Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution

Abstract

2We describe a new light-sheet microscopy method for fast, large-scale and imaging methods were developed to overcome this problem [9][10][11][12][13][14][15] . Recent 50 advances along this line was able to push the imaging speed to ~3 days per 51 . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/240770 doi: bioRxiv preprint first posted online Dec. 29, 2017; 3 mouse brain at synaptic resolution 14 , or ~2.4 hours per mouse brain but at a 52 reduced resolution 15 . Still, it is highly desirable to be able to obtain high 53 resolution images at very high speed for high volume tasks such as whole 54 brain mapping of many animals or of large primates. 56The past decade has also seen various tissue clearing techniques that 57 greatly improve the transparency of brain samples 16 The basic configuration of our system consists of an illumination objective 91 through which a scanning light-beam enters a thick slice sample at a 45-92 degree angle to the sample surface, and an imaging objective positioned 93 perpendicular to the illumination plane (Fig. 1a,b (Supplementary Fig. 1). The geometry and working distance of 98 the objectives constrain the imageable sample thickness to ~300 µm (Fig. 1b). 99The light path is configured such that the effective imaging area on the object 100 plane (corresponding to half of the camera sensor, ~1000 X 2000 pixels) 101covers an oblique section of ~420 µm X 1000 µm using a 20X imaging 102 objective with system magnification reduced to ~13X (Fig. 1c). 104To achieve fast volumetric imaging over a large range, we configured the (Fig. 1c, d). This approach has also been used in the we eliminate virtually all motion blur and achieve high image quality 118 indistinguishable from stationary imaging, with dendritic spines of cortical 119 neurons clearly visible ( Fig. 1e-g). In principle, motion blur can also be 120 reduced using strobe light illumination in a regular light-sheet configuration. 121This however requires much higher light power to achieve similar illumination. 122The synchronized beam scan is thus also optimized in light efficiency. Supplementary Fig. 2a). 134We also developed a system to automatically detect the edges of all mounted 135 brain slices, so that the imaging system can directly move to the target based color VISoR imaging and cell counting in different brain areas (Fig. 3). In the 174 paraventricular nucleus (PVN) of hypothalamus where CRH neurons were 175 observed to form a dense cluster (Fig. 3a,b), more than half of these neurons (Fig. 3b,c, Supplementary Video 6). This is consistent with the role of CRH 180 neurons of the PVN in stress response 28 . Interestingly, in the medial 181 amygdala (MeA) where CRH neurons were more dispersed (Fig. 3d), the . 29, 2017; 9 stimulated animal, with 104 CRH neurons being activated out of 1018 total 184 CRH neurons and 569 total activated neurons (Fig. 3d,e, Supplementary 1...

High‐throughput light sheet tomography platform for automated fast imaging of whole mouse brain

“…It can image a whole mouse brain at a voxel size of 0.75 × 0.75 × 2.5 μm within 14 hours. Bi et al recently reported a technique called VISoR [21]. In this technique, the mouse brain is first sectioned into 300 μm thick slices.…”