Measuring capillary flow dynamics using interlaced two-photon volumetric scanning

Two photon microscopy and optical coherence tomography (OCT) are two standard methods for measuring flow speeds of red blood cells in microvessels, particularly in animal models. However, traditional two photon microscopy lacks the depth of field to adequately capture the full volumetric complexity of the cerebral microvasculature and OCT lacks the specificity offered by fluorescent labeling. In addition, the traditional raster scanning technique utilized in both modalities requires a balance of image frame rate and field of view, which severely limits the study of RBC velocities in the microvascular network. Here, we overcome this by using a custom two photon system with an axicon based Bessel beam to obtain volumetric images of the microvascular network with fluorescent specificity. We combine this with a novel scan pattern that generates pairs of frames with short time delay sufficient for tracking red blood cell flow in capillaries. We track RBC flow speeds in 10 or more capillaries simultaneously at 1 Hz in a 237 µm × 237 µm × 120 µm volume and quantified both their spatial and temporal variability in speed. We also demonstrate the ability to track flow speed changes around stalls in capillary flow and measure to 300 µm in depth.