmonic (THG) contrasts. First, propose experimental scheme color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band hemoglobin. We show that there strong three-photon (3P) resonance related to hemoglobin THG signals from zebrafish human RBCs, sensitive RBC state. demonstrate our implementation enables specific detection flowing RBCs embryos dynamics with single-cell resolution microsecond pixel times. Moreover, it can be implemented 3P microscope RBC-specific contrast depths exceeding 600 µm live adult brain. Our results establish new multiphoton extending palette deep-tissue microscopy.