3D Printer Generated Tissue iMolds for Cleared Tissue Using Single- and Multi-Photon Microscopy for Deep Tissue Evaluation

Abstract

BackgroundPathological analyses and methodology has recently undergone a dramatic revolution. With the creation of tissue clearing methods such as CLARITY and CUBIC, groups can now achieve complete transparency in tissue samples in nano-porous hydrogels. Cleared tissue is then imagined in a semi-aqueous medium that matches the refractive index of the objective being used. However, one major challenge is the ability to control tissue movement during imaging and to relocate precise locations post sequential clearing and re-staining.MethodsUsing 3D printers, we designed tissue molds that fit precisely around the specimen being imaged. First, images are taken of the specimen, followed by importing and design of a structural mold, then printed with affordable plastics by a 3D printer.ResultsWith our novel design, we have innovated tissue molds called innovative molds (iMolds) that can be generated in any laboratory and are customized for any organ, tissue, or bone matter being imaged. Furthermore, the inexpensive and reusable tissue molds are made compatible for any microscope such as single and multi-photon confocal with varying stage dimensions. Excitingly, iMolds can also be generated to hold multiple organs in one mold, making reconstruction and imaging much easier.ConclusionsTaken together, with iMolds it is now possible to image cleared tissue in clearing medium while limiting movement and being able to relocate precise anatomical and cellular locations on sequential imaging events in any basic laboratory. This system provides great potential for screening widespread effects of therapeutics and disease across entire organ systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-017-0057-2) contains supplementary material, which is available to authorized users.

Hydrogel-Tissue Chemistry: Principles and Applications

“…Data collection on this timescale is associated with problems ranging from photobleaching to simple microscope overoccupancy, but rapid development of advanced light-sheet imaging, which offers orders-of-magnitude improvement in speed (29, 41, 44, 107, 115, 130, 131, 143), addressed this acquisition problem. Subsequent HTC-focused work included stochastic electrotransport (59); super-resolution-STED microscopy (137); adaptive optics (105); HTC sample handling chambers (44, 92, 93, 135); custom ETC and staining chambers (59, 71); and microfluidic chip-based embedding, clearing, and labeling (13).…”

Advances and perspectives in tissue clearing using CLARITY

“…While most chamber designs are freely and commercially available, setting up ETC can be expensive. 3Dprinting of shared designs may significantly reduces costs for experimenters and allow for organ-specific designs (Sulkin et al, 2013;Miller and Rothstein, 2017); e.g. a tissue cutting matrix (Tyson et al, 2015) and imaging chambers (idisco.info/idisco-protocol).…”