Three-dimensional multimodal sub-diffraction imaging with spinning-disk confocal microscopy using blinking/ fluctuating probes

Three-dimensional imaging cannot be achieved easily using previously developed localization super-resolution techniques. Here, we present a three-dimensional multimodal sub-diffraction imaging technique with spinning-disk （SD） confocal microscopy called 3D-MUSIC, which not only has all the advantages of SD confocal microscopy such as fast imaging speed, high signal-to-noise ratio, and optical-sectioning capability, but also extends its spatial resolution limit along all three dimensions. Both axial and lateral resolution can be improved simul- taneously by virtue of the blinking/fluctuating nature of modified fluorescent probes, exemplified with the quantum dots. Further, super-resolution images with dual modality can be obtained through super-resolution optical fluctuation imaging （SOFI） and bleaching/blinking-assisted localization microscopy （BALM）. Therefore, fast super-resolution imaging can be achieved with SD-SOFI by capturing only 100 frames while SD-BaLM yields high-resolution imaging.

Mirror-enhanced super-resolution microscopy

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation,super-resolution microscopy.STimulated Emission Depletion(STED)nanoscopy offers lateral super-resolution using a donut-beam depletion,but its axial resolution is still over 500 nm.Total internal reflection fluorescence microscopy is widely used for single-molecule localization,but its ability to detect molecules is limited to within the evanescent field of~100 nm from the cell attachment surface.We find here that the axial thickness of the point spread function(PSF)during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror.The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially,which enables axial super-resolution with all laser-scanning microscopes.Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen.With no additional complexity,the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED,which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments.The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens,which cannot tolerate high laser power.