In the work by Kazmi, et al a two-photon lifetime microscopy technique that utilizes PtP-C343 is combined with laser speckle contrast imaging (LSCI) to examine the vascular networking impact on intravascular oxygen tension. Two-photon lifetime microscopy enables deeper tissue penetration imaging due to the near-infrared wavelength of excitation light facilitating oxygen detection through PtP-C343 at increased depths over single photon lifetime microscopy. LSCI enables imaging of blood flow characterization, but is typically surface weighted. Thus combining these methods enables quantification of tissue oxygen tension at depth in live animals. Following development and optimization of the combined imaging system it was utilized to image oxygenation of microvasculature through window chambers in murine brains. Experiments were performed to quantify baseline pO2 in descending arterioles, where substantial pO2 gradients were observed with arteriole descent into the cortex. Photothrombotic clotting was used to occlude the entire lumen of an observed vessel; imaging demonstrated that intravascular pO2 immediately upstream of the clot remained consistent with baseline measurements. However, downstream there was a significant drop in pO2 compared to baseline measurements. Additional experiments were performed to examine observation of gradient reversal in the primary arteriole, which were readily quantifiable using the imaging system. This paper demonstrates the construction and validation of a combined two-photon fluorescence and phosphorescence lifetime microscope with laser speckle contrast imaging to image photothrombosis and its effect on tissue pO2. Cerebral microvasculature is used to demonstrate this technique, but similar interrogations could be performed on other highly perfused tissues with a feasible method for optical accessibility.
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