Breaking the Diffraction Limit: Progress and Prospects of Super-Resolution Fluorescence Microscopy in Biomedical and...

The emergence of super-resolution microscopy (SRM) has had a significant impact on the research field of diffraction-limited optical fluorescence microscopy, which has a span of applications from fundamental biophotonics to advanced materials science. SRM is a powerful technique capable of producing ultra-high-resolution images of organic, semiconductor materials and live cells by overcoming the conventional diffraction limit. In this manuscript, we discuss various strategies for improving the spatial and temporal resolution of SRM techniques without causing photodamage to the sample, which is a common issue when samples are exposed to high-intensity depletion beams. The problem of background noise is also a major limitation of all SRM techniques. In order to address these limitations, we propose various strategies to improve optical design, optical alignments, tuning of wavelengths of excitation and depletion laser beams, fluorescent probes, and microscopy modalities. Moreover, SRM techniques are reviewed and compared with each other as well as with conventional microscopy techniques in terms of imaging properties, spatiotemporal resolution, and fluorescent labels for cancer pathology applications. The continued integration of advanced probe design, adaptive optics, and AI-driven image reconstruction can serve as a future road-map to revolutionize SRM, enabling real-time, non-invasive, and ultra-high-resolution imaging across complex biological and material systems.