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

Júlia Ferrer Ortas; Pierre Mahou; Sophie Escot; Chiara Stringari; Nicolas B. David; Laure Bally-Cuif; Nicolas Dray; Michel Négrerie; Willy Supatto; Emmanuel Beaurepaire

doi:10.1038/s41377-022-01064-4

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

Original Articles

- Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy
- Light: Science & Applications Vol. 12, Issue 2, Pages: 168-182(2023)
- Affiliations：
  
  1.Laboratory for Optics and Biosciences, CNRS, INSERM, École polytechnique, IP Paris, 91128, Palaiseau, France
  2.Zebrafish Neurogenetics Unit, team supported by Ligue Nationale contre le Cancer, Institut Pasteur, CNRS, 75015, Paris, France
- Author bio：
  
  Emmanuel Beaurepaire (emmanuel.beaurepaire@polytechnique.edu)
- Funds：
- DOI：10.1038/s41377-022-01064-4
  CLC：
- Published：28 February 2023，
  
  Published Online：26 January 2023，
  
  Received：31 May 2022，
  
  Revised：9 December 2022，
  
  Accepted：22 December 2022
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Ortas, J. F. et al. Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy. Light: Science & Applications, 12, 168-182 (2023).
DOI：

Ortas, J. F. et al. Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy. Light: Science & Applications, 12, 168-182 (2023). DOI： 10.1038/s41377-022-01064-4.

摘要

Abstract

Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work

we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First

we propose a novel experimental scheme for color TSFG microscopy

which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs

and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover

it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue microscopy.

关键词

Keywords

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