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
- Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy
- 光：科学与应用 2023年12卷第2期页码：168-182
- 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
  中图分类号：
- 纸质出版日期：2023-2-28，
  
  网络出版日期：2023-1-26，
  
  收稿日期：2022-5-31，
  
  修回日期：2022-12-9，
  
  录用日期：2022-12-22
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Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy[J]. 光：科学与应用, 2023,12(2):168-182.

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).
Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy[J]. 光：科学与应用, 2023,12(2):168-182. DOI： 10.1038/s41377-022-01064-4.

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

references

D. Kleinfeld , 等 . Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex . Proc. Natl Acad. Sci. USA , 1998 . 95 15741 -15746 . DOI:10.1073/pnas.95.26.15741http://doi.org/10.1073/pnas.95.26.15741 .

E. Chaigneau , 等 . Two-photon imaging of capillary blood flow in olfactory bulb glomeruli . Proc. Natl Acad. Sci. USA , 2003 . 100 13081 -13086 . DOI:10.1073/pnas.2133652100http://doi.org/10.1073/pnas.2133652100 .

A. Y. Shih , 等 . Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain . J. Cerebral Blood Flow Metabol. , 2012 . 32 1277 -1309 . DOI:10.1038/jcbfm.2011.196http://doi.org/10.1038/jcbfm.2011.196 .

O. S. Finikova , 等 . Oxygen microscopy by two-photon-excited phosphorescence . ChemPhysChem , 2008 . 9 1673 -1679 . DOI:10.1002/cphc.200800296http://doi.org/10.1002/cphc.200800296 .

S. Sakadžić , 等 . Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue . Nat. Methods , 2010 . 7 755 -759 . DOI:10.1038/nmeth.1490http://doi.org/10.1038/nmeth.1490 .

J. Lecoq , 等 . Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels . Nat. Med. , 2011 . 17 893 -898 . DOI:10.1038/nm.2394http://doi.org/10.1038/nm.2394 .

T. V. Esipova , 等 . Oxyphor 2P: a high-performance probe for deep-tissue longitudinal oxygen imaging . Cell Metabol. , 2019 . 29 736 -744.e7 . DOI:10.1016/j.cmet.2018.12.022http://doi.org/10.1016/j.cmet.2018.12.022 .

Y. Zeng , 等 . In vivo micro-vascular imaging and flow cytometry in zebrafish using two-photon excited endogenous fluorescence . Biomed. Optics Express , 2014 . 5 653 -663 . DOI:10.1364/BOE.5.000653http://doi.org/10.1364/BOE.5.000653 .

P. Theer , W. Denk . On the fundamental imaging-depth limit in two-photon microscopy . J. Optical Soc. Am. A , 2006 . 23 3139 -3149 . DOI:10.1364/JOSAA.23.003139http://doi.org/10.1364/JOSAA.23.003139 .

N. G. Horton , 等 . In vivo three-photon microscopy of subcortical structures within an intact mouse brain . Nat. Photon. , 2013 . 7 205 -209 . DOI:10.1038/nphoton.2012.336http://doi.org/10.1038/nphoton.2012.336 .

D. G. Ouzounov , 等 . In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain . Nat. Methods , 2017 . 14 388 -390 . DOI:10.1038/nmeth.4183http://doi.org/10.1038/nmeth.4183 .

K. Guesmi , 等 . Dual-color deep-tissue three-photon microscopy with a multiband infrared laser . Light Sci. Appl. , 2018 . 7 12 DOI:10.1038/s41377-018-0012-2http://doi.org/10.1038/s41377-018-0012-2 .

M. Yildirim , 等 . Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy . Nat. Commun. , 2019 . 10 177 DOI:10.1038/s41467-018-08179-6http://doi.org/10.1038/s41467-018-08179-6 .

L. Streich , 等 . High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy . Nat. Methods , 2021 . 18 1253 -1258 . DOI:10.1038/s41592-021-01257-6http://doi.org/10.1038/s41592-021-01257-6 .

Y. Barad , 等 . Nonlinear scanning laser microscopy by third harmonic generation . Appl. Phys. Lett. , 1997 . 70 922 -924 . DOI:10.1063/1.118442http://doi.org/10.1063/1.118442 .

M. Müller , 等 . 3D microscopy of transparent objects using third-harmonic generation . J. Microscopy , 1998 . 191 266 -274 . DOI:10.1046/j.1365-2818.1998.00399.xhttp://doi.org/10.1046/j.1365-2818.1998.00399.x .

D. Oron , 等 . Depth-resolved structural imaging by third-harmonic generation microscopy . J.Struct. Biol. , 2004 . 147 3 -11 . DOI:10.1016/S1047-8477(03)00125-4http://doi.org/10.1016/S1047-8477(03)00125-4 .

N. Olivier , 等 . Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy . Science , 2010 . 329 967 -971 . DOI:10.1126/science.1189428http://doi.org/10.1126/science.1189428 .

M. J. Farrar , 等 . In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy . Biophys. J. , 2011 . 100 1362 -1371 . DOI:10.1016/j.bpj.2011.01.031http://doi.org/10.1016/j.bpj.2011.01.031 .

D. Débarre , 等 . Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy . Nat. Methods , 2006 . 3 47 -53 . DOI:10.1038/nmeth813http://doi.org/10.1038/nmeth813 .

B. Weigelin , G. J. Bakker , P. Friedl . Third harmonic generation microscopy of cells and tissue organization . J. Cell Sci. , 2016 . 129 245 -255. .

C. K. Sun , 等 . Higher harmonic generation microscopy for developmental biology . J. Struct. Biol. , 2004 . 147 19 -30 . DOI:10.1016/j.jsb.2003.10.017http://doi.org/10.1016/j.jsb.2003.10.017 .

G. J. Tserevelakis , 等 . Label-free imaging of lipid depositions in C. elegans using third-harmonic generation microscopy . PLoS One , 2014 . 9 e84431 DOI:10.1371/journal.pone.0084431http://doi.org/10.1371/journal.pone.0084431 .

H. Lim , 等 . Label-free imaging of Schwann cell myelination by third harmonic generation microscopy . Proc. Natl Acad. Sci. USA , 2014 . 111 18025 -18030 . DOI:10.1073/pnas.1417820111http://doi.org/10.1073/pnas.1417820111 .

S. Witte , 等 . Label-free live brain imaging and targeted patching with third-harmonic generation microscopy . Proc. Natl Acad. Sci. USA , 2011 . 108 5970 -5975 . DOI:10.1073/pnas.1018743108http://doi.org/10.1073/pnas.1018743108 .

H. J. Liu , 等 . In vivo deep-brain blood flow speed measurement through third-harmonic generation imaging excited at the 1700-nm window . Biomed.Optics Express , 2020 . 11 2738 -2744 . DOI:10.1364/BOE.389662http://doi.org/10.1364/BOE.389662 .

D. M. Chow , 等 . Deep three-photon imaging of the brain in intact adult zebrafish . Nat. Methods , 2020 . 17 605 -608 . DOI:10.1038/s41592-020-0819-7http://doi.org/10.1038/s41592-020-0819-7 .

Akbari, N. et al. Whole-brain optical access in small adult vertebrates with two- and three-photon microscopy. bioRxiv (in the press).

G. O. Clay , 等 . Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin . J. Opt. Soc. Am. B , 2006 . 23 932 -950 . DOI:10.1364/JOSAB.23.000932http://doi.org/10.1364/JOSAB.23.000932 .

C. H. Yu , 等 . Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye . Opt. Lett. , 2008 . 33 387 -389 . DOI:10.1364/OL.33.000387http://doi.org/10.1364/OL.33.000387 .

C. F. Chang , C. H. Yu , C. K. Sun . Multi-photon resonance enhancement of third harmonic generation in human oxyhemoglobin and deoxyhemoglobin . J. Biophoton. , 2010 . 3 678 -685 . DOI:10.1002/jbio.201000045http://doi.org/10.1002/jbio.201000045 .

U. Tripathy , 等 . Optimization of malaria detection based on third harmonic generation imaging of hemozoin . Anal. Bioanal. Chem. , 2013 . 405 5431 -5440 . DOI:10.1007/s00216-013-6985-zhttp://doi.org/10.1007/s00216-013-6985-z .

H. Segawa , 等 . Electronically resonant third-order sum frequency generation spectroscopy using a nanosecond white-light supercontinuum . Opt. Express , 2014 . 22 10416 -10429 . DOI:10.1364/OE.22.010416http://doi.org/10.1364/OE.22.010416 .

A. A. Lanin , 等 . Three-photon-resonance-enhanced third-harmonic generation for label-free deep-brain imaging: In search of a chemical contrast . J. Raman Spectrosc. , 2019 . 50 1296 -1302 . DOI:10.1002/jrs.5566http://doi.org/10.1002/jrs.5566 .

G. Campargue , 等 . Multiorder nonlinear mixing in metal oxide nanoparticles . Nano Lett. , 2020 . 20 8725 -8732 . DOI:10.1021/acs.nanolett.0c03559http://doi.org/10.1021/acs.nanolett.0c03559 .

A. M. Hanninen , 等 . High-resolution infrared imaging of biological samples with third-order sum-frequency generation microscopy . Biomed. Opt. Express , 2018 . 9 4807 -4817 . DOI:10.1364/BOE.9.004807http://doi.org/10.1364/BOE.9.004807 .

P. Mahou , 等 . Multicolor two-photon tissue imaging by wavelength mixing . Nat. Methods , 2012 . 9 815 -818 . DOI:10.1038/nmeth.2098http://doi.org/10.1038/nmeth.2098 .

Xu, C. & Webb, W. W. in Topics in Fluorescence Spectroscopy (ed Lakowicz, J. R. ) 471–540 (Springer, 2002).

P. Mahou , 等 . Metrology of multiphoton microscopes using second harmonic generation nanoprobes . Small , 2017 . 13 1701442 DOI:10.1002/smll.201701442http://doi.org/10.1002/smll.201701442 .

Boyd, R. W. Nonlinear Optics. 3rd edn. (Elsevier, 2008).

J. X. Cheng , X. S. Xie . Green's function formulation for third-harmonic generation microscopy . J. Opt. Soc. Am. B , 2002 . 19 1604 -1610 . DOI:10.1364/JOSAB.19.001604http://doi.org/10.1364/JOSAB.19.001604 .

D. Débarre , W. Supatto , E. Beaurepaire . Structure sensitivity in third-harmonic generation microscopy . Opt. Lett. , 2005 . 30 2134 -2136 . DOI:10.1364/OL.30.002134http://doi.org/10.1364/OL.30.002134 .

J. Morizet , 等 . Modeling nonlinear microscopy near index-mismatched interfaces . Optica , 2021 . 8 944 -951 . DOI:10.1364/OPTICA.421257http://doi.org/10.1364/OPTICA.421257 .

A. C. Millard , 等 . Third-harmonic generation microscopy by use of a compact, femtosecond fiber laser source . Appl. Opt. , 1999 . 38 7393 -7397 . DOI:10.1364/AO.38.007393http://doi.org/10.1364/AO.38.007393 .

V. Barzda , 等 . Visualization of mitochondria in cardiomyocytes . Opt. Express , 2005 . 13 8263 -8276 . DOI:10.1364/OPEX.13.008263http://doi.org/10.1364/OPEX.13.008263 .

A. E. Tuer , 等 . Nonlinear multicontrast microscopy of hematoxylin-and-eosin-stained histological sections . J. Biomed. Opt. , 2010 . 15 026018 DOI:10.1117/1.3382908http://doi.org/10.1117/1.3382908 .

R. D. Schaller , J. C. Johnson , R. J. Saykally . Nonlinear chemical imaging microscopy: near-field third harmonic generation imaging of human red blood cells . Anal. Chem. , 2000 . 72 5361 -5364 . DOI:10.1021/ac000699rhttp://doi.org/10.1021/ac000699r .

M. Rehberg , 等 . Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy . PLoS One , 2011 . 6 e28237 DOI:10.1371/journal.pone.0028237http://doi.org/10.1371/journal.pone.0028237 .

B. Weigelin , G. J. Bakker , P. Friedl . Intravital third harmonic generation microscopy of collective melanoma cell invasion: principles of interface guidance and microvesicle dynamics . IntraVital , 2012 . 1 32 -43 . DOI:10.4161/intv.21223http://doi.org/10.4161/intv.21223 .

S. Dietzel , 等 . Label-free determination of hemodynamic parameters in the microcirculaton with third harmonic generation microscopy . PLoS One , 2014 . 9 e99615 DOI:10.1371/journal.pone.0099615http://doi.org/10.1371/journal.pone.0099615 .

S. J. Ahn , 等 . Label-free assessment of hemodynamics in individual cortical brain vessels using third harmonic generation microscopy . Biomed. Opt. Express , 2020 . 11 2665 -2678 . DOI:10.1364/BOE.385848http://doi.org/10.1364/BOE.385848 .

C. K. Chen , T. M. Liu . Imaging morphodynamics of human blood cells in vivo with video-rate third harmonic generation microscopy . Biomed. Opt. Express , 2012 . 3 2860 -2865 . DOI:10.1364/BOE.3.002860http://doi.org/10.1364/BOE.3.002860 .

C. Zhang , D. L. Zhang , J. X. Cheng . Coherent Raman scattering microscopy in biology and medicine . Ann. Rev. Biomed. Eng. , 2015 . 17 415 -445 . DOI:10.1146/annurev-bioeng-071114-040554http://doi.org/10.1146/annurev-bioeng-071114-040554 .

R. M. White , 等 . Transparent adult zebrafish as a tool for in vivo transplantation analysis . Cell Stem Cell , 2008 . 2 183 -189 . DOI:10.1016/j.stem.2007.11.002http://doi.org/10.1016/j.stem.2007.11.002 .

S. Grillitsch , 等 . The influence of environmental $$P_{O_2}$$ on hemoglobin oxygen saturation in developing zebrafish Danio rerio . J. Exp. Biol. , 2005 . 208 309 -316 . DOI:10.1242/jeb.01410http://doi.org/10.1242/jeb.01410 .

D. Traver , 等 . Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants . Nat. Immunol. , 2003 . 4 1238 -1246 . DOI:10.1038/ni1007http://doi.org/10.1038/ni1007 .

J. Karlsson , J. Von Hofsten , P. E. Olsson . Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development . Mar. Biotechnol. , 2001 . 3 522 -527 . DOI:10.1007/s1012601-0053-4http://doi.org/10.1007/s1012601-0053-4 .

S. H. Huang , C. H. Yu , Y. L. Chien . Light-addressable measurement of in vivo tissue oxygenation in an unanesthetized zebrafish embryo via phase-based phosphorescence lifetime detection . Sensors. , 2015 . 15 8146 -8162 . DOI:10.3390/s150408146http://doi.org/10.3390/s150408146 .

G. J. Bakker , 等 . Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy . eLife , 2022 . 11 e63776 DOI:10.7554/eLife.63776http://doi.org/10.7554/eLife.63776 .

D. Débarre , N. Olivier , E. Beaurepaire . Signal epidetection in third-harmonic generation microscopy of turbid media . Opt Express , 2007 . 15 8913 -8924 . DOI:10.1364/OE.15.008913http://doi.org/10.1364/OE.15.008913 .

M. R. Wang , 等 . Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain . Biomed. Opt. Express , 2018 . 9 3534 -3543 . DOI:10.1364/BOE.9.003534http://doi.org/10.1364/BOE.9.003534 .

T. Y. Wang , C. Xu . Three-photon neuronal imaging in deep mouse brain . Optica , 2020 . 7 947 -960 . DOI:10.1364/OPTICA.395825http://doi.org/10.1364/OPTICA.395825 .

Y. Hontani , 等 . Deep-tissue three-photon fluorescence microscopy in intact mouse and zebrafish brain . J. Vis. Exp. , 2022 . 179 e63213 .

D. G. Ouzounov , 等 . GCaMP6 ΔF/F dependence on the excitation wavelength in 3-photon and 2-photon microscopy of mouse brain activity . Biomed. Opt. Express , 2019 . 10 3343 -3352 . DOI:10.1364/BOE.10.003343http://doi.org/10.1364/BOE.10.003343 .

S. Clavreul , 等 . Cortical astrocytes develop in a plastic manner at both clonal and cellular levels . Nat. Commun. , 2019 . 10 4884 DOI:10.1038/s41467-019-12791-5http://doi.org/10.1038/s41467-019-12791-5 .

N. Dray , 等 . Large-scale live imaging of adult neural stem cells in their endogenous niche . Development , 2015 . 142 3592 -3600. .

G. L. Pedroso , 等 . Blood collection for biochemical analysis in adult zebrafish . J. Visualized Exp. , 2012 . 63 e3865 .

Westerfield, M. The Zebrafish Book. The Zebra Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). 5th edn. (University of Oregon, Eugene, 2007).

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