ABSTRACT Lightsheet microscopy offers distinct capability advantages for the imaging of live intact biological samples and optically cleared fixed samples. Rapid 3D optical sectioning with little photodamage enables lightsheet systems to excel at the imaging of large organs or whole embryos. The Zeiss Lightsheet Z.1 upgrades we propose in this application for higher refractive index clearing solutions, larger sample chambers and longer working distance, larger field of view objectives will transform researchers’ capabilities to collect images of larger samples such as cleared whole mouse brain or cleared whole mouse embryos. The availability of the upgrade hardware and software for our Zeiss lightsheet system would enhance many projects investigating fundamental developmental biology and human disease models. The current instrument is housed in a Duke School of Medicine building within the Light Microscopy Core Facility space. The upgrade parts would be installed onto to the Zeiss system which has been in operation for 3.5 years. In addition to the major and minor users’ research described here, the system and the upgrades would serve the entire research base at Duke University and Medical School where the impact and use would be widespread. The user group has experience with lightsheet imaging and sample preparation for the Zeiss system at Duke, along with other forms located at other institutions. The general high-levels of imaging expertise and specific experience with the Zeiss lightsheet are well placed to immediately and effectively implement this upgrade equipment to expand the Zeiss lightsheet system for an important array of basic research. These upgrades proposed will significantly contribute to new research capabilities related to NIH funded developmental biology, neuroscience, and cell biology questions relevant to human disorders as outlined in the research statement.