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 simultaneously 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.