Abstract
Tissue clearing of gross anatomical samples was first described over a century ago and has only recently found widespread use in the field of microscopy. This renaissance has been driven by the application of modern knowledge of optical physics and chemical engineering to the development of robust and reproducible clearing techniques, the arrival of new microscopes that can image large samples at cellular resolution and computing infrastructure able to store and analyze large data volumes. Many biological relationships between structure and function require investigation in three dimensions and tissue clearing therefore has the potential to enable broad discoveries in the biological sciences. Unfortunately, the current literature is complex and could confuse researchers looking to begin a clearing project. The goal of this Primer is to outline a modular approach to tissue clearing that allows a novice researcher to develop a customized clearing pipeline tailored to their tissue of interest. Further, the Primer outlines the required imaging and computational infrastructure needed to perform tissue clearing at scale, gives an overview of current applications, discusses limitations and provides an outlook on future advances in the field.
🔬 Techniques
🔭 Microscopes
🧬 Organisms
💻 Software
✨ Fluorophores
🧪 Sample Preparation
🏭 Microscope Brands
🧪 Reagent Suppliers
💻 Software Details
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📊 Figures
Figure 1:
An overview of the components of a tissue clearing experiment.
Tissue clearing workflows are numerous, making them difficult to summarize. However, workflows can be defined as a series of modules (fixation, pre-treatment, delipidation, labelling and refractive in...
Figure 2:
Concept of hydrogel embedding.
( a ) Tissue biomolecules are fixed chemically or physically to a hydrogel mesh generated in situ , and then cell membranes are removed during delipidation to enable chemical transport and optical tra...
Figure 3:
Protocols for delipidation.
A summary of methods for delipidating tissue using the most common detergents and solvents. Methodology example represents clearing of a murine brain. CHAPS, (3-((3-cholamidopropyl) dimethylammonio)-1...
Figure 4:
Mechanisms of delipidation.
(a) The long non-polar tails of ionic detergents such as sodium dodecyl sulfate (SDS) or Triton X100 intercalate with membrane lipids. If the detergent concentration is high enough, large micelles wil...
Figure 5:
Examples of the clearing process.
(a) Four pieces of mouse skeletal muscle at various stages of the clearing process. From left to right: muscle extracted from the mouse and fixed in paraformaldehyde (PFA); muscle tissue extracted fro...
Figure 6:
Whole-organ cell profiling using the latest CUBIC-L/R+ protocol.
Volume-rendered and single-plane images of mouse organs of 8-week-old C57BL/6N male mice that were cleared using CUBIC-L/R+. ( a,b ) The organs were stained with propidium iodide (PI) and individual c...
Figure 7:
Example of neuronal staining in the mouse brain.
( a ) 3D rendering of an entire mouse expressing GFP downstream of the thy1 promoter 122 in a subset of cells. The brain was cleared using a CLARITY protocol. Major fibre tracks are clearly visible. (...
Figure 8:
Representative results of vDISCO panoptic imaging.
a, 3D rendering of a Thy1-GFPM mouse after vDISCO whole-body immunolabelling/clearing and imaging by light-sheet microscopy. Neuronal fibres expressing GFP are enhanced by anti-GFP nanobodies conjugat...
Figure 9:
Example of vascular staining in the mouse brain.
(a ) 3D render of an entire mouse brain that was perfused with CM-DiI lipophilic dye prior to fixation. After fixation, the brain was cleared using a modified iDISCO protocol and imaged via light shee...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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