A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales
May 10th, 2022
The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.
Our protocol would facilitate investigation of neuronal structures from circuit to component scales which is essential for better understanding of brain functions. Our clearing technique, ScaleSF exerts potent clearing capability, as well as a high level of tissue preservation that is required for simultaneous visualization of both large and small scale structures. Our protocol is especially effective in the brain where neuronal cells exhibit elaborate processes, tremendous links, and arrange specialized fine subcellular structures.
Before beginning the experiment, prepare Sca/eS solutions as shown in this table and cover the samples with foil. Start the procedure by adding eight milliliters each of ScaleS0 and ScaleS4 solutions to two separate wells of a six well cell culture plate and pre-warm the plate to 37 degrees Celsius in an incubator. Transfer the brain slices to the pre-warmed ScaleS0 solution with a spatula and incubate the slices for two hours at 37 degrees Celsius in a shaking incubator at 90 RPM.
Next, transfer the permeabilized brain slices in eight milliliters of PBS minus in a six well cell culture plate and wash for 15 minutes by keeping in an orbital shaker at 40 to 60 RPM. Repeat this step twice. Then, transfer the brain slices in eight milliliters of pre-warmed ScaleS4 solution and incubate for eight to 12 hours at 90 RPM at 37 degrees Celsius.
For chamber preparation, 3D print the chamber frame and microscope stage adapters. Attach the chamber frame to a coverslip using a pressure sensitive adhesive. Prepare 1.5%agarose in ScaleS4D25(0)solution.
Mix the solution and microwave it until the agarose is fully dissolved. Then allow the solution to cool to 37 degrees Celsius. Mount the cleared brain slice onto the bottom coverslip of the imaging chamber with a spatula.
Remove the excess solution from the cleared slice using a clean tissue. Add the ScaleS4 gel to the brain slice using a micropipette to fill the imaging chamber. Place another coverslip on top with forceps followed by a piece of tissue paper and a glass slide.
Transfer the imaging chamber to a refrigerator at four degrees Celsius. Place metal weights on the glass slide and leave them for 30 minutes. After the incubation, remove the material from the imaging chamber and wipe off excess gel.
Place the imaging chamber in a 60 millimeter glass Petri dish and attach the rim of the imaging chamber at multiple points to the dish with a pressure sensitive adhesive. Pour the ScaleS4 solution into the dish and shake gently for one hour at 40 to 60 RPM at 20 to 25 degrees Celsius. Substitute with fresh solution and remove air bubbles on the gel surface by gently scraping the surface using a pipette tip.
Mount the imaging chamber on a microscope stage. Prepare a confocal laser scanning microscope equipped with a multi-immersion objective lens of 2.5 millimeter working distance, 16x magnification and 0.60 numerical aperture. Turn on all the relevant imaging equipment, such as the workstation, microscope, scanner, lasers, mercury lamp, and launch an imaging software.
Set the correction collar of the multi-immersion objective lens to 1.47. Immerse the objective lens in the solution and let it approach the slice slowly. Remove all air bubbles trapped on the tip of the objective lens.
Find regions of interest in cleared tissues using epifluorescence. To set image acquisition parameters, first, determine the bit depth for image acquisition as the size of the image increases with the bit. Then set the detection wavelength according to the emission spectrum.
Set the XY resolution, scan speed, and pinhole size. Also adjust the laser power, detector amplifier gain, and offset until a suitable image is obtained. Determine the tiling size based on the size of the ROI, ensuring that the entire length and width of the ROI are captured.
Navigate through the tissue in all planes and set the start and endpoints of the stack. Set the Z step size, according to the desired Z resolution. Collect the images and process them using image analysis software.
Using this protocol, optical clearing of a mouse brain slice of one millimeter thickness was achieved. EGFP expression in the cerebral cortex of PV-FGL mice showed that the fluorescence and structural integrity of the tissues was preserved. Refractive index mismatch induced aberrations caused a noticeable loss of image brightness and resolution of EGFP positive neurons.
These neurons were clearly visualized with the confocal laser scanning microscope when the correction collar was adjusted to match this ScaleS4 solution. 3D reconstruction of EGFP labeled neurons in the mouse primary somatosensory cortex was done in a one millimeter thick brain slice. Individual dendritic arbors decorated with dendritic spines were seen at higher magnification.
Axon terminal arborization and axonal boutons were also observed in the slices. Reflective index matching between imaging fluid and object lens is critical for 3D imaging in cleared tissues. Our technique would facilitate integrating neuron structure from circuit to component scales, providing insights into neuron mechanisms, information of processing in the brain.
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Overview
This study presents a detailed protocol for neuronal imaging in brain slices using a novel tissue clearing method called ScaleSF. The technique allows for high-resolution visualization of neuronal structures, facilitating investigations from circuit to component scales, crucial for understanding brain function.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Imaging Techniques
Background
- Neuronal cells display intricate processes and specialized structures.
- Tissue clearing techniques enhance imaging capabilities in neuroscience.
- ScaleSF provides effective clearing without compromising tissue integrity.
- Detailed imaging methods are essential for elucidating neuronal mechanisms.
Purpose of Study
- To develop a reliable protocol for imaging neuronal structures in brain slices.
- To utilize the ScaleSF technique for simultaneous visualization of various neuronal scales.
- To enhance understanding of neuronal mechanisms and information processing in the brain.
Methods Used
- Ex vivo brain slices were utilized for imaging studies.
- The biological model involved mouse brain tissue, specifically PV-FGL mice.
- No multiomics workflows were mentioned.
- Key steps included solution preparation, incubation, and confocal microscopy setup.
- 3D reconstruction of neuronal structures was performed using confocal laser scanning microscopy.
Main Results
- The ScaleSF method preserved fluorescence and structural integrity of brain tissues.
- Clear visualization of EGFP-positive neurons allowed for detailed imaging of dendritic and axonal structures.
- Findings emphasize the importance of refractive index matching for effective imaging.
- 3D imaging successfully captured the complexity of neuronal morphologies.
Conclusions
- This study demonstrates the effectiveness of ScaleSF in neuronal imaging.
- Enhanced imaging techniques will enable better understanding of neuronal circuits and mechanisms.
- The protocol opens avenues for detailed exploration of neuronal processes in health and disease.
Frequently Asked Questions
Executive Industry Relevance
High-resolution neuronal imaging across mesoscopic to microscopic scales is critical for target validation and mechanistic de-risking in CNS drug discovery. The ScaleSF tissue clearing protocol enables robust visualization of neuronal circuits and subcellular structures, supporting predictive confidence in early discovery and translational neuroscience pipelines. This capability enhances portfolio decision-making by providing detailed structural insights essential for hypothesis-driven R&D.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of neuronal connectivity and circuit architecture for target validation.
- Supports mechanistic de-risking by preserving fine subcellular features during imaging.
- Facilitates predictive confidence in CNS target selection through comprehensive structural mapping.
Screening & Assay Development
- Prepares optically cleared, structurally intact brain slices for quantitative imaging assays.
- Standardizes imaging conditions to ensure reproducibility and comparability across experiments.
- Delivers high-content, scalable imaging outputs suitable for downstream screening workflows.
Translational & Preclinical Research
- Aligns structural imaging with disease-relevant neuronal models for translational continuity.
- Enables 3D reconstruction of neuronal morphologies to inform preclinical validation.
- Supports risk-adjusted advancement by providing detailed anatomical context for biomarker studies.
Pipeline & Workflow Integration
This tissue clearing and imaging protocol integrates into the discovery continuum from early target validation through preclinical research in neuroscience portfolios.
- Discovery Biology: Provides high-resolution visualization for hypothesis testing and pathway clarification in neural circuits.
- Screening: Delivers reproducible, quantitative imaging outputs for assay development and compound evaluation.
- Analytics: Enables 3D image acquisition and analysis of neuronal structures for robust data comparison.
- Translational Research: Bridges discovery and preclinical stages by supporting disease-relevant structural studies.
- Enterprise Reuse: Offers a standardized, reusable protocol for diverse CNS imaging applications.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in CNS research.
- Operational Value: Enhances standardization, reproducibility, and scalability of neuronal imaging workflows.
- Strategic Value: Improves go/no-go decisions and capital efficiency by providing detailed structural data early in the pipeline.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of CNS programs.
Implementation Considerations
- Requires expertise in tissue clearing, confocal microscopy, and image analysis.
- Demands access to multi-immersion objective lenses and compatible imaging chambers.
- Necessitates cross-team standardization of imaging parameters and sample preparation.
- Adaptation may be needed for different tissue types or disease models.
- Precise refractive index matching is critical for optimal imaging quality.
