High throughput detection of capillary stalling events with Bessel beam two-photon microscopy.

Significance: Brief disruptions in capillary flow, commonly referred to as capillary "stalling," have gained interest recently for their potential role in disrupting cerebral blood flow and oxygen delivery. Approaches to studying this phenomenon have been hindered by limited volumetric imaging rates and cumbersome manual analysis. The ability to precisely and efficiently quantify the dynamics of these events will be key in understanding their potential role in stroke and neurodegenerative diseases, such as Alzheimer's disease. Aim: Our study aimed to demonstrate that the fast volumetric imaging rates offered by Bessel beam two-photon microscopy combined with improved data analysis throughput allows for faster and more precise measurement of capillary stall dynamics. Results: We found that while our analysis approach was unable to achieve full automation, we were able to cut analysis time in half while also finding stalling events that were missed in traditional blind manual analysis. The resulting data showed that our Bessel beam system was captured more stalling events compared to optical coherence tomography, particularly shorter stalling events. We then compare differences in stall dynamics between a young and old group of mice as well as a demonstrate changes in stalling before and after photothrombotic model of stroke. Finally, we also demonstrate the ability to monitor arteriole dynamics alongside stall dynamics. Conclusions: Bessel beam two-photon microscopy combined with high throughput analysis is a powerful tool for studying capillary stalling due to its ability to monitor hundreds of capillaries simultaneously at high frame rates.

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.