Super-Resolution Imaging and Shared Management: A Protocol for Confocal Microscopy with Multiplex Detection

Super-resolution microscopy (SRM) overcomes the diffraction limit of conventional light microscopy, enabling nanoscale visualization of subcellular structures. However, the widespread adoption of techniques such as stimulated emission depletion (STED) microscopy, photoactivated localization microscopy (PALM) / stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM) can be hindered by factors such as high phototoxicity, specialized sample preparation, or complex operation. Here, we present a comprehensive protocol that leverages a laser-scanning confocal platform integrated with a multiplexed super-resolution detector. This system, combined with computational processing, enables a straightforward transition between confocal and super-resolution imaging within the same workflow. The protocol provides a user-friendly approach for fast, high-resolution, and low-phototoxicity imaging. The detailed method encompasses multimodal imaging capabilities, including multicolor fluorescence, differential interference contrast (DIC), three-dimensional Z-stack reconstruction, time-lapse live-cell imaging, large-area tiling, and various super-resolution modes offered by the multiplexed array detector. Systematic comparisons demonstrate that the super-resolution modes achieve a lateral resolution of approximately 120 nm, representing about a two-fold improvement over the diffraction limit of conventional wide-field microscopy and a significant enhancement in both resolution and signal-to-noise ratio (4-8 fold) compared to standard confocal operation on the same platform. The discussion also covers key points of a shared resource management model essential for supporting the sustainable operation of this technology. This integrated guide serves as a valuable resource for both new and experienced researchers and facility managers aiming to adopt and maximize the potential of this accessible form of advanced imaging in life sciences research.