Abstract
Tissue clearing enables us to observe thick tissue at a single cell resolution by reducing light scattering and refractive index matching. However, imaging of a large volume of tissue for 3D reconstruction requires a great deal of time, cost, and efforts. Few methods have been developed to transcend these limitations by mechanical compression or isotropic tissue shrinkage. Tissue shrinkage significantly lessens the imaging burden; however, there is an inevitable trade-off with image resolution. Here, we have developed the “BrainFilm” technique to compress cleared tissue at Z-axis by dehydration, without alteration of the XY-axis. The Z-axis compression was approximately 90%, and resulted in substantial reduction in image acquisition time and data size. The BrainFilm technique was successfully used to trace and characterize the morphology of thick biocytin-labelled neurons following electrophysiological recording and trace the GFP-labelled long nerve projections in irregular tissues such as the limb of mouse embryo. Thus, BrainFilm is a versatile tool that can be applied in diverse studies of 3D tissues in which spatial information of the Z-axis is dispensable.
Highlights
Tissue clearing enables us to observe thick tissue at a single cell resolution by reducing light scattering and refractive index matching
We introduce a novel method to reduce the volume of image acquisitions applicable to polymer-based tissue clearing techniques
Cleared brain slices were sandwiched within a compressing tool that we have named “BrainFilm kit.”
Summary
Tissue clearing enables us to observe thick tissue at a single cell resolution by reducing light scattering and refractive index matching. Tissue shrinkage may cause distortion of the sample structure and reduce spatial resolution/ accuracy of the images, the application of this technique is limited to non-quantitative morphometric analyses[11,12,13]. Times and high computing power, and isotropic tissue shrinkage can reduce these efforts to obtain large volume information. This technique has a trade off with isotropic image resolution. We introduce a novel method to reduce the volume of image acquisitions applicable to polymer-based tissue clearing techniques. Our BrainFilm technique can be applied to trace single neuronal profiles or axonal projections in a large volume of tissue, and can be applied in various studies such as transgenic animal phenotyping
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