RSM: A Breakthrough in Super-Resolution Imaging Technology

By Muhammad OsamaReviewed by Louis CastelJul 25 2025

A recent study published in the journal Cell Reports Physical Science introduced a novel super-resolution imaging technique called resonant multi-focal scanning microscopy (RSM). The objective was to enhance optical imaging by achieving a twofold improvement in resolution compared to conventional wide-field methods, while also enabling fast, high-contrast volumetric imaging across extended depths and varying fields of view.

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Advancement in Super-Resolution Microscopy

Super-resolution microscopy has revolutionized biological imaging by overcoming the diffraction limit of conventional optical microscopes, allowing visualization of subcellular structures with nanoscale clarity. Techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED), and image scanning microscopy (ISM) have significantly advanced spatial resolution while minimizing phototoxicity.

However, broader adoption of these methods is limited by complex instrumentation, difficult alignment procedures, and low volumetric imaging throughput, particularly when applied to large samples. Traditional axial scanning can also be slow and susceptible to drift, reducing its utility for high-throughput tissue imaging. These limitations highlight the need for super-resolution approaches that offer high resolution, deep volumetric imaging, and ease of use.

Development and Design of the RSM System

Researchers developed the RSM technique to enhance conventional epi-fluorescence microscopy. They integrated standard optical components, including a 100× objective lens, an electrically tunable lens (ETL), and a microlens array (MLA). The MLA produces a patterned array of diffraction-limited spots, enabling simultaneous imaging within the sample.

The ETL operates in resonant mode, rapidly oscillating to achieve axial scanning over a 20 μm range, with validated imaging depths up to 15 μm. A motorized stage continuously translates the sample at 10 μm/s, synchronized with ETL oscillations and image acquisition at 100 frames per second, minimizing motion blur and enhancing image clarity.

RSM combines stationary multi-focal excitation, continuous lateral scanning, and rapid axial refocusing, all without mechanical movement. The resonantly driven ETL, positioned at the conjugate pupil plane, enables fast and stable axial scanning while avoiding mechanical drift. The excitation pattern is tilted at 4° to ensure full lateral coverage during scanning.

The image reconstruction process involves four steps, including linear tracking to stabilize raw data, calibration and alignment of the multi-focal excitation pattern, pixel reassignment for a 1.4× intermediate resolution gain, and final two-dimensional (2D) deconvolution to achieve a full twofold improvement over wide-field microscopy. This workflow produces high-contrast, super-resolved images over extended areas and depths.

Performance Validation and Imaging Capabilities

The study validated RSM’s performance through a series of biological imaging experiments. In bovine pulmonary artery endothelial cells, RSM improved resolution, with decorrelation analysis estimating approximately 164 nm compared to 280 nm for wide-field imaging. It resolved microtubule filaments ranging from 150 to 170 nm in width, with separations as small as 160 nm. Compared to SIM and spinning disk confocal microscopy, RSM delivered superior lateral resolution and axial range without requiring mechanical refocusing.

RSM also enabled volumetric imaging of green fluorescent protein (GFP)-labeled mitochondria in U2-OS cells across a 2-μm depth range, revealing structures not visible in conventional imaging. In 16-μm-thick mouse kidney tissue, RSM maintained seamless depth penetration and enhanced contrast, capturing glomerular morphology throughout.

Additionally, RSM demonstrated an unlimited acquisition range by continuously scanning beyond the sensor’s typical field of view. In clinical samples, it successfully imaged 5-μm formalin-fixed paraffin-embedded (FFPE) breast cancer tissue sections, resolving sub-diffraction human epidermal growth factor receptor 2 (HER2) clusters (approximately 100 clusters/mm²), compared to less than 20% detection with wide-field methods. Multi-color imaging of HER2 and mitochondrial markers showed minimal signal overlap and distinct spatial distributions (correlation coefficient of 0.25), highlighting RSM’s multiplexing ability.

Overall, these results confirm RSM’s capability to produce high-contrast, super-resolved images across extended depths and areas in various specimens. This advancement offers valuable insights into tissue organization, disease progression, and diagnostic potential.

Implications for Biological and Clinical Research

This research has significant implications for clinical and biomedical fields, enabling high-resolution imaging of thick biological samples and thereby improving the understanding of disease mechanisms and the therapeutic process. The compatibility of RSM with standard epi-fluorescence microscopes and FFPE samples allows easy integration into existing clinical routines. Its flexibility makes it suitable for use in processes like pathology, tissue biology, drug screening, and spatial omics. By providing detailed sub-diffraction imaging and fast, three-dimensional (3D) scans over large sample areas and depths, RSM simplifies tissue analysis and supports advances in disease research and treatment development.

Conclusion and Future Directions

In summary, RSM represents a significant advancement in optical imaging by combining optical simplicity, computational efficiency, and user accessibility. The technique utilizes tunable electro-optics and synchronized sample movement to capture high-resolution images over large depths and areas while maintaining super-resolution quality.

Future work should focus on applying faster image reconstruction methods, such as non-iterative or deep learning-based deconvolution, to improve processing speed and throughput. Additionally, combining RSM with enhanced optical designs and computational tools could further improve volumetric imaging performance.

Overall, RSM offers a practical solution to overcome current limitations in super-resolution microscopy. It has strong potential to advance biological research and clinical diagnostics by providing detailed, high-resolution imaging across substantial sample volumes, ultimately supporting discoveries and improved healthcare outcomes.

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