Abstract
Occlusions in single cortical microvessels lead to a reduction in oxygen supply, but this decrement has not been able to be quantified in three dimensions at the level of individual vessels using a single instrument. We demonstrate a combined optical system using two-photon phosphorescence lifetime and fluorescence microscopy (2PLM) to characterize the partial pressure of oxygen (pO2) in single descending cortical arterioles in the mouse brain before and after generating a targeted photothrombotic occlusion. Integrated real-time Laser Speckle Contrast Imaging (LSCI) provides wide-field perfusion maps that are used to monitor and guide the occlusion process while 2PLM maps changes in intravascular oxygen tension. We present the technique’s utility in highlighting the effects of vascular networking on the residual intravascular oxygen tensions measured after occlusion in three dimensions.
Highlights
The distribution of dissolved oxygen in the brain has proven difficult to examine at the microvascular level under baseline conditions let alone after selective flow alterations due to methodological shortcomings of classical oximetry techniques
Through the combination of two-photon fluorescence and phosphorescence lifetime microscopy with laser speckle contrast imaging and photothrombosis, we have presented an all-optical, non-contact system for interrogating oxygen tension and blood flow within the microcirculation
We highlight the utility of using 2PLM for examining a large range of absolute oxygen tensions with three dimensional sectioning
Summary
The distribution of dissolved oxygen in the brain has proven difficult to examine at the microvascular level under baseline conditions let alone after selective flow alterations due to methodological shortcomings of classical oximetry techniques. Techniques combining laser scanning microscopy and phosphorescence quenching for oxygen measurements have begun mapping microvascular oxygen tension These techniques have proven challenging for three dimensional imaging with conventional oxygen sensitive probes [6,7] due to very low two-photon absorption crosssections and skewed calibrations at high probe load. A new platinum-porphyrin based phosphorescent oxygen sensor, PtP-C343 [8] has been developed with a much larger two-photon cross-section, tuned oxygen sensitivity and greater molecular dispersion in biological environments. These properties combined with its phosphorescence efficiency make PtP-C343 an effectively brighter and stable molecular probe for two-photon microscopy, resulting in greater confidence in measurements and improved three-dimensional sectioning. We demonstrate a two-photon lifetime microscopy technique that utilizes this new oxygen sensor for examining the vascular networking impact on intravascular oxygen tension
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