By Owais AliMay 9 2024Reviewed by Lexie CornerMicroscopy has been essential in exploring the complexities of biological systems by visualizing structures at cellular and molecular levels. However, the inherent opacity of biological tissues limits light penetration and the depth of clear imaging.
Image Credit: arrowsmith2/Shutterstock.com
Optical clearing techniques address this limitation by enhancing tissue transparency, significantly improving microscopy capabilities, and expanding the possibilities for exploration in life sciences.
Principles of Optical Clearing
Proteins, lipids, and other molecules that form cells and tissues have high refractive indices compared to the cytosol (the fluid inside cells). This mismatch in refractive indices causes light to scatter through tissue, limiting penetration depth and degrading image quality by reducing contrast and resolution.
Optical clearing techniques minimize these mismatches by adjusting the refractive indices of tissue components to match that of the surrounding medium or embedding material.
By reducing the refractive index differences, light scattering is minimized, allowing light to propagate through the tissue with minimal obstruction. As a result, the tissues become more transparent, enabling deeper imaging and improved visualization of intricate structures that were previously obscured.1
Techniques and Methods
Over the past two decades, researchers have developed numerous optical clearing techniques, each with its unique approach and applications. These methods can be broadly categorized into three main groups: solvent-based clearing, aqueous clearing, and hydrogel embedding.
1. Solvent-Based Clearing
Solvent-based clearing techniques involve dehydrating the tissue using organic solvents and immersing it in a refractive index-matching solution. While effective in achieving tissue transparency, these methods can quench fluorescent proteins and potentially damage the tissue, limiting their applications in certain scenarios.2
Spalteholz's Technique
Spalteholz's technique laid the foundation for solvent-based optical clearing methods. It involves fixing tissues with formalin, bleaching with hydrogen peroxide, dehydrating with alcohol, and clearing with methyl salicylate and benzyl benzoate.
However, this approach causes significant tissue shrinkage and browning, bubble formation, and protein fluorescence quenching due to hydrogen peroxide, making it unsuitable for 3D cultures.
Benzyl Alcohol/Benzyl Benzoate (BABB)
Based on Spalteholz’s method, the BABB protocol utilizes a 1:2 mixture of benzyl alcohol and benzyl benzoate after dehydration with ethanol and hexane. This technique successfully clears various cancer spheroids, such as T47D and DLD1. It enables detailed light-sheet fluorescence microscopy (LSFM) imaging of hypoxic and dormant cells, confirming that spheroids mimic in vivo tumor environments.
3D Imaging of Solvent-Cleared Organs (3DISCO)
3DISCO improves on BABB, using graded tetrahydrofuran (THF) solutions for dehydration, followed by dichloromethane and dibenzyl ether for clearing. It reduces background fluorescence and increases signal intensity. Variations like iDISCO (immunolabeling-enabled) and uDISCO (ultimate) provide whole-organ immunolabeling and significant tissue shrinkage, respectively.3
2. Aqueous Clearing
Aqueous clearing techniques use hyper-hydrating aqueous solutions to clear tissues, offering reduced hazards and better preservation of endogenous fluorescent signals, making them suitable for various imaging modalities. However, they may be limited to smaller sample sizes and require longer processing times compared to solvent-based methods.2
Simple Clear Lipid-Exchange (Scale)
The scale method, a widely used aqueous clearing technique for brain sample imaging, uses solutions of urea, Triton X-100, and glycerol or sorbitol. Its optimal solution variant, ScaleA2 (4M urea, 10 % glycerol, 0.1 % Triton X-100), is effective but slow and causes swelling.
In contrast, the improved ScaleSQ variant, with higher concentrations of urea and sorbitol, reduces processing time and increases light penetration by 7.5 % in spheroids.3
3. Hydrogel-Embedding
Hydrogel-embedding methods (for example, CLARITY) involve embedding the tissue in a hydrogel matrix and removing lipids with detergents, followed by refractive index matching. They excel in preserving proteins, RNA, and DNA within the tissue, making them ideal for multiplexed labeling studies and fluorescent in situ hybridization (FISH) applications.2
Clear Lipid-Exchanged Acrylamide-Hybridized Rigid Imaging (CLARITY)
The original CLARITY method infuses tissues with a hydrogel solution, then removes lipids electrophoretically using SDS before clearing with a refractive index-matching solution. Modern derivatives, such as passive CLARITY (PACT) and perfusion-assisted agent release (PARS), simplify this process using high-concentration SDS and passive diffusion.
Microfluidic clearing of spheroids with CLARITY protocols reduces clearing time to two days, and osmotic pump cycles with pH alternation accelerate the process to five hours.3
Applications in Research and Medicine
In Vivo Imaging and Detecting Diabetes-Induced Changes
Optical clearing provides a noninvasive approach for studying diabetes-related changes in skin microvascular structures. By reducing light scattering with chemical agents, it enhances the performance of optical imaging techniques, such as optical coherence tomography and two-photon microscopy. This allows for improved detection of changes like collagen loss and structural alterations due to protein glycation, providing valuable insights into diabetes progression and severity.
Optical Clearing in PDT Treatment of Melanoma
Melanoma is characterized by high pigmentation. It poses significant challenges for light-based techniques and treatments due to melanin's strong light absorption and the high concentration of melanosome granules within the tumor, severely restricting light penetration into the lesions.
Optical clearing agents improve the photodynamic therapy (PDT) of melanoma by enhancing light penetration and distribution, maximizing the effectiveness of photosensitizers like Visudyne and Photodithazine, and resulting in complete tumor eradication.4
3D In Vitro Cancer Research Using Cleared Organoids/Spheroids
Organoids are 3D structures derived from human pluripotent or organ-restricted stem cells that replicate organ architectures and interactions. As they mimic in vivo cellular environments, they provide more accurate cancer models than traditional 2D cultures or animal models. However, imaging thick 3D organoids is challenging due to contamination and inconsistent tissue architecture.
Optical clearing techniques, coupled with fluorescence microscopy, have enabled high-resolution 3D imaging to characterize the cellular composition, spatial distribution, and interactions within cleared organoids. Recent clearing protocols, like fructose, urea, and glycerol for imaging (FUnGI), enhance the visualization of organoids for multi-color lineage tracing.
These advanced imaging methods enable researchers to understand cancer progression better and create a foundation for personalized medicine.1
Future Trends and Challenges in Optical Clearing
The integration of optical clearing with advanced microscopic techniques has greatly enhanced the visualization of large biological samples, including improved resolution, contrast, and penetration depth.
Current clearing techniques frequently cause cell damage due to fixation or solvent use. Thus, researchers are seeking methods that avoid cell damage, allowing time-course studies and real-time monitoring of cellular processes. They are also striving to increase imaging depth for whole-body imaging of small animals by minimizing light scattering and absorption in thick specimens.
With these advancements, optical clearing techniques will unlock new possibilities in biomedical imaging, offering deeper insights into complex biological structures and enabling innovative research and therapeutic applications.3
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References and Further Reading
- Brenna, C., Simioni, C., Varano, G., Conti, I., Costanzi, E., Melloni, M., Neri, LM. (2022). Optical tissue clearing associated with 3D imaging: application in preclinical and clinical studies. Histochemistry and Cell Biology. doi.org/10.1007/s00418-022-02081-5
- Fasoli, A., Florindo, C. (2022). Overview of Tissue Clearing Methods and Applications. [Online] Oxford Instruments. Available at: https://andor.oxinst.com/learning/view/article/overview-of-tissue-clearing-methods-and-applications
- Costa, EC., Silva, DN., Moreira, AF., Correia, IJ. (2019). Optical clearing methods: an overview of the techniques used for the imaging of 3D spheroids. Biotechnology and Bioengineering. doi.org/10.1002/bit.27105
- Tuchin, VV., Zhu, D., Genina, E. A. (2022). Handbook of tissue optical clearing: new prospects in optical imaging. [Online] Taylor & Francis Group. Available at: https://doi.org/10.1201/9781003025252
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