Abstract
We show that third harmonic generation (THG) microscopy using a 1-MHz train of 1,300-nm femtosecond duration laser pulses enabled visualization of the structure and quantification of flow speed in the cortical microvascular network of mice to a depth of > 1 mm. Simultaneous three-photon imaging of an intravascular fluorescent tracer enabled us to quantify the cell free layer thickness. Using the label-free imaging capability of THG, we measured flow speed in different types of vessels with and without the presence of an intravascular tracer conjugated to a high molecular weight dextran (2 MDa FITC-dextran, 5% w/v in saline, 100 µl). We found a ∼20% decrease in flow speeds in arterioles and venules due to the dextran-conjugated FITC, which we confirmed with Doppler optical coherence tomography. Capillary flow speeds did not change, although we saw a ∼7% decrease in red blood cell flux with dextran-conjugated FITC injection.
Highlights
Nonlinear microscopy is widely used in biomedical research to study the function and dynamic behavior of cells in both healthy and disease states [1,2,3]
In the first imaging session, the blood plasma was labeled by an intravenous injection of high molecular weight dextran with conjugated FITC (5% w/v 2 MDa FITC-Dextran in sterile saline, 100 μl)
The absorption length of the Third harmonic generation (THG) light is about 30 (90) μm for deoxygenated blood, so the THG generated from red blood cells (RBCs) near the top of the vessel can be significantly attenuated during propagation through the blood in vessels with diameters of even a few tens of micrometers
Summary
Nonlinear microscopy is widely used in biomedical research to study the function and dynamic behavior of cells in both healthy and disease states [1,2,3]. THG microscopy provides a 3D imaging modality that highlights optical interfaces. This THG signal is generated in the forward direction, so when imaging in scattering samples the epi-detected THG signal is the result of light that scatters back out of the sample. THG has been shown to provide high contrast imaging of a number of tissue structures, including myelinated axons in the brain [9] and spinal cord [10], lipid deposits in atherosclerotic plaques in aorta [11], muscle fiber sarcomeres [12], red blood cells (RBCs) in vessels of the ear in mice [13] and white blood cells in human skin [14]. When imaging the mouse cortex in vivo, the two dominant sources of THG contrast appear to be from myelinated axons [10] and RBCs in the vessels
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