CytoVista Clearing System and ProLong Glass Antifade Mountant

(See a list of the products featured in this article.)

The development of reliable 2D and 3D cell and tissue culture techniques and reagents has produced a plethora of options for studying the brain and other neural tissues. However, as the tissues and cultures become more complex, the experimental samples become more challenging to investigate. New approaches are needed for imaging spheroids, organoids, and thicker tissue sections.

One key challenge with imaging neural tissue is uneven light scattering caused by cell organelles and components such as nuclei, mitochondria, and membranes [1], as well as by lipids, which are heterogeneously distributed. It is this heterogeneity of the biological sample that contributes to the uneven scattering of light, making the sample appear milky white or opaque under the microscope [2]. Opaqueness is the result of an inherent mismatch between the refractive indices of the objective, medium, and cells or tissue. In fluorescence microscopy, opaqueness limits axial (z-dimension) resolution and focal depth during imaging. Therefore, it becomes imperative to match the refractive index of each component in order to capture high-quality images.

Optical clearing for sharp images

Several clearing techniques have been developed to match the refractive index of the sample to that of coverslips, immersion oil, and objectives. An effective clearing treatment for cells, spheroids, organoids, and tissue must meet specific criteria. First, it cannot change the overall morphology of the sample. Second, it must be compatible with immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC) techniques, including incubations with fixatives, permeabilization reagents, and antibodies. Third, the resulting refractive index from the clearing treatment needs to be closely matched to common microscope objectives, and the instrumentation needed to process and image the sample must be easily available in research laboratories. With the increased use of fluorescent proteins in neuronal studies, it is also important that clearing treatments do not diminish the fluorescence of these proteins [1].

CytoVista Clearing Reagents

Thermo Fisher Scientific recently released the Invitrogen CytoVista Clearing System (Table 1), a family of products developed to minimize the impact of refractive index mismatch when imaging cells, spheroids, organoids, or tissue. The CytoVista clearing workflow is compatible with most fluorophores, including fluorescent proteins, that are detected with common fluorescence imaging instruments such as widefield, confocal, and light sheet microscopes, and high-content analyzers. Features of the CytoVista family of tissue and 3D cell culture clearing reagents include rapid clearing of fluorescently labeled cells, spheroids, organoids, and tissue for 2D and 3D imaging, as well as minimal shrinkage, expansion, or other morphological changes to cells and tissue. In addition, cell and tissue clearing with CytoVista reagents does not require any special equipment and is compatible with IF, ICC, and IHC protocols. After the fluorescence analysis is complete, if needed, the cells or tissue clearing can be reversed and samples can be further analyzed for other histological studies.

The CytoVista 3D Cell Culture Clearing/Staining Kit clears fluorescently labeled 3D cultured cells such as organoids and spheroids, enabling the acquisition of sharp, bright images on samples up to a depth of 1,000 μm using fluorescence instrumentation (Figure 1). This kit includes CytoVista 3D Cell Culture Clearing Reagent, penetration buffer, wash buffer, blocking buffer, and antibody dilution buffer, and can be used to clear and image samples on microscope slides or in microplates or chambers. With this system, most samples can be cleared in as little as 30 minutes, depending on the thickness of the sample.

For thicker samples, the CytoVista Tissue Clearing/Staining Kit can be used to clear fluorescently labeled tissue up to 10 mm thick prior to fluorescence imaging. This kit contains both CytoVista Tissue Clearing Reagent and CytoVista Tissue Clearing Enhancer, which together can be used to clear most fluorescently labeled tissue types, as well as the buffers needed for IHC protocols. The clearing process is relatively fast, again depending on the thickness of the sample. For example, a whole mouse brain, which is approximately 8 mm thick, can be cleared in 24 hours, while a 1 mm section can be cleared in 2 hours.

Table 1. Selection guide for clearing reagents.

  ProLong Glass Antifade Mountant CytoVista 3D Cell Culture Clearing/Staining Kit CytoVista Tissue Clearing Reagent and Enhancer
Form Hard-setting, ready to use Soft-setting, ready to use Soft-setting, ready to use
Media type Aqueous Solvent Solvent
Refractive index ~1.52 after curing 1.48 Reagent alone: 1.50
With enhancer: 1.53
Sample archiving time frame Months to years Weeks to months Weeks to months
Imaging depth Up to 150 μm Up to 1 mm Up to 10 mm
3D cell culture Yes Yes No
Tissue sections up to 150 μm Yes No Yes
Tissue sections up to 10 mm No No Yes
Signal-to-noise ratio Best Good Good
Photobleaching protection Best None None
Mounted microscope slides Yes Yes Yes
Microplate imaging No Yes No
Sample preparation time Overnight to 4 days 30 min to a few hours 1–3 days
Reversibility/tissue recovery Yes Yes Yes
Cat. No. P36980 V11325 V11324
Clearing and fluorescent staining of brain spheroids

Figure 1. Clearing and fluorescent staining of brain spheroids. StemoniX 3D microBrain cortical neuronal spheroids (~500 μm diameter) were generated from induced pluripotent stem cell (iPSC)-derived neural progenitor cells. Neuronal bodies were stained with Invitrogen NeuroTrace Green Fluorescent Nissl Stain (green). Glial fibrillary acidic protein (GFAP) was detected with an anti-GFAP primary antibody and labeled with Invitrogen Alexa Fluor Plus 647 goat anti–rabbit IgG secondary antibody (red). Nuclei were stained with DAPI (blue). The tissue was cleared and mounted with the Invitrogen CytoVista 3D Cell Culture Clearing/Staining Kit. This image is a composite of z-stack imaging using the Thermo Scientific CellInsight CX7 LZR HCA Platform and HCS Studio Software.

ProLong Glass Antifade Mountant

Invitrogen ProLong Glass Antifade Mountant (Table 1) is a glycerol-based, hard-setting, ready-to-use mountant that can be applied directly to fluorescently labeled cells or tissue samples on microscope slides or coverslips. Prolong Glass mountant has a refractive index of 1.52 after curing, similar to that of glass coverslips, compatible immersion oil, and oil immersion objectives. Due to the close match in the refractive indices of the objective, medium, and cells or tissue, ProLong Glass mountant enables superior axial and lateral resolution in fluorescence imaging applications. ProLong Glass mountant also provides exceptional photobleaching protection across the visible to near-infrared spectra, and it is compatible with most organic dyes and fluorescent proteins. ProLong Glass mountant is ideal for producing bright, high-resolution z-stack, 3D, and 2D images of any cell or tissue sample up to 150 μm in thickness without adding extra steps to the fluorescence imaging workflow. Figure 2 shows the use of ProLong Glass mountant for imaging along the axial dimension of a rat brain section (100 μm thick) without loss of target resolution throughout the entire sample.

3D imaging using ProLong Glass Antifade Mountant

Figure 2. 3D imaging using ProLong Glass Antifade Mountant. Formalin-fixed, paraffin-embedded (FFPE) 100 μm rat brain sections were stained with Invitrogen β-3 tubulin primary antibody and Invitrogen Alexa Fluor 594 goat anti–mouse IgG secondary antibody (red), with overnight incubations for each antibody. Nuclei were stained with DAPI (blue). The stained tissue sections were placed on a coverslip, and Invitrogen ProLong Glass Antifade Mountant was added. The samples were allowed to air-dry uncovered overnight on a flat, dry, dark surface. The following day, ~30 μL of 100% glycerol was applied on the surface of the hardened mountant, microscope slides were placed on top, and the samples were left to cure for 1 hr. Sections were imaged on a Zeiss LSM 710 confocal microscope using a Plan-Apochromat 63x/1.4 NA oil immersion objective. Images were processed using Ziess ZEN software.

Choosing the right clearing solutions

The CytoVista Clearing System and ProLong Glass Antifade Mountant offer unique solutions for improving 2D and 3D imaging quality and resolution. Table 1 provides a selection guide to help you choose the right product for your experiment.