Multi-Scale Light-Sheet Fluorescence Microscopy for Fast Whole Brain Imaging

Abstract

Whole-brain imaging has become an increasingly important approach to investigate neural structures, such as somata distribution, dendritic morphology, and axonal projection patterns. Different structures require whole-brain imaging at different resolutions. Thus, it is highly desirable to perform whole-brain imaging at multiple scales. Imaging a complete mammalian brain at synaptic resolution is especially challenging, as it requires continuous imaging from days to weeks because of the large number of voxels to sample, and it is difficult to acquire a constant quality of imaging because of light scattering during in toto imaging. Here, we reveal that light-sheet microscopy has a unique advantage over wide-field microscopy in multi-scale imaging because of its decoupling of illumination and detection. Based on this observation, we have developed a multi-scale light-sheet microscope that combines tiling of light-sheet, automatic zooming, periodic sectioning, and tissue expansion to achieve a constant quality of brain-wide imaging from cellular (3 μm × 3 μm × 8 μm) to sub-micron (0.3 μm × 0.3 μm × 1 μm) spatial resolution rapidly (all within a few hours). We demonstrated the strength of the system by testing it using mouse brains prepared using different clearing approaches. We were able to track electrode tracks as well as axonal projections at sub-micron resolution to trace the full morphology of single medial prefrontal cortex (mPFC) neurons that have remarkable diversity in long-range projections.

Sparse imaging and reconstruction tomography for high-speed high-resolution whole-brain imaging

“…By taking advantage of sparse labeling, which is necessary for the reconstruction of fine axon collaterals using light microscopy, we have used the SMART system to image neurons in a variety of cortical regions within a few hours to a day, a much shorter time than previous methods ( Table S1 ) ( Economo et al., 2016 ; Gong et al., 2016 ; Li et al., 2010 ). This further demonstrates that real-time data analysis combined with instrument control can dramatically augment the performance of specific imaging systems ( Long et al., 2017 ; Peng et al., 2014b ; Zhang et al., 2021 ). There have been a number of alternative methods for whole-brain imaging, including knife-edge scanning microscopy, block-face two-photon or confocal microscopy, and light-sheet microscopy ( Dodt et al., 2007 ; Economo et al., 2016 ; Gong et al., 2016 ; Li et al., 2010 ; Migliori et al., 2018 ; Narasimhan et al., 2017 ; Ragan et al., 2012 ; Seiriki et al., 2017 ; Wang et al., 2019a ).…”

Smart imaging to empower brain-wide neuroscience at single-cell levels

“…Based on the light sheet microscopy, the entire-brain imaging at cellular resolution is achieved within a few hours for mouse brains [ 86 , 87 ] and 100 h for monkey brains [ 17 ]. In combination with online image analysis, the sparsity property of neuron structures is highly implemented to speed up the imaging procedure [ 70 , 71 , 88 ]. As a new trend, the miniaturized imaging setups are being developed to bring new chances for in vivo brain science [ 89 ].…”

“…With modules such as optical sectioning, image acquisition, data reconstruction and analysis, etc. all combined together, the past years have witnessed several integrated systems being developed for brain research [ 16 , 17 , 87 , 88 ]. This has led to many exciting discoveries and particularly leveraged the studies on NHP and human brains [ 1 , 2 , 17 ].…”

Application of light-sheet mesoscopy to image host-pathogen interactions in intact organs

“…One of the many applications of LSFM is to explore the tissue architecture of complex organs, such as the CNS, in health and disease. For example, similar approaches have been employed to resolve structural features of neuronal synapsis to reconstruct activity patterns and connectivity maps (Niedworok et al, 2012; Bèchet et al, 2020; Blutke et al, 2020; Perens et al, 2021; Zhang et al, 2021). However, to our knowledge, LSFM has not been implemented to study infectious diseases affecting the CNS at the mesoscale.…”