Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination

Abstract

Significance Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale (“mesoscopic”) imaging of neuronal activity with 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. Aim Develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. Approach Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm LEDs 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. Results We demonstrate 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. Conclusions Our widefield microscope design with single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter. ### Competing Interest Statement The authors have declared no competing interest.