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.
Focused ultrasound (FUS) is an emerging technology for non-invasive and controlled blood-brain barrier (BBB) opening for drug delivery successfully tested in clinical trials. To improve the safety of this method, we use optical imaging techniques to better understand the relationship between repeated FUS-BBB opening, neuroinflammation, and alteration of neurovascular coupling in an animal model. We perform 1- and 2-photon microscopy in awake mice to image neuronal activity hemodynamics. Before and following FUS treatment, optical imaging sessions access changes in neuronal activity and/or hemodynamics; FUS treatments are repeated several times to approximate a clinical schedule.
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