The ability to image intact tissues, now made possible with advances in clearing protocols ( Richardson and Lichtman, 2015 Matryba et al., 2019 Ueda et al., 2020 McCreedy et al., 2021), and also the desire to image naturally dynamic 3D biological systems with live-cell imaging are the two main forces driving this unusual technological variety. Researchers have adapted the basic light-sheet body plan to different applications with different lens geometries ( Huisken and Stainier, 2007 Dunsby, 2009 Wu et al., 2011, 2013 Tomer et al., 2012 Kumar et al., 2014, 2018 Voleti et al., 2016, 2019 Sapoznik et al., 2020), beam shaping strategies ( Keller et al., 2008 Planchon et al., 2011 Chen et al., 2014 Vettenburg et al., 2014 Liu et al., 2018 Chang et al., 2019), sample mounting and scanning techniques ( Bouchard et al., 2015 Royer et al., 2016 Wu et al., 2017 Fadero et al., 2018 Glaser et al., 2019), and contrast mechanisms ( Truong et al., 2011 Di Battista et al., 2019). Science and technology has always been a moving target, but the pace of light-sheet instrumentation and software development has been staggering.

Since light-sheet microscopy was introduced to the life and biomedical science communities in 1993 ( Voie et al., 1993) and more broadly in 2004 ( Huisken et al., 2004), there has been a virtual Cambrian explosion of light-sheet instrumentation and image analysis tools. We provide our perspective on efficient processes for tool selection and review current hardware and software commonly used in light-sheet image analysis, as well as discuss what ideal tools for the future may look like. Our goal is to provide a contextual framework of basic computing concepts that cell and developmental biologists can refer to when mapping the peculiarities of different light-sheet data to specific existing computing environments and image analysis pipelines. 6High Performance Research Computing, Texas A&M University, College Station, TX, United Statesįrom the combined perspective of biologists, microscope instrumentation developers, imaging core facility scientists, and high performance computing experts, we discuss the challenges faced when selecting imaging and analysis tools in the field of light-sheet microscopy.5Department of Biology and Biochemistry, University of Houston, Houston, TX, United States.4Department of Neurology, Baylor College of Medicine, Houston, TX, United States.

3Department of Biology, Texas A&M University, College Station, TX, United States.

2Microscopy and Imaging Center, Texas A&M University, College Station, TX, United States.

1Department of Biomedical Engineering, Texas A&M University, College Station, TX, United States.

Pritchard 1, Anindito Sen 2, Stan Vitha 2, Sreeja Sarasamma 4, Avery L.

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