RNA Under the Microscope: Techniques & Insights

RNA plays a crucial role in gene expression, cellular function, and disease mechanisms. Studying RNA at high resolution provides valuable insights into its spatial organization and interactions within cells. However, traditional imaging techniques often struggle to resolve fine RNA structures due to the small size of molecular components.

Recent advancements have improved RNA visualization by enhancing sample resolution and contrast, allowing researchers to explore RNA localization with unprecedented detail.

Principles of Expansion Microscopy

Expansion microscopy (ExM) has transformed RNA studies by physically enlarging biological specimens, enabling nanoscale resolution with conventional light microscopes. This technique bypasses the diffraction limit by embedding samples in a swellable hydrogel, which expands biological structures isotropically. By increasing the distance between molecular components, ExM reveals spatial RNA patterns previously obscured.

The process begins with anchoring biomolecules to a polymer network, preserving RNA spatially during expansion. A highly crosslinked polyacrylate hydrogel swells uniformly when exposed to water, increasing sample size by four to ten times and enhancing resolution. Unlike super-resolution microscopy, which relies on complex optics, ExM achieves high resolution through physical magnification, making it more accessible.

ExM is compatible with fluorescence in situ hybridization (FISH) and immunostaining, enabling subcellular mapping of RNA transcripts. This has been particularly useful for studying RNA-protein interactions, as expanded samples provide sufficient separation between molecular targets to resolve individual complexes. Studies show ExM can distinguish closely spaced RNA molecules that conventional microscopy would blur together.

Beyond improving resolution, ExM preserves the three-dimensional architecture of cells and tissues, making it ideal for investigating RNA localization in complex environments like neural networks or developing embryos. Maintaining spatial relationships during expansion allows researchers to study RNA dynamics in their native context. Iterative expansion, which involves multiple rounds of swelling, further enhances magnification and opens new possibilities for RNA organization studies.

Protocol Steps for Sample Preparation

Preparing a sample for ExM requires careful execution to preserve RNA integrity while ensuring effective expansion. Fixation stabilizes cellular structures and prevents RNA degradation. Paraformaldehyde (PFA) is commonly used due to its ability to crosslink nucleic acids and proteins while maintaining spatial organization. Some protocols include glutaraldehyde for enhanced structural preservation, though excessive crosslinking can hinder probe accessibility. Optimizing fixation conditions is crucial—over-fixation may impede hybridization, while under-fixation risks RNA diffusion and spatial loss.

Permeabilization enhances reagent penetration, allowing labeling probes and hydrogel components to reach intracellular RNA. Detergents like Triton X-100 or saponin disrupt lipid membranes, facilitating probe entry while minimizing structural disruption. Enzymatic treatments, such as proteinase K digestion, can increase accessibility, particularly when targeting RNA in dense compartments. The key challenge is balancing permeabilization efficacy with sample preservation, as excessive treatment may lead to RNA degradation.

Anchoring RNA to the hydrogel matrix ensures spatial registration during swelling. This is achieved using acrydite-modified oligonucleotides or methacryloyl-conjugated reagents, which covalently link RNA to the polymer network. Different anchoring strategies vary in efficiency and compatibility with downstream labeling techniques. Robust anchoring is critical—insufficient linkage can cause RNA displacement, compromising spatial accuracy.

Hydrogel polymerization solidifies the sample within a swellable matrix. Monomers such as sodium acrylate, acrylamide, and bisacrylamide polymerize into a stable gel, catalyzed by initiators like ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). Polymerization conditions, including temperature and reagent concentrations, must be optimized to ensure uniform gel formation and prevent artifacts.

Enzymatic digestion clears cellular components that may obstruct expansion. Proteinase K degrades proteins while preserving RNA-polymer linkage. This step is especially important for dense tissues, where residual material can impede uniform swelling. Digestion conditions, including enzyme concentration and incubation time, require precise calibration to prevent excessive degradation. Some protocols include detergents or chaotropic agents to enhance clearing efficiency without compromising RNA stability.

The final step involves controlled expansion of the hydrogel-embedded sample. Immersion in deionized water triggers swelling, increasing sample dimensions by four- to tenfold. The isotropic expansion maintains spatial relationships, allowing high-resolution RNA imaging. Multiple rounds of expansion, or iterative ExM, can further enhance resolution, though each cycle must be validated to confirm structural fidelity. Ensuring uniform swelling is essential, as uneven expansion can distort RNA localization.

Strategies for RNA Labeling

Labeling RNA for visualization requires sensitivity, specificity, and signal stability. Fluorescence in situ hybridization (FISH) remains a widely used technique, leveraging complementary probes to bind target sequences with high precision. Advances in probe design have improved signal-to-noise ratios, with single-molecule FISH (smFISH) detecting individual RNA transcripts. By using multiple short probes, each conjugated to a fluorophore, smFISH generates punctate signals corresponding to distinct RNA molecules, reducing background fluorescence while enhancing resolution. This approach is especially valuable for analyzing low-abundance transcripts.

Enzymatic amplification methods further refine RNA labeling by increasing fluorescence intensity without compromising spatial resolution. Hybridization chain reaction (HCR) and rolling circle amplification (RCA) introduce self-assembling fluorescent polymers that amplify probe signals. HCR uses metastable DNA hairpins that hybridize in a cascading reaction upon target recognition, generating extended fluorescent structures. RCA exploits circularized probes to produce long, repeated sequences that accumulate fluorescence over time. Both methods offer improved sensitivity, making them suitable for detecting rare RNA species while preserving spatial context.

Molecular beacons provide an alternative RNA labeling strategy with dynamic capabilities. These stem-loop structures fluoresce only upon hybridization with a complementary RNA sequence, minimizing background signal in live-cell imaging. Unlike fixed-sample techniques, molecular beacons allow real-time tracking of RNA localization and movement, offering insights into transcript dynamics. Photoactivatable probes expand these applications, enabling temporally controlled labeling to study RNA behavior in response to stimuli. These innovations have proven useful in studying RNA transport, particularly in neurons and other polarized cells where transcript localization is tightly regulated.

Multiplexed labeling strategies enable simultaneous visualization of multiple RNA species. Spectrally distinct fluorophores or barcoded probe sets differentiate RNA populations, allowing comparative transcript distribution analyses. Multiplexed error-robust FISH (MERFISH) enhances this approach by integrating combinatorial labeling with error-correction algorithms, vastly increasing the number of detectable RNA species. This has been instrumental in mapping cellular transcriptomes with spatial precision, revealing gene expression heterogeneity that bulk RNA sequencing would obscure. The ability to resolve multiple RNA types within a single cell provides new insights into cellular function, particularly in complex tissues.

Visualization of Spatial RNA Patterns

Mapping RNA distribution within cells has transformed our understanding of transcript localization and its functional implications. High-resolution imaging techniques reveal how RNA transcripts are positioned relative to cellular structures, offering insights into gene regulation, protein synthesis, and intracellular transport. The spatial arrangement of RNA is particularly significant in polarized cells, where localization dictates translation, enabling precise protein production control. For instance, in neurons, mRNA encoding synaptic proteins is trafficked to dendritic spines, where translation is spatially restricted to support synaptic plasticity. Similarly, in epithelial cells, transcripts localize to distinct membrane domains to regulate cell polarity and function.

Fluorescence microscopy combined with computational image analysis allows researchers to quantify RNA distribution patterns with high accuracy. Clustering algorithms and spatial autocorrelation metrics determine whether transcripts exhibit random dispersion, targeted localization, or distinct compartmentalization. This is particularly useful in identifying RNA granules—membraneless organelles that serve as hubs for transcript storage and processing. Advanced imaging pipelines now integrate machine learning to classify RNA spatial arrangements, distinguishing between diffuse cytoplasmic distributions and tightly regulated transcript hotspots.