Tissue Clearing New - Fluorescence Imaging - Tocris

Tissue clearing is achieved by refractive index matching of a sample tissue and the surrounding medium, rendering the sample optically transparent. Several clearing methods exist based on hydrogel embedding (CLARITY ^[1], aqueous hyper-hydration (Scale ^[2], CUBIC ^[3]) and solvent-based clearing methods (BABB ^[4], iDISCO ^[5]), Tissue Clearing Pro). Many of these existing clearing protocols have not been widely adopted due to the destructive, complex and time-consuming procedures required, and the lack of commercially available kits.

Tissue Clearing Pro is a solvent-based technique, which enables tissue samples to be fixed, labeled and cleared by following a rapid and straightforward protocol. The Tissue Clearing Pro technique is reversible, which means that follow-up 2D histological straining can be performed after 3D imaging of the tissue sample.

Tissue Clearing in Organoids and 3D Cell Cultures

Imaging organoids, microtissues, spheroids and 3D cell cultures can be problematic due to the opacity and thickness of these models, as light can only penetrate up to two to three cell layers deep. The outer layers of organoid tissue may differ considerably in morphology and function from the innermost cell layers. To fully understand the system being studied on all levels, obtaining clear images of all the layers is important. Using tissue clearing techniques, allows researchers to image the entire organoid or 3D cell culture model, providing a 3 to 4-fold increase in number of cells imaged.

Tissue clearing is especially important in drug discovery, as visual assessment of drug activity on the outer layers of organoids and 3D cell cultures are often extrapolated to the whole model, which can lead to overestimated drug potencies. For example, drug-induced cell proliferation or pathway inhibition in the outer cell layers is often greater than that observed in the inner cell layers.

Figure 1: Tissue Clearing Pro-Organoid (Cat. No 7390) was used to clear HepaRG NPC liver spheroids (~ 250 μm), capturing data from significantly more cells. Images were acquired following the Organoid tissue clearing protocol set out in the Guidebook.

Tissue Clearing Kits for Organoids and Whole Tissues

Tissue Clearing Pro-Organoid (Cat. No 7390) is a rapid, easy-to-use, reversible and non-destructive kit, which allows effective tissue clearing of organoids, 3D cell cultures and microtissues, giving unparalleled visualization of the desired labeled targets. This organoid tissue clearing kit allows 3D cell cultures up to 500 μm thick to be cleared in minutes, and is compatible with fluorescent protein, immunolabeling and small molecule staining techniques, as well as being suitable for use with all standard dyes and buffers. The kit comes with a getting started guide and a more comprehensive tissue clearing guidebook, providing detailed protocols.

The Tissue Clearing Pro (Cat. No. 7389) kit is designed for use with whole tissues. This kit can clear a whole mouse brain of up to 8 mm thickness in 24 hours; for sections with a thickness of 1 mm this can be achieved in 2 hours. Like Tissue Clearing Pro-Organoid, Tissue Clearing Pro is a rapid, easy to use, reversible and non-destructive kit.

Figure 2: Tissue Clearing Pro-Organoid kit (Cat. No 7390) was used on Open Liver HepaRG NP spheroids, enabling visualization of stained structures: Left DNA, MRPII and MDRI; Middle DNA, CD68, Albumin and Vimentin; Right DNA, panCK and CD31.

Tissue Clearing Pro-Organoid and Tissue Clearing Pro Benefits

• Rapid action (depending on tissue thickness: minutes to hours)
• Simple to use protocols
• Suitable for use with wide-field, confocal, and light sheet microscopes, and high-content analyzers
• Compatible with fluorescent protein, immunolabeling and small molecule dye staining techniques
• Suitable for use in high-throughput screening - compatible with flat-bottom plates as well as ultra-low attachment U-bottom plates
• Clearing is reversible, allowing further immunohistochemistry (IHC) and hematoxylin and eosin (H&E) investigations
• Relatively gentle technique, avoids tissue damage or shrinkage issues associated with BABB, 3DISCO, iDISCO, uDISCO and vDISCO techniques

Tissue Clearing is applicable across a wide range of research areas but is having a big impact in neuroscience. Tissue clearing has allowed neuroscientists to study light microscopy-based connectomics of cellular signaling networks in brains (as well as other tissue types) and image whole mouse brains, giving new insights into neural development, function and disease. For more details on the impact of tissue clearing in neuroscience, read Ueda et al, (2020) Tissue clearing and its applications in neuroscience. Nature Reviews Neuroscience 21 61. PMID: 31896771.

Tissue Clearing References

1. Wallace et al. (2013) Structural and molecular interrogation of intact biological systems. Nature 497 332. PMID: 23575631
2. Kurokawa et al. (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14 1481. PMID: 21878933
3. Susaki et al (2014) Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157 726. PMID: 24746791
4. Dodt et al. (2007) Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Methods 4 331. PMID: 17384643
5. Renier et al. (2014) IDISCO: IDISCO: A simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159 896. PMID: 25417164
6. Ueda et al, (2020) Tissue clearing and its applications in neuroscience. Nature Reviews Neuroscience 21 61. PMID: 31896771