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.

Microvasculature on a chip: study of the Endothelial Surface Layer and the flow structure of Red Blood Cells

“…We have thus been able to highlight the existence of a near-wall layer, extending ~4.5  μ m away from the apical membrane of the endothelial cells, that is depleted in RBC. Such a layer is reminiscent of the Cell-Free Layer (CFL) that has been observed both in vivo 63646566 and in vitro 556567, and has been documented to play an important role in e.g. oxygen and nitric oxide transport in arterioles65.…”

Third harmonic generation microscopy of cells and tissue organization

“…An additional signal, with variable efficiency, originates from basement membranes lining the vessels (Rehberg et al, 2011;Weigelin et al, 2012). The resulting time-lapse THG recordings of erythrocyte flow kinetics reflect hemodynamic parameters, including blood flow velocity, the shear stress between erythrocytes and the vessel wall, and flowdeduced rates and rhythmicity of the heart beat (Dietzel et al, 2014) (Fig. 3G,H).…”

“…Further, signal intensity strongly depends on excitation pulse length and laser repetition rate (with both inverse second-order dependence), excitation wavelength (third-order dependence) and the numerical aperture of the objective (second-order dependence) . In deep-tissue imaging applications, THG signals are thus strongly affected by light scattering and wavelength dispersion that is caused by out-of-focus structures and by increasing distortions of the focus in depth (Dietzel et al, 2014). The detection of THG in 3D tissues can be improved by using adaptive optics in order to achieve a sharp focus in light-scattering samples, which maximizes the signal intensity of SHG and THG (Thayil et al, 2011).…”

Unraveling the host's immune response to infection: Seeing is believing

“…Notably, the advancement of label‐free imaging technologies offers exciting new possibilities for the field of IVM. Second, and now third, harmonic generation, for example, allows the visualization of structures like collagen and blood flow without dyes …”