Imaging three-dimensional, subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy. However, trade-offs exist between axial resolution and other important technical indicators, such as temporal resolution, optical power density, and imaging process complexity. We report a new imaging modality, fluorescence interference structured illumination microscopy (FI-SIM), which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction. FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning. Moreover, the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.
Stimulated emission depletion (STED) nanoscopy is one of the most well-developed nanoscopy techniques that can provide subdiffraction spatial resolution imaging. Here, we introduce dual-modulation difference STED microscopy (dmdSTED) to suppress the background noise in traditional STED imaging. By applying respective time-domain modulations to the two continuous-wave lasers, signals are distributed discretely in the frequency spectrum and thus are obtained through lock-in demodulation of the corresponding frequencies. The background signals can be selectively eliminated from the effective signal without compromise of temporal resolution. We used nanoparticle, fixed cell, and perovskite coating experiments, as well as theoretical demonstration, to confirm the effectiveness of this method. We highlight dmdSTED as an idea and approach with simple implementation for improving the imaging quality, which substantially enlarges the versatility of STED nanoscopy.
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