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.
A recently developed strategy to generate optical nonlinearities from various emitters localized in multilayer core/shell optical nanocrystals represents significant progress and great potential.
Recently, a new technique called MINFLUX was promoted and attained ~1-nanometer precision. However, MINFLUX is incapable of discerning two molecules within the diffraction-limited region unless with the help of on-off switching scheme of SMLM which yet entails time-consuming processes. Here, we produce a novel kind of focal spot pattern, called sub-diffraction dark spot, to localize molecules within the sub-diffraction region of interest. In our proposed technique nominated as sub-diffracted dark spot localization microscopy (SDLM), multiple molecules within the diffraction-limited region could be distinguished without the requirement of fluorescent switches. We have numerically presented the SDLM modality and some impacts, like intensity, are investigated. Simulative localization framework has been implemented on randomly-distributed and specifically-structured samples. SDLM is evidenced to have high localization accuracy and stability in densely-packed fluorescent solution.
We have designed and built a time-gated continuous wave stimulated emission depletion (CW-STED) nanoscopy to visualize microstructures beyond the diffraction limit. An off-line time-gating detection was performed with the help of time-correlated single-photon counting technique. Experimental results showed that before time-gating, the resolution of our system was about 75 nm with a depletion beam (592 nm) power of 200 mW. By using the off-line time-gating detection, the resolution was further improved to 38 nm. Biological samples were also used to test the performance of our time-gated CW-STED, and a resolution of 70 nm was achieved with a depletion beam (592 nm) power of 85 mW. Detailed principles of time-gated CW-STED were discussed in the text. The time-gated STED provides a better resolution with finite laser power.
Stimulated emission depletion microscopy (STED) has been proved to be a feasible and straightforward method of breaking the conventional diffraction barrier in the far-field. In this paper, we design and setup a home-built high speed STED microscope. Both the excitation beam (488nm) and the depletion beam (592nm) are provided by continuous wave lasers. By using a pair of galvo mirrors (TILL Yanus IV Digital Scan Head), we realize a pixel dwell time down to 2 μs in the experiment which enables an acquisition speed of 2 frames per second in an imaging field of 5*5 μm with an individual pixel size of 10nm. The image acquisition process is controlled by the software Imspector. In the manuscript, we give a clear description on how to build the microscope and also conduct several experiments to evaluate its performance in practice. A spatial resolution of <100nm, which is well beyond the diffraction barrier has been demonstrated in both nanoparticles and biomedical samples. Featuring a superresolution together with a high imaging speed, our STED microscope has big potential to be widely applied in related scientific researches.
We experimentally demonstrated that the microsphere on the top surface of the sample can enhance the imaging
capability of microscope, and possible to arrive a resolution below the conventional diffractive limit. The principle of the
super-resolution was discussed, and mainly due to the transformation of surface wave to propagation wave. The
mechanism of super-resolution and the corresponding phenomenon are presented in the paper.
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