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Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a user-friendly system that can address diverse imaging applications with varied requirements in terms of resolution (mesoscopic to sub-micron), sample geometry (size, shape, and number), and compatibility with tissue-clearing protocols of different refractive indices. We present a hybrid system that combines a novel non-orthogonal dual-objective and conventional open-top light-sheet architecture for highly versatile multi-scale volumetric imaging. One sentence summary Glaser et al. describe a hybrid open-top light-sheet microscope to image cleared tissues at mesoscopic to sub-micron resolution and depths of up to 1 cm.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a user-friendly system that can address diverse imaging applications with varied requirements in terms of resolution (mesoscopic to sub-micron), sample geometry (size, shape, and number), and compatibility with tissue-clearing protocols of different refractive indices. We present a hybrid system that combines a novel non-orthogonal dual-objective and conventional open-top light-sheet architecture for highly versatile multi-scale volumetric imaging. One sentence summary Glaser et al. describe a hybrid open-top light-sheet microscope to image cleared tissues at mesoscopic to sub-micron resolution and depths of up to 1 cm.

Bdellovibrio bacteriovorus is a Gram-negative predator of other Gram-negative bacteria. Interestingly, Bdellovibrio bacteriovorus 109J cells grown in coculture with Escherichia coli ML-35 prey develop into a spatially organized two-dimensional film when located on a nutrient-rich surface. From deposition of 10 μl of a routine cleared coculture of B. bacteriovorus and E. coli cells, the cells multiply into a macroscopic community and segregate into an inner, yellow circular region and an outer, off-white region. Fluorescence in situ hybridization and atomic force microscopy measurements confirm that the mature film is spatially organized into two morphologically distinct Bdellovibrio populations, with primarily small, vibroid cells in the center and a complex mixture of pleomorphic cells in the outer radii. The interior region cell population exhibits the hunting phenotype while the outer region cell subpopulation does not. Crowding and high nutrient availability with limited prey appear to favor diversification of the B. bacteriovorus population into two distinct, thriving subpopulations and may be beneficial to the persistence of B. bacteriovorus in biofilms.