Generation of Three-dimensional Printed Biological Innovative Mold for Optically Transparent Tissue Specimens

Summary

Tissue innovative Molds (iMolds) have been developed to reduce specimen movement, structurally support the specimen being imaged, and allow for repeated imaging on precise anatomical locations using optically-transparent samples.

Abstract

Histology continues to evolve in modern day experimentation. Recently, groups have been able to make tissue and bones transparent by removing lipids and other biological species that prevent deep tissue imaging. Now, groups can robustly "clear" the tissue or specimen and perform a wide array of microscopy such as single-photon, multiphoton, and light-sheet to evaluate their biological target of interest. This has eliminated the need to freeze, cyro-slice, and cyro-preserve tissue, greatly limiting human prone error. However, current tissue clearing methods still lack a robust system to mount and structurally support unique specimens. This leads to issues with tissue movement during imaging, the lack of reliability when re-staining and re-evaluating specimens, and working with abnormally shaped specimens such as tumors. To overcome these obstacles, the innovative Mold (iMold) was generated. iMolds are first created from images of the specimen followed by three-dimensional (3D) printed molds that support the unique tissue structure being imaged and re-imaged. This leads to a dramatic reduction in tissue movement while imaging and allows for scientists to robustly re-evaluate areas of interest that have been re-stained or need to be re-imaged. Furthermore, iMolds can be created in a short time period and generated for multiple samples in any imaging format (i.e. slide, cell culture plate). This allows users to scan multiple organs or specimens on one slide or cell culture dish. In closing, iMolds can be utilized by any research laboratory studying cleared specimens while also reducing costs, time, and tissue movement compared to other current methodologies.

Introduction

Whole tissue clearing methods have recently become a widely-used method in histology. First the tissue and organ are cleared resulting in the specimen becoming optically-transparent, then this is followed by staining and imaging. With these techniques scientists are able to image at high resolution entire organs and thick specimens. In addition, many tissue clearing methodologies allow for repeated staining. This provides users the ability to develop 3D renderings of structural and functional relationships across a large volume of area.

The 3D renderings that are produced have application in many fields of science. For example, scientists c....

Protocol

1. Clear the Tissue or Bone Specimens Following Established Clearing Methods

1. Follow this protocol or another established tissue-clearing method: for passive CLARITY begin by making hydrogel monomer (HM) solution included of 1% (wt/vol) acrylamide, 0.0125 - 0.05% (wt/vol) bisacrylamide, 4% paraformaldehyde, 1x PBS, deionized water, and 0.25% of the thermal initiator into a 50-mL tube³.
2. Perform a trans-cardial perfusion with HM and incubate the tissue overnight or 2 days at 4 &.......

Discussion

Optical clearing methods are continuing to increase the understanding of environmental niches and cellular heterogeneity. The tissue is maintained and stored in a hydrogel complex that allows biomacromolecules to permeate the matrix. This allows for groups to study immunofluorescence of proteins and nucleic acids either by endogenous fluorescence or post-hoc labeling, across large volumes of tissue.

However, a major caveat in the system comes when attempting to image the tissue. In current met.......

Materials

Name	Company	Catalog Number	Comments
Acrylamide	Sigma	A9099
bisacrylamide	VWR	0172-50G
paraformaldehyde	Electron Microscopy Sciences	15714-S
PBS	Quality Biological	119-069-131
thermal initiator	Wako Chemicals	VA-044
sodium dodecyl sulfate	Bio-Rad	161-0302
boric acid	Sigma	B-7660
Triton X-100	Sigma	X100
focus clear	CEDARLANE	F101-KIT
Google Sketchup
Camera			we used an iPhone 7 camera
3D Printer	M3D	Micro +
PLA plastic roll for 3D Printer	M3D	3D Ink	no catalog number listed, we use black PLA filament