Clearing Reagents for Imaging 3D Cell Culture and Tissue

On this page:

• CytoVista Reagent Selection Tables
• See applications of 3D culture clearing and imaging

The imaging of organoids, spheroids and thick tissue presents new challenges inherent in the samples themselves. Because of the opaqueness of tissue and the opaqueness of 3D cell cultures, it is harder to obtain sharp images with great clarity using standard imaging reagents and techniques. A fluorescently labeled thick specimen must be “cleared” so that the sample is more transparent for easier 3D imaging. The Invitrogen CytoVista Clearing Reagents are optimized for tissue up to 10 mm thick, and spheroids and organoids of varying thickness.

When staining adherent cells, all that is required is the addition of a simple staining buffer, but a fluorescently labeled thick specimen must be optically “cleared” to make it more transparent for easier three-dimensional imaging. Ideally clearing reagents should have minimum effect on a sample’s morphology or reagent fluorescent sensitivity. The CytoVista reagents for 3D imaging provide both optical clearing and mounting functions to meet these needs.

These CytoVista clearing reagents provide:

• Rapid clearing—1 mm thick samples can be cleared in as little as 2 hours and whole mouse brains of 8 mm thickness can be cleared in as little as 24 hours
• Gentle optical clearing—Minimal effect of detection sensitivity or sample morphology is observed after fluorescence labeled–tissue is cleared for 3D imaging
• Workflow compatibility—Easily adapt use of the reagents to IHC workflows without additional equipment or instruments
• Platform compatibility—Works with most fluorophores and standard fluorescence imaging instruments
• Flexibility—Store optically cleared tissue samples and cultures for weeks with minimal effect on sample morphology or fluorophore brightness and specificity
• Reversibility—For precious samples, the clearing can be reversed and the tissue can be processed and sectioned for histology studies such as hematoxylin and eosin (H&E) staining
  
  

Figure 2. Obtain clearer images of spheroids with CytoVista 3D Cell Culture Clearing Reagent. The CytoVista 3D Cell Culture Clearing Reagent cleared HepG2 spheroids to minimize light scattering, resulting in a sharper image and a more comprehensive cell count. By accurately counting dead cells at the core of the spheroid, more viable cells can be analyzed. Confocal z-slices of (A) uncleared and (B) cleared HepG2 spheroids. Blue = DAPI; Green = LIVE/DEAD Fixable Green Dead Cell Stain.

Figure 4. Easily image thick tissue samples using the CytoVista 3D Cell Culture Clearing Reagent. (A) A rat brain sample, 1 mm thick, was labeled with an anti-parvalbumin antibody. Captured with 10x air objective on an upright confocal microscope. (B) A mammary tumor sample, 1 mm thick obtained from a PyMT-transfected mouse, was labeled with an anti-Ki-67 FITC conjugated monoclonal antibody to study cell proliferation labeling. Captured with 10x air objective on an upright confocal microscope.

Figure 5. Obtain clear, sharp images from whole mouse brain samples using the CytoVista Tissue Clearing / Staining Kit.
(A) Parvalbumin-tdTomato transgenic whole mouse brain, captured with light sheet microscope. (B) Whole mouse brain cleared with CytoVista Tissue Clearing / Staining Kit and imaged with light sheet microscope.

Learn about techniques and methods for using an antifade, mountant or clearing reagent