Label-free determination of hemodynamic parameters in the microcirculaton with third harmonic generation microscopy.

Steffen Dietzel,A Katharina Nekolla,Joachim Pircher,Markus Rehberg,Ulrich Pohl,Mazhar Gull,André W Brändli,Marc Van Zandvoort

doi:10.1371/journal.pone.0099615

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.

Highlights

Blood flow velocity is an important parameter in investigations of the hemodynamics of the microcirculation [1,2] and various microscopic methods have been developed to measure it [2,3]
With the current work we investigated if Third Harmonic Generation (THG) microscopy can be a feasible and reliable tool for a label-free intravital characterization of the microcirculation in established animal models
We found that such a characterization is possible, e.g. by blood flow velocity measurements using THG imaging or THG line scanning and we describe several possibilities to evaluate such measurements

Summary

Introduction

Blood flow velocity is an important parameter in investigations of the hemodynamics of the microcirculation [1,2] and various microscopic methods have been developed to measure it [2,3]. The speed of laser scanning microscopes was usually too slow to record whole images at a frequency that would allow tracking of individual flowing cells. Velocity values for labeled particles are calculated from two images recorded with a short delay by cross-correlation. This procedure requires modeling that includes the application of several correction factors, e.g. for different magnifications and vessel diameters [14]. An approach that applied cross-correlation to scan lines from two-photon excited fluorescent dextran recently allowed blood velocity measurements of up to 84 mm/s in pathological arteriovenous shunts in the mouse brain [15]

Methods

Results

Conclusion