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Confocal fluorescence microscopy is often used in brain imaging experiments, however conventional confocal microscopes are limited in their field of view, working distance, and speed for high resolution imaging. We report here the development of a novel high resolution, high speed, long working distance, and large field of view confocal fluorescence microscope (H 2 L 2 -CFM) with the capability of multi-region and multifocal imaging. To demonstrate the concept, a 0.5 numerical aperture (NA) confocal fluorescence microscope is prototyped with a 3 mm × 3 mm field of view and 12 mm working distance, an array of 9 beams is scanned over the field of view in 9 different regions to speed up the acquisition time by a factor of 9. We test this custom designed confocal fluorescence microscope for future use with brain clarification methods to image large volumes of the brain at subcellular resolution. This multi-region and multi-spot imaging method can be used in other imaging modalities, such as multiphoton microscopes, and the field of view can be extended well beyond 12 mm × 12 mm.

High resolution, high speed, long working distance, large field of view confocal fluorescence microscope