Achieving localization with molecular precision has been of great interest for extending fluorescence microscopy to nanoscopy. MINFLUX pioneers this transition through point spread function (PSF) engineering, yet its performance is primarily limited by the signal-to-background ratio. Here we demonstrate theoretically that two-photon MINFLUX (2p-MINFLUX) could double its localization precision through PSF engineering by nonlinear effect. Cramér-Rao Bound (CRB) is studied as the maximum localization precision, and CRB of two-photon MINFLUX is halved compared to single-photon MINFLUX (1p-MINFLUX) in all three dimensions. Meanwhile, in order to achieve same localization precision with 1p-MINFLUX, 2p-MINFLUX requires only 1/4 of fluorescence photons. Exploiting simultaneous two-photon excitation of multiple fluorophore species, 2p-MINFLUX may have the potential for registration-free nanoscopy and multicolor tracking.
The pixel size of a charge-coupled device (CCD) camera plays a major role in the image resolution, and the square pixels are attributed to the physical anisotropy of the sampling frequency. We synthesize the high sampling frequency directions from multiple frames acquired with different angles to enhance the resolution by 1.4× over conventional CCD orthogonal sampling. To directly demonstrate the improvement of frequency-domain diagonal extension (FDDE) microscopy, lens-free microscopy is used, as its resolution is dominantly determined by the pixel size. We demonstrate the resolution enhancement with a mouse skin histological specimen and a clinical blood smear sample. Further, FDDE is extended to lens-based photography with an ISO 12233 resolution target. This method paves a new way for enhancing the image resolution for a variety of imaging techniques in which the resolution is primarily limited by the sampling pixel size, for example, microscopy, photography, and spectroscopy.
Open-3DSIM is an open-source reconstruction platform for three-dimensional structured illumination microscopy. We demonstrate its superior performance for artifact suppression and high-fidelity reconstruction relative to other algorithms on various specimens and over a range of signal-to-noise levels. Open-3DSIM also offers the capacity to extract dipole orientation, paving a new avenue for interpreting subcellular structures in six dimensions (xyzθλt). The platform is available as MATLAB code, a Fiji plugin and an Exe application to maximize user-friendliness.
Stimulated emission depletion (STED) microscopy allows high lateral and axial resolution, long term imaging in living cells. Here we review recent technical advances in STED microscopy, with emphasis on resolution and measurement range of XYZt four dimensions. Different STED technical advances and novel STED probes are discussed with their respective application in biological subcellular imaging. This review may serve as a practical guide for choosing a suitable approach to the advanced STED super‐resolution imaging.
The effect of cascade amplified depletion in lanthanide upconversion systems boosts their own emission bands inhibition, which facilitates multi-color nanoscopy with only one pair of low-power NIR CW lasers.
STimulated Emission Depletion (STED) microscopy attains super‐resolution in biological imaging beyond the diffraction limit. Here, we give a concise protocol to construct a dual‐pulse STED setup with one super‐continuum laser. Moreover, a flexible and dismountable Bessel modulation module is introduced for potential 2D‐stack STED imaging. Experiments and notices are introduced in detail, with discussion on some important check‐points for STED, such as detector saturation. Finally, the results validate the system working.
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