title: Label-free determination of hemodynamic parameters in the microcirculaton with third harmonic generation microscopy.
abstract: Determination of blood flow velocity and related hemodynamic parameters is an important aspect of physiological studies which in many settings requires fluorescent labeling. Here we show that Third Harmonic Generation (THG) microscopy is a suitable tool for label-free intravital investigations of the microcirculation in widely-used physiological model systems. THG microscopy is a non-fluorescent multi-photon scanning technique combining the advantages of label-free imaging with restriction of signal generation to a focal spot. Blood flow was visualized and its velocity was measured in adult mouse cremaster muscle vessels, non-invasively in mouse ear vessels and in Xenopus tadpoles. In arterioles, THG line scanning allowed determination of the flow pulse velocity curve and hence the heart rate. By relocating the scan line we obtained velocity profiles through vessel diameters, allowing shear rate calculations. The cell free layer containing the glycocalyx was also visualized. Comparison of the current microscopic resolution with theoretical, diffraction limited resolution let us conclude that an about sixty-fold THG signal intensity increase may be possible with future improved optics, optimized for 1200-1300 nm excitation. THG microscopy is compatible with simultaneous two-photon excited fluorescence detection. It thus also provides the opportunity to determine important hemodynamic parameters in parallel to common fluorescent observations without additional label.
authors.affiliation: Walter-Brendel-Zentrum für Experimentelle Medizin, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Dietzel
authors.affiliation: Walter-Brendel-Zentrum für Experimentelle Medizin, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Pircher
authors.affiliation: Walter-Brendel-Zentrum für Experimentelle Medizin, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: A Katharina
authors.initials: AK
authors.lastname: Nekolla
authors.affiliation: Walter-Brendel-Zentrum für Experimentelle Medizin, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: Mazhar
authors.initials: M
authors.lastname: Gull
authors.affiliation: Walter-Brendel-Zentrum für Experimentelle Medizin, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: André W
authors.initials: AW
authors.lastname: Brändli
authors.affiliation: Walter-Brendel-Zentrum für Experimentelle Medizin, Ludwig-Maximilians-Universität München, München, Germany; SyNergy, Munich Cluster for Systems Neurology, München, Germany; Deutsches Zentrum für Herz-Kreislaufforschung e.V., München, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Pohl
authors.affiliation: Walter-Brendel-Zentrum für Experimentelle Medizin, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Rehberg
mesh_terms: D000818:Animals; D001783:Blood Flow Velocity; D003911:Dextrans; D004431:Ear, External / Q000098:blood supply; D004912:Erythrocytes / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D019276:Glycocalyx / Q000648:ultrastructure; D006339:Heart Rate; D006454:Hemoglobins / Q000737:chemistry; D007814:Larva; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008833:Microcirculation; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D008863:Microspheres; D018482:Muscle, Skeletal / Q000098:blood supply; D014982:Xenopus laevis / Q000254:growth & development
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D006454:Hemoglobins; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate