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 625nm 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.