How LifeCanvas tissue clearing preserves maximum fluorescent protein signal and tissue integrity

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Kit-Yi Yam, Ph.D.

Kit-Yi Yam, Ph.D.

Scientific Consultant for LifeCanvas Technologies

Principles of tissue clearing

Clearing enables deep-tissue imaging by rendering tissue transparent and reducing light scattering, while preserving spatial distribution of biomolecules and tissue components. Lipid membranes are a major cause of light scattering, limiting imaging depth. Hence, a common approach for tissue clearing is lipid removal followed by refractive index matching between the tissue and the immersion medium used for imaging. One way to extract lipids is by using detergents such as sodium dodecyl sulfate (SDS). However, passive diffusion of SDS is a time-consuming process, and can lead to severe loss of tissue integrity and biomolecular information when applied to formaldehyde-fixed tissues without additional protection (such as SHIELD or synthetic hydrogel).

LifeCanvas tissue processing pipeline, tissue clearing, light sheet imaging, cell detection image analysis

LifeCanvas’ holistic end-to-end workflow, from tissue preservation, active clearing, immunolabeling, and refractive index-matching, to light sheet imaging and image analysis.

How does active clearing with SmartClear work?

SmartClear Pro II is based on active clearing using stochastic electrotransport (PNAS 2015). A rotating electric field is used to actively drive SDS micelles through the tissue, resulting in rapid, uniform and nondestructive clearing of samples. SmartClear is a batch processing tissue-clearing device, consisting of a dual-buffer and membrane system. While one container contains one type of buffer (A) to pump through the clearing chamber and hydrate the membranes, the other comprises another buffer (B) that is distributed across the electrodes on the other side of the membranes. The position of these membranes between the electrodes and the clearing chamber is critical to preventing the formation of black precipitates, caused by breakdown products from SDS action on tissues. The device also prevents other common complications, such as tissue browning and large bubble formation.

Did you know?

SmartClear buffers and membranes are reusable! These buffers and membranes have a lifetime of 10 days. Afterwards, they can be discarded as non-hazardous waste.

Start SmartClear tissue clearing in 5 minutes

SmartClear is a user-friendly device with quick and easy installation and loading.

Our SmartClear device allows simultaneous processing of up to 8 mouse brain hemispheres (or equivalent size samples), using a multi-purpose sample holder to achieve cleared samples within 3-4 days.

Did you know?

LifeCanvas offers several different sample holders that are compatible with almost any type of tissue! Contact us if you have a unique sample and we will customize a holder for you.

There are two settings: beginner and expert mode. It is recommended to clear samples containing endogenous fluorescence at a lower temperature (42°C for buffer A) using the “gentle” setting in beginner mode, whereas samples without endogenous fluorescence can be cleared at higher temperatures (50°C for buffer A) using the “fast” setting in expert mode.

After clearing, samples can be stored in PBS + 0.02% sodium azide at 4°C until proceeding to either immunolabeling or index matching and imaging. Note that cleared SHIELD tissues appear slightly (10-15%) expanded due to the active lipid removal process, but shrink back to normal size and are optically transparent after index matching.

Why should I use SmartClear tissue clearing?

SmartClear tissue clearing is fast, reliable and cost-effective, allowing uniform and simultaneous clearing for multiple samples. Our nanoporous membranes prevent tissue contamination, browning, bubble formation, black precipitates, and tissue deformation. Both membranes and buffers are reusable and can be used for 10 days of continuous clearing and ensure maximum preservation of fluorescent protein signals. Uniform and reliable tissue clearing is critical for optimal antibody diffusion during immunolabeling and, most importantly, for high-quality light sheet imaging.

"The killer application of optically clearing tissue samples is, for us, that it enables high-throughput study of learning-induced gene expression patterns in the brain. The SmartClear II Pro has made this possible in practice by being well-designed, reliable, and easy to fit into our workflow. The unit clears our mouse hemibrains effectively and rapidly, without browning, and LifeCanvas technical support has been very responsive both regarding the units themselves and in discussing best practices for tissue clearing and sample preparation."
Thomas Cleland headshot
Thomas Cleland, Ph.D.
Cornell University

Multiple studies have benefited from using SmartClear in several types of tissues, including mouse (intestine, kidney, whole brain, spleen, liver, heart, embryo, spinal cord, colon), rat (brains and lungs), human, (skin and lungs) as well as cerebral organoids (see Park et al., 2018; Saritas et al., 2018; Martorell et al., 2019; Roy et al., 2019; Sakaguchi et al., 2019; Swaney et al., 2019; Yun et al., 2019; Foster et al., 2021; Muñoz-Castañeda et al., 2020; Helseth et al., 2021; Luchsinger et al., 2021; Ogujiofor et al., 2021; Pop et al., 2021; Sweeney et al., 2021; Trautmann et al., 2021).

New to tissue clearing?

Clearing using the SmartClear device is an order of magnitude faster than passive clearing. However, we also offer the option of passive clearing using the Passive Clearing Kit, consisting of a SHIELD preservation kit, Passive Clearing Buffer, and EasyIndex. This is an easy way to adopt the tissue clearing technology without the use of specialized equipment, while achieving similar uniform, reliable, and robust results with maximum fluorescent protein signal preservation and tissue integrity (see Bharat et al., 2020; Bharat et al., 2021; Kwon et al., 2020). 

The LifeCanvas Team will further assist you with technical and scientific support and provide feedback in judging tissue clarity to ensure optimal outcomes. LifeCanvas also designed the New User Startup Program for new users to receive personalized guidance, and benefit from free reagents and discounts.

Conclusion

LifeCanvas Technologies, founded by the inventor of CLARITY, strives to be at the forefront of new technological advances, driving research forward with novel reagents, products, and imaging solutions. The LifeCanvas Team consists of scientific experts who are here to help you with every step of your sample’s journey – from tissue preservation, clearing, and labeling, to imaging and analysis.

Click below to request a demo of our SmartClear device or learn more!

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