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.
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