3D image of rat brain section using ProLong Glass

Advanced clearing technologies for imaging thick tissue sections and 3D cell culture models—including spheroids, organoids, and whole organs—have helped to understand disease states and related mechanisms in a variety of biological disciplines. Benefits provided by high content analysis, confocal, laser scanning, wide field microscopy, and others have helped actualize volume fluorescence microscopy to capture 3D images of whole tissues or organs. However, interference from inherent autofluorescence and other biomolecular aberrations can still limit imaging depth in more complex models. Such aberrations may require ‘clearing’ of a sample by removing the opaque, milky features, as they are often described.

What is tissue clearing?

3D model of tissue clearing key takeaways

  • Match the refractive index to minimize the light scattering through the specimen and microscope objective.
  • Clearing should help provide reproducible results and allow for long term archiving of samples.

Successful clearing is the process of optically resolving a sample or complex biological material, such as whole organs, large tissue, and cellular models, with minimal changes to morphology and without compromising the ability for immunolabeling or fluorescence imaging detection. (Figure 1) The clearing method should be easy to implement, with reproducible results, and help enable the archiving of samples for weeks, if not years.

Mouse brain clearing using SlowFade Glass

Figure 1. Mouse brain clearing with SlowFade Glass. Clearing of 1-mm mouse brain section by refractive index matching with SlowFade Glass at time intervals of 0, 16, and 48 hours.

Methods to decrease image opaqueness

Refractive index matching is ideal for obtaining the best fluorescence images, but it isn’t always possible. Mismatching among mediums can cause loss of imaging resolution, as light needs to travel through the sample itself, a mounting media, glass coverslip, oil, and a microscope objective. Just as important, however, is limiting the amount of variable scattering of light from cellular membranes, lipids, and molecules of the specimen. This heterogeneity of scattering among the cellular components leads to an increase in opaqueness of an image (1,2). A denser makeup of lipids, trafficking organelles, and other subcellular molecules will increase lateral, or non-forward, light scattered (2,3). Non-forward light scattering in situ will often not pass through the specimen, as it is exacerbated by the continuous, pinball like, interactions of scattered light with neighboring molecules. Through the multiplicity of scattering, refraction, and absorbance the light’s energy is reduced or ultimately lost, leading to a distorted and white, non-translucent image. Thus, the goal of any clearing reagent and mountant is to optically clear the sample by matching the refractive index to minimizing the light scattering through the specimen and to the microscope objective.

Types of clearing

Chemical clearing key takeaways

  • Different specimens and applications require different clearing or mounting approaches.
  • Aqueous based–slower, useful to protect from photobleaching, and does not require dehydration.
  • Solvent based–faster, useful for specimens above 500 µM and whole organs.

The practice of optical clearing has taken different approaches, primarily being divided into chemical and matrix-based approaches, each having their own advantages and disadvantages. Discussed below is a comparison of chemical solutions, which can either be aqueous- or solvent-based, with the goal of achieving a highly resolved 3D image for immunolabeling, including immuno-cytochemistry, immuno-histochemistry, or immunofluorescence.

Aqueous-based clearing approaches, rather than solvents, are generally used to avoid dehydration and toxicity, which can destroy the integrity of a sample. Traditionally, researchers made refractive index matching solutions (RIMS) with sugar or glycerol for simple, passive immersion. This may be preferred with thinner or smaller samples, because they are easier to clear and can maintain fluorescent protein emission. Such immersion techniques however usually achieve less than 1.5 refractive index and can take days to achieve clearing, resulting in often poorer image quality when compared to traditional solvent approaches, due to refractive index mismatching between the cleared sample, the glass coverslip, and immersion oil (glass and oil have an RI of 1.51) (1,3). As sugar or glycerol solutions may take extended periods for clearing, a sample can experience considerable shrinkage while losing lipid content, while solutions such as ProLong Glass and SlowFade Glass control morphological alterations and loss of lipid content while achieving a higher refractive index of 1.52.

Optical clearing for multiple sample types with varying depth

Choosing the right clearing approach and reagents that meet both the needs of the researcher and biological specimen can be difficult when considering time, toxicity, morphological effects, and compatibility with either immunolabeling or fluorescent protein applications. In addition, choosing the correct approach must consider sample type and thickness so that there is minimal shrinkage of the sample and preservation of lipid content and fluorescence, so that researchers can ask important questions related to cell signaling, membrane trafficking, and adhesion.

Thermo Fisher now offers both aqueous refractive index matching solutions (RIMS), with perfect 1.52 refractive index, and also solvent-based optical clearing techniques. Aqueous based RIMS solutions, such as ProLong Glass and SlowFade Glass, are perfect for optical clearing up to 0.5 mm thick specimen–as they do not require dehydration, they have minimal to no effect on specimen morphology. For specimens as thick as 0.5 mm, overnight incubation is required for complete optical clearing. Both also have antifade chemistry, which allow for sharp and bright images across the whole spectrum. Importantly, almost all major fluorophores, including organic dyes, fluorescent proteins, and phalloidins are compatible.

CytoVista Clearing Reagents are based on advanced solvent chemistry and requires some degree of dehydration for optimal clearing. Solvent approaches are much faster than aqueous clearing reagents, as a 0.5 mm thick sample requires only 30 minutes, a 1 mm thick sample takes only 4 hours, and whole mouse brain in 24 hours to clear. CytoVista 3D clearing reagent is specially formulated to be gentle with spheroids and organoids in order to keep the integrity of those fragile cellular structures, while CytoVista Tissue clearing reagent can clear tissue up to 1 mm thick. It can clear almost any soft tissue such as brain, lung, prostate, heart, intestine, liver, and many others. If this dehydration is done with butanol, then these reagents are compatible with fluorescent proteins as well. If sample is precious and needed for H&E after fluorescent microscopy, then this clearing is reversible, giving back the intact tissue, ready for H&E staining with minimal effect on integrity or morphology of tissue.

Clearing and antifade mounting reagents for 3D imaging

  ProLong Glass (curing) SlowFade Glass (non-curing) CytoVista Reagents (non-curing)
General specifications
Form Ready-to-use, curing (hard-setting) mountant

For immediate imaging of samples up to 150 µm thick
Ready-to-use non-curing (soft-set) mountant

For immediate imaging of samples up to 500 µm thick
Ready-to-use, non-curing (soft-setting) family of reagents

3D Cell Culture clearing and staining kit for immediate imaging of 3D cell culture (organoids and spheroids up to 1 mm thick)

Tissue clearing and staining kit for immediate imaging of thick tissue (up to 10 mm)
Refractive index 1.52 (after curing) 1.52 1.48 (organoids/spheroids up to 1 mm thick)

1.53 (tissue samples up to 10 mm thick)
What type of imaging am I planning?
Specimen type Animal cells/tissue (fixed) Animal cells/tissue (fixed) Animal cells/tissue (fixed)
Fixed-cell imaging, immediate imaging  
Fixed-cell, long-term imaging    
What is the thickness of my sample?
Up to 10 µm    
Up to 150 µm    
Up to 500 µm    
Up to 1 mm    
Up to 10 mm    

Additional information about curing and non-curing product options

General guidance for clearing different sample types

Uses for CytoVista Tissue Clearing Reagent

  • For easier-to-clear tissues, such as brain, lung, intestine, and skin, with less than 250 µm thickness.
  • For tissues thicker than 250 µm or difficult-to-clear tissues, such as kidney, liver, heart, and placenta, as well as tissues 500 µm to 10 cm thick (e.g., heart, liver, etc.).
  • Not recommended for hard tissues, such as bone, cartilage, and connective tissue.
  • Provided in complete ICC/IHC staining kit, where all of the required buffers are provided and specially formulated to assist antibodies to penetrate thick tissues.
  • Clearing can be enhanced by incubating the sample with the CytoVista Tissue Clearing Enhancer.

Uses for CytoVista 3D Cell Culture Clearing Reagent

  • For organoids and spheroids up to 1,000 µm thickness.
  • For microplate imaging, including high-content imaging.
  • Not compatible with most 3D cell culture matrices. We recommend harvesting the cells from the matrix to enhance the penetration of antibodies and other staining reagents.
  • Provided in complete ICC/IHC staining kit, where all of the required buffers are provided and specially formulated to assist antibodies to penetrate thick tissues.
  • We recommend cutting thicker tissues into 2 mm thick sections using a device such as the CytoVista 2 mm Coronal Mouse Brain Slicer or the 2 mm Sagittal Mouse Brain Slicer.

See CytoVista Reagent User Guide

Uses for SlowFade Glass Soft-set Antifade Mountants

  • For imaging of samples up to 500 µm thick.
  • Compatible with ICC, IHC, fluorescent proteins, nuclear dyes, phalloidin conjugates, and other fluorescent reagents typically used in fluorescent microscopy.
  • For multiplexed 3D imaging.
  • Since it is an aqueous approach, no dehydration is required.
  • Not recommended for lipophilic membrane stains because it contains glycerol, which may interfere with the use of lipophilic membrane stains such as DiI.

See SlowFade Antifade Mountant User Guide

Uses for ProLong Glass Antifade Mountant

  • For sample imaging up to 150 µm depth, while providing superior photobleaching protection.
  • For extended slide storage (months to years), although storage time is partially dependent upon specimen type, fluorophores used, and storage temperature.
  • For multiplexed 3D imaging.
  • Since it is an aqueous approach, no dehydration is required.

See ProLong Antifade Mountant User Guide

References