Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood

Ilyas Saytashev; Rachel Glenn; Gabrielle A Murashova; Sam Osseiran; Dana Spence; Conor L Evans; Marcos Dantus

doi:10.1364/BOE.7.003449

Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood

Biomed Opt Express. 2016 Aug 12;7(9):3449-3460. doi: 10.1364/BOE.7.003449. eCollection 2016 Sep 1.

Authors

Ilyas Saytashev¹, Rachel Glenn¹, Gabrielle A Murashova¹, Sam Osseiran², Dana Spence¹, Conor L Evans³, Marcos Dantus⁴

Affiliations

¹ Department of Chemistry, Michigan State University, 578 S Shaw Ln., East Lansing, MI 48824, USA.
² Harvard-MIT Division of Health Sciences and Technology, 77 Massachusetts Avenue E25-519, Cambridge, MA 02139, USA; Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA.
³ Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA.
⁴ Department of Chemistry, Michigan State University, 578 S Shaw Ln., East Lansing, MI 48824, USA; Department of Physics and Astronomy, Michigan State University, 567 Wilson Rd., East Lansing, MI 48824, USA.

Abstract

Red blood cells (RBC) in two-photon excited fluorescence (TPEF) microscopy usually appear as dark disks because of their low fluorescent signal. Here we use 15fs 800nm pulses for TPEF, 45fs 1060nm pulses for three-photon excited fluorescence, and third harmonic generation (THG) imaging. We find sufficient fluorescent signal that we attribute to hemoglobin fluorescence after comparing time and wavelength resolved spectra of other expected RBC endogenous fluorophores: NADH, FAD, biliverdin, and bilirubin. We find that both TPEF and THG microscopy can be used to examine erythrocyte morphology non-invasively without breaching a blood storage bag.

Keywords: (180.0180) Microscopy; (180.4315) Nonlinear microscopy; (180.5810) Scanning microscopy.

Grants and funding

R21 EB016379/EB/NIBIB NIH HHS/United States