In the last few years there has been a significant increase in the number of researchers that want to incorporate tissue clearing and 3D histology into their bio-imaging workflows. While there are numerous tissue clearing techniques and versions of these techniques, adopting tissue clearing has been challenging for many researchers as it requires an expertise in tissue clearing and 3D imaging – we at Visikol are working to make this process easier.

As a company, we provide researchers with both easy-to-use tissue clearing reagents and kits as well as 3D tissue imaging services using confocal and light sheet microscopy. Through this webinar, Visikol CEO Michael Johnson, PhD provides an overview of how tissue clearing works as well as practical considerations for adopting tissue clearing into your research.

If you are interested in imaging your tissues in 3D, reach out to us today,

Tissue Clearing Resources

Which tissue clearing technique is best for me? 

This question unfortunately does not have an easy answer and will depend entirely upon your specific research question. Each tissue clearing technique has advantages and no technique is best for all applications. However, you can narrow down the best clearing technique for your application by considering four different tissue clearing properties using the table above.

1) Molecular Labeling Technique

Tissue clearing techniques are compatible with either only immunolabeling or fluorescent protein (FP) or both. Each approach to tissue clearing has advantages and disadvantages whereas approaches that are compatible with both immunolabeling and FP such as FocusClear™, Sucrose, ClearT2, TDE, CUBIC, CLARITY have their own specific disadvantages such as cost, tissue shrinkage, toxicity, slow clearing, tissue swelling and complexity, respectively. While techniques that are compatible with immunolabeling can be used with FP through using anti-FP immunolabels, this might increase overall processing time when compared to techniques that are compatible with FP.

2) Processing Time

Overall tissue processing and clearing time is a major driver for which technique should be chosen for an application as this will directly impact the rate at which the technique can be adopted. The time required to render a tissue transparent will depend on several factors including; tissue type, temperature, tissue size, animal age and tissue preparation method (e.g. fixed or fresh). Additionally, each clearing technique will react differently to each one of these parameters whereas solvent based techniques can easily render fatty tissues transparent and lipid removal techniques like CLARITY cannot.

3) Ease-of-use

The use of protein expansion/denaturation clearing techniques (e.g. Scale) and solvent based techniques (e.g. Visikol HISTO, i/3/uDISCO, BABB) are relatively simple wherein tissues are placed in varying solutions until the tissues are rendered transparent and are able to be visualized on the microscope. These processes lend themselves for high-throughput applications and can be successfully executed by any laboratory technician. Conversely, hydrogel-based approaches (e.g. CLARITY) require embedding tissues in acrylamide hydrogels which is a significantly more challenging process. Though companies (Logos Biosystem, LifeCanvas Technologies) have developed CLARITY devices to automate such processing, these techniques require expensive equipment (>$15,000) and are challenging.

4) Validation

Most researchers who are using tissue clearing for basic research do not need to be concerned with validation whereas researchers and clinicians using tissue clearing for applied research or on clinical tissues do need to consider validation. For some applications researchers need to be certain that the 3D renderings acquired from tissues are indicative of the tissue’s molecular and morphological properties and not the tissue clearing process. Therefore, these 3D renderings need to be correlated to the traditional histological methods that have been developed over the last century that serve as the foundation for tissue characterization. The challenge in validating these approaches is that while they can be used to acquire 3D renderings of tissues, many of the processes tend to significantly alter cellular morphology, leading to the expansion or shrinkage of tissue. Additionally, some clearing processes (e.g. CLARITY) require the removal of cellular components (e.g. lipids) from tissues to render tissues transparent. Combined, these factors lead to irreversible damage to the cellular morphology of tissues which necessitates parallel validation for these tissue clearing approaches.

Therefore, the need for validation is a significant consideration for some researchers whereas the need to validate 3D histology requires parallel 2D histological processing or a tissue clearing technique that preserves tissue morphology, does not cause damage to tissue structures, and is reversible. It has been shown that Visikol HISTO is reversible as opacity can be returned to tissues following imaging such that 2D histology can be conducted after 3D imaging with the same tissue [1].

Common questions and problems with tissue clearing

1) I have cleared and labeled tissue – now what?

The second part of the 3D histology process is to image tissues that have been rendered transparent with a confocal, light sheet or two-photon microscope. These imaging modalities also have their own advantages and disadvantages and the modality you should use is based upon your specific research question. For example, light sheet microscopy is very good at acquiring 3D images from large whole tissues while it is limited in super high resolution imaging compared to confocal microscopy.

2) You cannot see greater than 500 to 1,000 microns with air-matched objective on a confocal instrument

Commonly, researchers will encounter a problem in which it appears that their labels have not penetrated their tissues. However, the majority of the time this is due to a misunderstanding about the limitations of ones instrument. Because of differences in refractive index between objectives and solutions, traditional air objectives are limited to approx. 500 to 1,000 microns in imaging depth even if they have very long working distances. To see deeper, a researcher can section their tissue into multiple 1-2 mm sections, purchase an RI-matched dipping objective (>$10,000) or use a light sheet microscope.

3) Will your tissue clearing solution (e.g. Visikol HISTO, BABB, i/3DISCO) destroy your dipping objective?

The short answer is that you cannot be sure unless you ask your manufacturer, but just don’t try it with your expensive objective. Most objectives are currently designed for water, glycerol or oil and should not be placed in a solvent (Visikol HISTO, BABB, i/3DISCO). To use a solvent based clearing technique with your objective you can use a double chambered cuvette wherein the outer chamber is filled with the medium matching your lens (e.g. glycerol) and the inner chamber with Visikol HISTO. For light sheet microscopes, the sample can be placed in a cuvette within the imaging cuvette or you can use the Ultramicroscope that is compatible with all solvents.

References

[1] Royen, M. E., Verhoef, E. I., Kweldam, C. F., Cappellen, W. A., Kremers, G. J., Houtsmuller, A. B., & Leenders, G. J. (2016). Three‐dimensional microscopic analysis of clinical prostate specimens. Histopathology, 69(6), 985-992.

[2] Richardson, D. S., & Lichtman, J. W. (2015). Clarifying tissue clearing. Cell, 162(2), 246-257.

[3] Berke, I. M., Miola, J. P., David, M. A., Smith, M. K., & Price, C. (2016). Seeing through musculoskeletal tissues: improving in situ imaging of bone and the lacunar canalicular system through optical clearing. PloS one, 11(3), e0150268.

[4] Pan, C., Cai, R., Quacquarelli, F. P., Ghasemigharagoz, A., Lourbopoulos, A., Matryba, P., … & Ertürk, A. (2016). Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nature Methods.

Light sheet fluorescence microscopy (LSFM) is a powerful tool in the life science researcher’s toolkit. While the old histopathology workflow of physical sectioning and slide mounting still remains relevant, LSFM continues to shift the paradigm of imaging whole tissues, organs and organisms (via optical sectioning of cleared specimens). With powerful tools like LSFM and the Visikol® HISTO™ clearing reagents, researchers can capture high content images without damaging or distorting tissue morphology.

Light sheet microscopes work by illuminating samples not with a one-dimensional laser point, but with a two dimensional sheet of light. When two of the three spatial dimensions are illuminated at once, the image capturing speeds can be orders of magnitude faster than single point scanning platforms like confocal. Less illuminations means less risk of photobleaching precious samples. The excitation laser sheet is emitted perpendicularly to the objective to allow for the collection of all emission within this illumination plane.

The light sheet is great at avoiding out of focus signals, which increases sensitivity by contrast optimization. One of the greatest advantages of LSFM is the ability to 3D image specimens generally considered too large for conventional fluorescence microscopes. From organoids to whole animals, the flexibility in specimen range makes this platform adaptable for many sample sizes. LSFM utilizes both air and immersion objectives for both high throughput 4x scans and high resolution 20x scans.

Light sheet fluorescence microscopy is not just for large samples. 3D cell culture models are also amenable to LSFM scanning. A spheroid for example has a small size but a great deal of complexity, with the interactions between neighboring cells often determining the relevancy of in vivo disease states. LSFM can rapidly map these interactions without sacrificing the resolution needed to glean real insight into the significant changes in structure of these models. Structural fidelity is a critical goal of many disease model-based biotechnologies, including more cutting-edge platforms 3D bio-printing. Biological structures made by 3D bio-printing must have tight tolerances at microscopic scales. The required quality control of such models make suitable for optical sectioning with LSFM.

Visikol has launched light sheet microscopy services in partnership with Bruker. We are now offering a range of services from neural network mapping to quantifying volumes of vasculature in any organ. If your research could benefit from a greater understanding of the structural conditions present in your in-vivo specimens, Visikol’s imaging and digital pathology expertise paired with Bruker’s Luxendo MuVi SPIM LSFM platforms can provide those insights and more.

Reach out today to speak with one of our knowledgeable scientists who will find the best way to apply Visikol’s leading imaging capabilities to accomplishing your research goals.

Hampton, NJ – March 9th, 2021 – Visikol today announced its partnership with Bruker to use Bruker’s flagship MuVi SPIM CS Light-Sheet Microscopy platform to provide its clients with an even wider range of 3D tissue imaging services. The expanded services will now allow researchers to image larger tissues in 3D, such as entire mouse brains.

Since its inception in 2016, Visikol has assisted thousands of researchers in transitioning from two-dimensional to three-dimensional imaging through its contract research services and Visikol® HISTO™ reagents. Initially, the company began selling its Visikol HISTO reagents and kits primarily to academic researchers but quickly realized that there was also a large unmet need in the marketplace for advanced imaging services. The use of tissue clearing combined with advanced microscopy (e.g., confocal, light-sheet, 2-photon) and fluorescent labeling to image tissues in 3D requires a deep understanding of multiple complex fields, and even if researchers are successful in acquiring large image data sets, many times they struggle to transform these into useful insights. Over the last few years, Visikol has helped to fill this gap in the marketplace for 12 of the top 20 pharmaceutical companies and has built out a suite of advanced 3D image analysis tools.

“Prior to this partnership with Bruker, we relied heavily upon confocal microscopy for our 3D imaging services, which provides a high level of 3D resolution but is greatly limited in the amount of tissue volume that can be imaged,” said Visikol CSO Tom Villani, Ph.D. “The addition of light-sheet microscopy to our portfolio of imaging solutions means that we can now image much larger tissues and address novel questions that we could not evaluate before, such as mapping whole mouse brains in 3D or sampling tumors in 3D for heterogeneity.”

Light-sheet microscopy is an incredibly powerful tool for imaging 3D biological samples. Its inherent optical sectioning capabilities enable imaging of cleared and fluorescently labeled tissues with unprecedented optical resolution and volumetric imaging speed, yielding  dramatically reduced photo-bleaching compared to confocal microscopy. Through this partnership with Bruker, Visikol will now employ the industry-leading MuVi SPIM CS, which is compatible with a variety of tissue clearing techniques (e.g., BABB, Scale, CUBIC, SeeDB, CLARITY, Visikol HISTO) and can be tuned over a broad range of refractive indices. Visikol is well-versed in every tissue-clearing approach and is able to use these various techniques to effectively image fluorescent proteins, immunofluorescent markers, and other chemical dyes in 3D.

“We are very excited by this partnership with Visikol and the ability to provide more researchers access to our technology through this unique service offering,” added Lars Hufnagel, Ph.D., GM of Bruker’s Light-Sheet Business. “The MuVi SPIM CS pushes the boundary of light-sheet imaging of cleared tissues in terms of optical resolution and imaging speed while maintaining easy system operation and convenient sample access and handling.”

Visikol has already begun to offer these light-sheet imaging services to its clients at highly competitive rates for both industry customers and academic researchers. With many researchers unable to access core facilities due to COVID19 restrictions, Visikol is discounting these services for its academic customers to help provide increased access to these capabilities. To leverage light-sheet imaging, researchers can either send Visikol fixed tissues for clearing or already fixed, labeled, and cleared samples for imaging.

About Bruker Corporation

Bruker is enabling scientists to make breakthrough discoveries and develop new applications that improve the quality of human life. Bruker’s high-performance scientific instruments and high-value analytical and diagnostic solutions enable scientists to explore life and materials at molecular, cellular, and microscopic levels. In close cooperation with our customers, Bruker is enabling innovation, improved productivity, and customer success in life science molecular research, in applied and pharma applications, in microscopy and nanoanalysis, and in industrial applications, as well as in cell biology, preclinical imaging, clinical phenomics and proteomics research and clinical microbiology. For more information, please visit www.bruker.com.

About Visikol

Visikol is a contract services company that is focused on accelerating drug discovery and development through the use of its imaging, digital pathology and advanced cell culture assay services. The company provides end-to-end preclinical services that include both 2D and 3D in vitro models and assays, 3D whole mount tissue imaging, multiplex imaging, high content imaging, digital pathology and custom drug discovery solutions. Visikol’s expertise is in transforming tissues and cells into large image-based data sets that can be mined for actionable insights such that pharmaceutical and biotech companies can make more quantitative and informed decision during the drug development process. Additionally, Visikol manufactures and sells a suite of tissue clearing reagents and 3D immuno-labeling kits. These products allow researchers to easily and rapidly image whole tissues and 3D cell culture models in 3D instead of traditional 2D sectioning. For more information about Visikol or its services, please visit www.visikol.com.