Project Summary Optical recording of calcium transients from large number of neurons is an indispensable technique for comprehending neuronal ensembles during brain processes. While two-photon, confocal, and light sheet microscopes have effectively recorded from many neurons, these devices come at a high cost. This project aims to create an affordable device for imaging calcium transients, using pinhole illumination to create optical sectioning, which will reduce background light (F0) and consequently increasing the optical signal (dF/F0). The device will be simple enough to share as an open-source tool or as a modification of existing brightfield fluorescent microscopes. The project's significance lies in its capacity to identify a substantially greater number of neurons compared to widefield fluorescent imaging. Additionally, the open-source and low-cost approach is likely to attract a broader user base. Our proposed designs are supported by preliminary experiments and offer several advantages: 1. Elimination of mechanical scanning components, granting significant cost reduction compared to spinning disk confocal microscopes. 2. Using ordinary CMOS cameras, eliminating the need for expensive scientific-grade CMOS and EMCCD cameras. 3. High light flux, enabling the use of LED light sources, reducing the expense associated with high-power laser light sources. The proposed device will be constructed by modifying an existing brightfield fluorescent microscope, enabling us to test the designs with a modest grant, without requiring additional capital equipment. If successful, we anticipate the ability to simultaneously record from hundreds of neurons at a cost less than one-tenth of a two-photon microscope. In SA1, we introduce the patterned pinhole illuminator which can decrease background light and enhance optical signals of neuronal calcium transients in densely labeled brain tissue with GCaMP6f. Planar pinhole patterns on a glass disk and pinholes created by microlens arrays will be tested. In SA2, we propose a fiber optics-based pinhole illuminator, a coherent fiber optics bundle installed at the plane of the field diaphragm. Each fiber within the bundle will function as an illuminating pinhole when one end of the fiber is connected to a 470 nm LED chip. These LEDs can be electronically switched on and off to create a scanning pinhole pattern across the imaging field without the need for moving parts. This fiber optics-based pinhole illuminator would have a potential to simultaneous recording of up to four times more neurons compared to the designs in SA1. since it fully illuminates the entire imaging field.