This supplemental grant will remove the significant bottleneck in the 3-D imaging of cleared brains. We request a Keyence BZ-X810 automated fluorescent microscope ($182,860). The considerable advantage of this microscope is that it uses a structured UV light, which performs the optical sectioning as a confocal microscope does, while at a significantly faster speed by using a CCD sensor instead of point scanning, resulting in 20x faster scanning in BZ-X810 comparing with Leica SP8 confocal microscope, which we are currently using. The current confocal setting (Leica SP8) would spend 40 hours per brain, but the proposed system can scan it in 2 hours, and much less photobleaches fluorescent signals of the samples. The scanning speed is a significant bottleneck for the proposed Specific Aim 2-2, “addressing if the dopaminergic and/or GABAergic nervous systems are dysregulated in cavefish comparing with surface fish, and whether these neural disregulations are adjusted under the ketogenic diet treatment”. We plan to scan 116 brains in the Aim 2-2 as a minimum; thus, 232 hrs (10 full-days in BZ-X810) vs. 4,640 hrs (over 6 months in Leica SP8) of scannings. We are able to reduce the duration of 3-D scanning with the current SP8 system by restricting the imaging field. However, since we have discovered the brain-wide hyperactivities in the asocial cavefish population, restricting the imaging fields will significantly limit our ability to identify the activity signatures of neurons associated with stereotypic repetitive behavior. Above all, the requested equipment will remove the current bottleneck in the 3-D imaging of cleared brains. In addition, since this microscope is easy to use, we also offer spot uses of this BZ-X810 to microscope users. After our quick survey, we have heard from a few potential users about quick 3-D scans of their samples. Our department chair is highly favorable to support this supplemental grant, too.