Continuous imaging of large-volume tissues with a machinable optical clearing method at subcellular resolution

Abstract

Optical clearing methods are widely used for three-dimensional biological information acquisition in the whole organ. However, the imaging quality of cleared tissues is often limited by ununiformed tissue clearing. By combining tissue clearing with mechanical sectioning based whole organ imaging system, we can reduce the influence of light scattering and absorption on the tissue to get isotropic and high resolution in both superficial and deep layers. However, it remains challenging for optical cleared biological tissue to maintain good sectioning property. Here, we developed a clearing method named M-CUBIC (machinable CUBIC), which combined a modified CUBIC method with PNAGA (poly-N-acryloyl glycinamide) hydrogel embedding to transparentize tissue while improving its sectioning property. With high-throughput light-sheet tomography platform (HLTP) and fluorescent micro-optical sectioning tomography (fMOST), we acquired continuous datasets with subcellular resolution from intact mouse brains for single neuron tracing, as well as the fine vascular structure of kidneys. This method can be used to acquire microstructures of multiple types of biological organs with subcellular resolutions, which can facilitate biological research.

Three-dimensional mapping in multi-samples with large-scale imaging and multiplexed post staining

“…The array-fMOST has the potential to be combined with the embedding scheme of tissue clearing and PNAGA to deepen the imaging monolayer scanning depth and slice thickness 33 . On the premise of stably acquiring continuous 3D data, this will improve the acquiring efficiency by reducing the time spent on imaging and cutting.…”

Precision vibratome for high-speed ultrathin biotissue cutting and organ-wide imaging

“…Recently, another high elastic material, i.e., the hydrogel, has also been widely used in the tissue imaging field, such as expansion microscopy ( Gotz et al., 2020 ) and optical clearing ( Chung and Deisseroth, 2013 ; Yang et al., 2014 ). The superior biocompatibility and permeability make it ideal for large-volume tissue embedding ( Zhou et al., 2020 ). Through online optical clearing methods that combine hydrogel embedding with high-frequency vibration cutting, potentially it can be used to obtain complete 3D data of a marmoset or even human brain at a consistent submicron resolution ( Wu et al., 2019 ).…”

Advanced high resolution three-dimensional imaging to visualize the cerebral neurovascular network in stroke

“…The combination of rabies virus markers with fluorescent MOST (fMOST) can show the input of single neurons in the whole brain, providing a reliable option for optogenetics. The imaging speed was also improved, and the entire mouse brain could be imaged in an hour at a voxel size of 1.30×1.30×0.92 μm 3 with a section thickness of 40 μm, without optical clearance [117]. The drawback is that the data set is too large and thus makes data processing more difficult, but this is the inevitable result of this kind of idea.…”