In the relentless pursuit of observing intricate biological structures hidden deep within tissues, optical microscopy has long faced formidable challenges. Traditional super-resolution methods often falter when confronted with the complexities of scattering and background fluorescence inherent in thick, heterogeneous specimens. A groundbreaking development now promises to shift this paradigm. Researchers from Peking University, led by Professor Peng Xi, have unveiled an innovative imaging modality termed Confocal² Spinning-Disk Image Scanning Microscopy (C²SD-ISM), which masterfully combines advanced hardware with novel computational strategies to significantly enhance imaging fidelity and resolution in deep tissue environments.
Conventional super-resolution techniques, including STED, SIM, and SMLM, offer remarkable resolution under optimal conditions yet struggle as imaging depth increases. STED microscopy relies on a doughnut-shaped depletion beam whose spatial integrity is compromised by tissue scattering, undermining resolution. SIM’s dependence on structured stripe illumination patterns makes it vulnerable to distortion and artifacts when photons scatter. SMLM, while powerful for localizing single molecules, suffers from poor localization accuracy in environments rife with background fluorescence and optical aberrations. These limitations have restricted their efficacy in thick biological tissues, which are opaque and highly scattering.
Image Scanning Microscopy (ISM), an evolution of confocal microscopy, enhances spatial resolution by approximately twofold beyond the diffraction limit through pixel reassignment and deconvolution techniques. However, ISM’s conventional implementations encounter inherent constraints—particularly a compromise between spatial and temporal resolution during data acquisition, impeding its applicability in live or volumetric deep-tissue studies. Addressing this, the research team previously introduced Multi-Confocal ISM (MC-ISM), improving temporal resolution but still contending with background interference and fidelity reduction at significant depths.
The newly introduced C²SD-ISM pushes these boundaries by integrating a spinning-disk confocal microscope with a digital micromirror device (DMD) and a sophisticated adaptive reconstruction algorithm named dynamic pinhole array pixel reassignment (DPA-PR). This dual-confocal architecture physically filters out-of-focus fluorescence through the spinning disk, establishing the first confocal layer, effectively enhancing optical sectioning and mitigating background noise at the hardware level. Simultaneously, the DMD projects sparse multifocal excitation patterns, enabling efficient spatial encoding vital for the second confocal layer powered by the DPA-PR computational engine.
Unlike conventional ISM reconstruction algorithms that assume idealized Gaussian excitation and detection point spread functions (PSFs), C²SD-ISM confronts real-world complexities head-on. Systemic aberrations and the ubiquitous Stokes shift—differences between excitation and emission wavelengths—distort PSF profiles, undermining reconstruction accuracy. The DPA-PR algorithm innovatively constructs a virtual 5×5 detector array from subsets of raw image stacks by extracting multiple offset sub-images. Spatial offsets are precisely quantified using phase cross-correlation methods, allowing for high-fidelity reassignment that preserves structural integrity and brightness linearity within reconstructed images.
Demonstrating formidable prowess, C²SD-ISM achieves lateral resolution down to 144 nanometers within dense tissue specimens—a substantial improvement over standard confocal microscopy. This precision is maintained even in heavily scattering environments, as validated using mouse kidney tissue sections. The system’s capability to maintain high visibility of excitation foci under dense multifocal excitation patterns is critical, substantiated by comparative analyses showing superior local contrast and reduced computation overhead, requiring six times fewer raw images than conventional multifocal structured illumination microscopy (MSIM).
One of the standout features of C²SD-ISM lies in its volumetric imaging capabilities. The technology facilitates three-dimensional reconstructions over volumes as large as 66.5 × 66.5 × 12 micrometers with axial step sizes as fine as 150 nanometers. This fine sampling preserves exquisite detail and spatial continuity across complex cellular architectures. The application to EGFP-labeled zebrafish vasculature exemplifies its scalability and robustness. Over a mosaic volume reaching nearly 3 millimeters in lateral dimensions, the system delivers markedly enhanced spatial resolution compared to conventional confocal microscopy, revealing delicate vascular networks with unprecedented clarity.
Further expanding its versatility, C²SD-ISM leverages the DMD’s programmability to perform projection-based Structured Illumination Microscopy, addressing key SIM limitations in thick tissues. The spinning disk’s physical rejection of out-of-focus fluorescence dramatically improves stripe modulation contrast, permitting 3D imaging of fungal samples to depths and resolutions unattainable by traditional SIM approaches. Achieving approximately a 1.68-fold resolution enhancement, this mode breaks the longstanding depth barrier, facilitating detailed volumetric observation in optically challenging samples.
C²SD-ISM’s elegant synergy of hardware optimization and intelligent algorithm design culminates in an imaging platform that harmonizes resolution enhancement, imaging depth penetration, and signal fidelity. This triumvirate is critical for advancing biological microscopy beyond its conventional boundaries, enabling more reliable investigations of cellular and tissue-scale phenomena. Moreover, the system’s high throughput, multicolor capabilities, and adaptability render it an exceptionally practical tool for diverse research domains, from neurobiology to developmental biology and pathology.
Forecasting future developments, the research team envisions augmenting C²SD-ISM with deep learning algorithms for denoising and adaptive optics for aberration correction. Such integrations could unlock even deeper penetration depths and larger volumetric datasets with minimized phototoxicity. Particularly, the adaptive illumination strategies enabled by DMD programmability open avenues for real-time feedback-controlled microscopy—ushering in ‘intelligent’ imaging platforms that minimize photodamage while maximizing information throughput during live-cell or in vivo studies.
A testament to its technological maturity and industrial applicability, elements of the C²SD-ISM framework have already been commercialized in the Nova-SD spinning-disk confocal system. This commercial instrument boasts native lateral resolutions around 230 nanometers, exceptionally high imaging speeds reaching 2000 frames per second, seven-channel excitation options, and expansive fields of view up to 25 millimeters, underscoring the practical impact of this research on mainstream biological imaging.
In the spirit of open science, the researchers have made critical components freely available, including simulation codes for artifact-free spinning-disk imaging, mask designs for disk fabrication, super-resolution reconstruction software for multifocal excitation leveraging DPA-PR, and comprehensive hardware control packages. This commitment facilitates global collaboration and accelerates adoption across imaging centers worldwide, ensuring that C²SD-ISM’s benefits extend beyond the originating lab.
By surmounting entrenched obstacles to deep tissue super-resolution imaging, the Confocal² Spinning-Disk Image Scanning Microscopy stands poised to revolutionize optical microscopy. Its capacity to deliver unprecedented resolution and fidelity at significant depths, coupled with efficient data acquisition and adaptability, heralds a new chapter for biomedical research—empowering scientists to unravel the complexities of life with clarity and precision previously thought unattainable.
Subject of Research: Advanced optical microscopy techniques for super-resolution imaging in complex biological tissues
Article Title: High-fidelity tissue super-resolution imaging achieved with confocal² spinning-disk image scanning microscopy
Web References: https://doi.org/10.1038/s41377-025-01930-x
Image Credits: Liang, Q., Ren, W., Jin, B. et al.
Keywords
Optics, Super-resolution microscopy, Confocal microscopy, Spinning-disk microscopy, Digital micromirror device, Image scanning microscopy, Deep tissue imaging, Adaptive optics, Structured illumination microscopy
