SignificanceWidefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale (“mesoscopic”) imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts.AimWe aim to develop a system for mesoscopic imaging of two fluorescent and two reflectance channels.ApproachExcitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm light-emitting diodes positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector.ResultsWe demonstrate the performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake, head-fixed mice with a curved “crystal skull” window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRABACh3.0. Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes.ConclusionsOur widefield microscope design with a single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.
SignificanceBrief 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.AimOur 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.ResultsWe 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.ConclusionsBessel 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.
We present the use of intrinsic optical signal imaging to monitor the progression of ischemic stroke in the acute and chronic phases of recovery. An optimized mouse photothrombosis stroke model is used to induce stroke in the sensory forelimb area and longitudinal recovery is assessed using resting state functional connectivity as well as forelimb stimulation. These functional measures are then correlated with behavioral outcome assessed with a forelimb asymmetry test. Obtaining measures in both the acute and chronic phases of recovery will enable better interpretation of human functional neuroimaging stroke studies and allow their use in understanding acute stroke.
We combined a home-built two photon microscope with a Bessel focus capable of volumetric imaging with a custom scan pattern that allowed us to generate image pairs with a time delay of ~12 ms. This approach allows us to track red blood cell (RBC) flow along individual capillaries and therefore calculate RBC speed. We demonstrate the ability to measure flow speeds in capillaries across a 475x475x150 µm volume which will enable us to monitor network wide changes in capillary flow.
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