Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing
April 20th, 2021
Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.
This protocol will allow researchers to answer important questions about neuronal patterns of connectivity and spatial organization that underlies function within a circuit. The main advantage of this technique is that it enables visualization of neuronal morphologies located deep within intact three-dimensional tissue. This method is straightforward and thus easily reproducible.
First-time experimenters should be comfortable performing this technique. It is imperative to have a clear tissue sample in order to obtain high-quality data. The visual demonstration of appropriate tissue clarity and clearing techniques will enable easily reproducible results.
To begin, place the hydrogel-submerged brain tissue in a vacuum incubator for three hours at 37 degrees Celsius with minus 90 kilopascal vacuum. Leave the tube top unscrewed to allow the vacuum to form properly. Wash the tissue with PBS for 10 minutes at room temperature with gentle shaking.
Place the polymerized tissue sample in the electrophoresis chamber, keeping track of the orientation of the tissue within the chamber. Fill the chamber and reservoir with the supplied electrophoresis SDS buffer. Run the sample at 70 volts, one ampere, and 35 degrees Celsius with constant current for about two hours per milliliter of tissue.
Check the sample periodically for sufficient clearing of brain tissue by removing it from the chamber and replacing it in the same orientation. After the sample has finished clearing, wash it in PBS overnight at room temperature, replacing the PBS with fresh PBS as often as possible. Following the final wash in PBS, wash the tissue for five minutes in deionized water at room temperature three times.
The tissue will become opaque and may expand. Incubate the tissue in the refractive index matching solution for at least four hours at room temperature. During the incubation, construct a suitable housing chamber to image the sample.
Start by using a glass slide as a base for mounting, then lay down either rubber or plastic spacers and secure them with super glue, making sure that there aren't any holes. Place the clear tissue into the mounting chamber pre-filled with refractive index matching solution. Securely mount the tissue by placing a glass coverslip on top and sealing it with nail polish.
Image this tissue by adding a drop of refractive index matching mounting solution directly on top of the glass. To construct a large tissue imaging chamber for tissues with more than a five millimeter thickness, use a 10 centimeter glass dish with a high wall. Place a 50 milliliter conical tube in the center, making sure that the diameter of the tube is large enough to accept the barrel of the objective lens used.
Make 3%agarose in water and pour it in the space between the glass dish and the conical tube, then allow it to cool for one hour. This will form a ring of solid agarose. Securely adhere the tissue to the bottom of the chamber using super glue and fill the chamber with refractive index matching solution, which may start to polymerize unless preserved from air and stored at four degrees Celsius.
Acquire the image using a confocal microscope. Turn on all the relevant imaging equipment. Place the sample on the stage.
Carefully approach the immersion media with the objective and form a continuous column of media. Using epifluorescence, find an appropriate imaging field. Start by setting the resolution and scan speed settings.
If imaging using a confocal microscope, fully close the pinhole to obtain the smallest optical section and thus the best Z resolution. Gradually increase the laser power or sensor gain until a suitable image is obtained with a high signal-to-noise ratio. If utilizing standard EGFP or tdTomato two-color imaging, set the light collection settings.
Set the Z-stack parameters based on the observed start and end points of the tissue. Set the step size based on the desired Z resolution. Smaller step sizes will yield a greater Z resolution, but may lead to sample bleaching.
When satisfied with the image acquisition settings, acquire the image. Ensure that the image has a high signal-to-noise ratio and shows distinct boundaries of structures. After image acquisition, the representative cell morphology was analyzed using embedded statistics and classifying scripts within the analysis software.
The collected data reflects that Neuron 2 has larger than dendritic structure with a higher density of spines compared to Neuron 1. To substantiate this result, standard Scholl analysis was performed, which affirms that Neuron 2 is more dendritically complex than Neuron 1 as denoted by the increased number of Scholl intersections at 50 to 100 micrometers from the soma. Neuron 1 contains a larger proportion of spines with more filopodia-like shapes and is an immature spine subtype.
Spines with defined heads, called mushroom spines, likely contain more developed and mature synapses. Thus, Neuron 2 is more mature with a higher density of mature spines. After clearing, the tissue chunks can be stained using traditional immunohistochemistry.
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Overview
This article presents a detailed protocol for processing, visualizing, and analyzing thick slices of labeled neural tissue using CLARITY tissue clearing, confocal microscopy, and image analysis. The method enables high-resolution imaging of neuronal morphologies deep within intact three-dimensional brain tissue, facilitating the study of neuronal connectivity and substructure organization in both healthy and diseased states.
Key Study Components
Area of Science
- Neuroscience
- Imaging Technology
- Neuroanatomy
Background
- Brain activity is determined by neuronal connectivity and morphology.
- Advances in imaging and tissue clearing have improved the ability to study thick brain slices at high resolution.
- CLARITY enables visualization of neuronal structures deep within tissue.
- Image processing software allows rapid analysis of large datasets.
Purpose of Study
- To provide a reproducible protocol for clearing, imaging, and analyzing thick neural tissue slices.
- To facilitate the study of neuronal connectivity patterns and morphologies.
- To enable comparison of healthy and diseased brain tissue at the morphological level.
Methods Used
- Hydrogel embedding and vacuum incubation of brain tissue.
- Electrophoretic tissue clearing using SDS buffer.
- Refractive index matching and mounting for imaging.
- Confocal microscopy for high-resolution imaging.
- Image analysis including Scholl analysis and spine classification.
Main Results
- The protocol allows clear visualization of neuronal morphologies deep within tissue.
- Neuron 2 exhibited more complex dendritic structure and higher density of mature (mushroom) spines compared to Neuron 1.
- Neuron 1 had more filopodia-like, immature spines.
- Scholl analysis confirmed greater dendritic complexity in Neuron 2.
- The method is straightforward and reproducible for first-time users.
Conclusions
- This protocol enables detailed morphological analysis of neurons in thick brain tissue.
- It supports studies of neuronal connectivity and maturation.
- The approach is accessible and can be adopted by researchers new to tissue clearing and imaging.
Frequently Asked Questions
Executive Industry Relevance
High-resolution imaging of intact neuronal dendrites using CLARITY tissue clearing enables detailed morphological analysis critical for early neuroscience drug discovery. This approach supports predictive confidence in target validation by revealing connectivity patterns and substructure changes relevant to disease models. Integrating reproducible 3D imaging workflows strengthens translational continuity from discovery through preclinical research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of neuronal connectivity and spatial organization in intact tissue.
- Supports functional target validation by quantifying dendritic complexity and spine maturation.
- Facilitates mechanistic de-risking through direct visualization of disease-relevant neuronal changes.
Screening & Assay Development
- Prepares validated 3D neural tissue systems for downstream compound screening.
- Standardizes imaging and analysis protocols for reproducible quantitative outputs.
- Enables scalable assessment of neuronal morphology for screening readiness.
Translational & Preclinical Research
- Aligns morphological readouts with disease-relevant biomarkers in preclinical models.
- Provides continuity from discovery imaging to preclinical validation of neuronal endpoints.
- Supports risk-adjusted advancement decisions based on quantitative structural data.
Pipeline & Workflow Integration
This method integrates into the neuroscience discovery continuum, bridging early target validation, phenotypic screening, and preclinical model assessment.
- Discovery Biology: Supports hypothesis testing and pathway clarification by visualizing intact neuronal networks.
- Screening: Delivers reproducible, quantitative imaging outputs for compound evaluation.
- Analytics: Provides statistical measurements such as Scholl analysis and spine classification for condition comparison.
- Translational Research: Connects morphological changes to disease models and biomarker strategies.
- Enterprise Reuse: Establishes a standardized, reusable imaging and analysis workflow for neuroscience R&D teams.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neuronal target validation.
- Operational Value: Promotes standardization, reproducibility, and scalability in 3D tissue imaging.
- Strategic Value: Improves go/no-go decisions and capital efficiency by providing robust morphological endpoints.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of neuroscience assets.
Implementation Considerations
- Requires expertise in tissue clearing, confocal microscopy, and image analysis software.
- Needs access to vacuum incubators, electrophoresis chambers, and high-resolution imaging platforms.
- Demands cross-team standardization of sample preparation and imaging protocols.
- Adaptable to various neural tissue models and labeling strategies.
- Dependent on achieving sufficient tissue clarity and signal-to-noise for reliable quantification.
