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Despite the prevalence of superresolution (SR) microscopy, quantitative live-cell SR imaging that maintains the completeness of delicate structures and the linearity of fluorescence signals remains an uncharted territory. Structured illumination microscopy (SIM) is the ideal tool for live-cell SR imaging. However, it suffers from an out-of-focus background that leads to reconstruction artifacts. Previous post hoc background suppression methods are prone to human bias, fail at densely labeled structures, and are nonlinear. Here, we propose a physical model-based Background Filtering method for living cell SR imaging combined with the 2D-SIM reconstruction procedure (BF-SIM). BF-SIM helps preserve intricate and weak structures down to sub-70 nm resolution while maintaining signal linearity, which allows for the discovery of dynamic actin structures that, to the best of our knowledge, have not been previously monitored.

A main determinant of the spatial resolution of live-cell super-resolution (SR) microscopes is the maximum photon flux that can be collected. To further increase the effective resolution for a given photon flux, we take advantage of a priori knowledge about the sparsity and continuity of biological structures to develop a deconvolution algorithm that increases the resolution of SR microscopes nearly twofold. Our method, sparse structured illumination microscopy (Sparse-SIM), achieves ~60-nm resolution at a frame rate of up to 564 Hz, allowing it to resolve intricate structures, including small vesicular fusion pores, ring-shaped nuclear pores formed by nucleoporins and relative movements of inner and outer mitochondrial membranes in live cells. Sparse deconvolution can also be used to increase the three-dimensional resolution of spinning-disc confocal-based SIM, even at low signal-to-noise ratios, which allows four-color, three-dimensional live-cell SR imaging at ~90-nm resolution. Overall, sparse deconvolution will be useful to increase the spatiotemporal resolution of live-cell fluorescence microscopy. The resolution of fluorescence microscopy is increased by incorporating prior information into deconvolution algorithms.

In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). The local quality of the images may vary on multiple scales and these differences can lead to misconceptions. However, current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) to quantify reconstruction quality down to the SR scale. To visually pinpoint regions with low reliability, we combine a filtered rFRC with a modified resolution scaled error map (RSM), providing a comprehensive and concise map for further examination. We demonstrate their performances on different SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Beyond that, as a model-independent assessment, we show its applications in evaluating the local restoration qualities for the deep-learning methods.

The emergence of super-resolution (SR) fluorescence microscopy has rejuvenated the search for new cellular sub-structures. However, SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment. Thus, we developed SR fluorescence-assisted diffraction computational tomography (SR-FACT), which combines label-free three-dimensional optical diffraction tomography (ODT) with two-dimensional fluorescence Hessian structured illumination microscopy. The ODT module is capable of resolving the mitochondria, lipid droplets, the nuclear membrane, chromosomes, the tubular endoplasmic reticulum, and lysosomes. Using dual-mode correlated live-cell imaging for a prolonged period of time, we observed novel subcellular structures named dark-vacuole bodies, the majority of which originate from densely populated perinuclear regions, and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane. This work demonstrates the unique capabilities of SR-FACT, which suggests its wide applicability in cell biology in general., Microscopy: New tool furthers our understanding of living cells Scientists from China have developed a new microscopy tool that could help to further expand our understanding of cell biological processes in live cells. Although the emergence of superresolution (SR) fluorescence microscopy is revolutionising the life sciences, the technique can only be used to observe a handful of biomolecules simultaneously and cannot provide a holistic map of the cellular landscape. Furthermore, the imaging of live cells in three dimensions (3D) over time also remains very challenging. By combining two different techniques, a team of Chinese researchers led by Liangyi Chen and Kebin Shi from Peking University has created a novel dual-mode high-speed SR microscopy technique. The tool is capable of revealing previously unseen sub-cellular structures and dynamic processes, allowing both the cellular landscape and molecular identity in live cells to be observed in 3D over prolonged periods.

Zhou et al. identify the mechanism of autophagosome (AP) closure. They show that Rab5 GTPase regulates an interaction between the ESCRT subunit Snf7 and Atg17 to bring ESCRT to APs where it catalyzes AP closure. These findings highlight the convergence of the endocytic and autophagic pathways at this step., In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17–Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17–Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

The imaging rate of structured illumination microscopy (SIM) reached 188 Hz recently. As the exposure time decreases, the camera detects fewer virtual photons, while the noise level remains the same. As a result, the signal-to-noise ratio (SNR) decreases sharply. Furthermore, the SNR decreases further because of photobleaching and phototoxicity. This decreased quality of SIM raw data may lead to surprising artifacts with various causes, which may confuse a new user of SIM microscopy. We summarize three significant possible sources of severe artifacts in reconstructed super-resolution (SR) images. Ultrafast motion of a biological sample or an uneven illumination pattern is the most difficult to be identified. The estimated parameter could also be incorrect, leading to artifact of regular patterns. Furthermore, reconstruction with the Wiener method generates stochastic artifacts due to the amplification of noise during the deconvolution process. To deal with these problems, we have established a protocol to reconstruct ultrafast SIM raw data obtained in low SNR conditions. First, we checked the quality of the raw data with the ImageJ plugin SIMcheck before reconstruction. Then, a modified parameter estimation method was used to improve the precision of the parameters. Finally, an iterative algorithm was used for SIM reconstruction under low signal-to-noise ratio conditions. This procedure effectively suppressed the artifacts in the super-resolution images reconstructed from raw data of low signal-to-noise ratio.

The human kinome comprises 538 kinases playing essential functions by catalyzing protein phosphorylation. Annotation of subcellular distribution of the kinome greatly facilitates investigation of normal and disease mechanisms. Here, we present Kinome Atlas (KA), an image-based map of the kinome annotated to 10 cellular compartments. 456 epitope-tagged kinases, representing 85% of the human kinome, were expressed in HeLa cells and imaged by immunofluorescent microscopy under a similar condition. KA revealed kinase family-enriched subcellular localizations and discovered a collection of new kinase localizations at mitochondria, plasma membrane, extracellular space, and other structures. Furthermore, KA demonstrated the role of liquid-liquid phase separation in formation of kinase condensates. Identification of MOK as a mitochondrial kinase revealed its function in cristae dynamics, respiration, and oxidative stress response. Although limited by possible mislocalization due to overexpression or epitope tagging, this subcellular map of the kinome can be used to refine regulatory mechanisms involving protein phosphorylation.

Among the hypomyelinating leukodystrophies, Pelizaeus–Merzbacher disease (PMD) is a representative disorder. The disease is caused by different types of PLP1 mutations, among which PLP1 duplication accounts for ~ 70% of the mutations. Previous studies have shown that PLP1 duplications lead to PLP1 retention in the endoplasmic reticulum (ER); in parallel, recent studies have demonstrated that PLP1 duplication can also lead to mitochondrial dysfunction. As such, the respective roles and interactions of the ER and mitochondria in the pathogenesis of PLP1 duplication are not clear. In both PLP1 patients’ and healthy fibroblasts, we measured mitochondrial respiration with a Seahorse XF Extracellular Analyzer and examined the interactions between the ER and mitochondria with super-resolution microscopy (spinning-disc pinhole-based structured illumination microscopy, SD-SIM). For the first time, we demonstrated that PLP1 duplication mutants had closer ER-mitochondrion interfaces mediated through structural and morphological changes in both the ER and mitochondria-associated membranes (MAMs). These changes in both the ER and mitochondria then led to mitochondrial dysfunction, as reported previously. This work highlights the roles of MAMs in bridging PLP1 expression in the ER and pathogenic dysfunction in mitochondria, providing novel insight into the pathogenicity of mitochondrial dysfunction resulting from PLP1 duplication. These findings suggest that interactions between the ER and mitochondria may underlie pathogenic mechanisms of hypomyelinating leukodystrophies diseases at the organelle level.

The spatial resolutions of live-cell super-resolution microscopes are limited by the maximum collected photon flux. Taking advantage of a priori knowledge of the sparsity and continuity of biological structures, we develop a deconvolution algorithm that further extends the resolution of super-resolution microscopes under the same photon budgets by nearly twofold. As a result, sparse structured illumination microscopy (Sparse-SIM) achieves ~60 nm resolution at a 564 Hz frame rate, allowing it to resolve intricate structural intermediates, including small vesicular fusion pores, ring-shaped nuclear pores formed by different nucleoporins, and relative movements between the inner and outer membranes of mitochondria in live cells. Likewise, sparse deconvolution can be used to increase the three-dimensional resolution and contrast of spinning-disc confocal-based SIM (SD-SIM), and operates under conditions with the insufficient signal-to-noise-ratio, all of which allows routine four-color, three-dimensional, ~90 nm resolution live-cell super-resolution imaging. Overall, sparse deconvolution may be a general tool to push the spatiotemporal resolution limits of live-cell fluorescence microscopy.

Impaired insulin release is a hallmark of type 2 diabetes and is closely related to chronically elevated glucose concentrations, known as “glucotoxicity.” However, the molecular mechanisms by which glucotoxicity impairs insulin secretion remain poorly understood. In addition to known kiss-and-run and kiss-and-stay fusion events in INS-1 cells, ultrafast Hessian structured illumination microscopy (Hessian SIM) enables full fusion to be categorized according to the newly identified structures, such as ring fusion (those with enlarged pores) or dot fusion (those without apparent pores). In addition, we identified four fusion intermediates during insulin exocytosis: initial pore opening, vesicle collapse, enlarged pore formation, and final pore dilation. Long-term incubation in supraphysiological doses of glucose reduced exocytosis in general and increased the occurrence of kiss-and-run events at the expense of reduced full fusion. In addition, hyperglycemia delayed pore opening, vesicle collapse, and enlarged pore formation in full fusion events. It also reduced the size of apparently enlarged pores, all of which contributed to the compromised insulin secretion. These phenotypes were mostly due to the hyperglycemia-induced reduction in syntaxin-1A (Stx-1A) and SNAP-25 protein, since they could be recapitulated by the knockdown of endogenous Stx-1A and SNAP-25. These findings suggest essential roles for the vesicle fusion type and intermediates in regulating insulin secretion from pancreatic beta cells in normal and disease conditions.

The emergence of super-resolution (SR) fluorescence microscopy has rejuvenated the search for new cellular sub-structures. However, SR fluorescence microscopy achieves high contrast at the cost of the lack of a holistic view of their interacting partners and surrounding environment. Thus we develop SR fluorescence-assisted diffraction computational tomography (SR-FACT), which combines label-free three-dimensional optical diffraction tomography (ODT) with two-dimensional fluorescence Hessian structured illumination microscopy. The ODT module is capable of resolving mitochondria, lipid droplets, the nuclear membrane, chromosomes, the tubular endoplasmic reticulum and lysosomes. Using dual-mode correlated live cell imaging for prolonged period of time, we observe the dynamics of a novel subcellular structure named dark-vacuole bodies. These works demonstrate the unique capabilities of SR-FACT, which suggest its wide applicability in cell biology in general.

Cytoskeletal‐based molecular motors produce force perpendicular to their direction of movement. However, it remains unknown whether and why motor proteins generate sidesteps movement along their filamentous tracks in vivo. Using Hessian structured illumination microscopy, we located green fluorescent protein (GFP)‐labeled intraflagellar transport (IFT) particles inside sensory cilia of live Caenorhabditis elegans with 3–6‐nanometer accuracy and 3.4‐ms resolution. We found that IFT particles took sidesteps along axoneme microtubules, demonstrating that IFT motors generate torque in a living animal. Kinesin‐II and OSM‐3‐kinesin collaboratively drive anterograde IFT. We showed that the deletion of kinesin‐II, a torque‐generating motor protein, reduced sidesteps, whereas the increase of neck flexibility of OSM‐3‐kinesin upregulated sidesteps. Either increase or decrease of sidesteps of IFT kinesins allowed ciliogenesis to the regular length, but changed IFT speeds, disrupted axonemal ninefold symmetry, and inhibited sensory cilia‐dependent animal behaviors. Thus, an optimum level of IFT kinesin sidestepping is associated with the structural and functional fidelity of cilia.

A main determinant of the spatial resolution of live-cell super-resolution (SR) microscopes is the maximum photon flux that can be collected. To further increase the effective resolution for a given photon flux, we take advantage of a priori knowledge about the sparsity and continuity of biological structures to develop a deconvolution algorithm that increases the resolution of SR microscopes nearly twofold. Our method, sparse structured illumination microscopy (Sparse-SIM), achieves ~60-nm resolution at a frame rate of up to 564 Hz, allowing it to resolve intricate structures, including small vesicular fusion pores, ring-shaped nuclear pores formed by nucleoporins and relative movements of inner and outer mitochondrial membranes in live cells. Sparse deconvolution can also be used to increase the three-dimensional resolution of spinning-disc confocal-based SIM, even at low signal-to-noise ratios, which allows four-color, three-dimensional live-cell SR imaging at ~90-nm resolution. Overall, sparse deconvolution will be useful to increase the spatiotemporal resolution of live-cell fluorescence microscopy.

To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.

Despite the wide application of super-resolution (SR) microscopy in biological studies of cells, the technology is rarely used to monitor functional changes in live cells. By combining fast spinning disc-confocal structured illumination microscopy (SD-SIM) with loading of cytosolic fluorescent Ca2+ indicators, we have developed an SR method for visualization of regional Ca2+ dynamics and related cellular organelle morphology and dynamics, termed SR calcium lantern imaging. In COS-7 cells stimulated with ATP, we have identified various calcium macrodomains characterized by different types of Ca2+ release from endoplasmic reticulum (ER) stores. Finally, we demonstrated various roles of mitochondria in mediating calcium signals from different sources; while mitochondria can globally potentiate the Ca2+ entry associated with store release, mitochondria also locally control Ca2+ release from the neighboring ER stores and assist in their refilling processes.

Many mitochondria-related diseases are associated with the mutation of mitochondrial DNA (mtDNA). Therefore, visualizing its dynamics in live cells is essential for the understanding of the function of mtDNA transcription and translation. By employing carbazole as the framework and designing a module for DNA minor-groove binding, here we have developed a novel fluorescent probe with a large Stokes shift (λab = 480 nm and λem = 620 nm), CNQ, for mtDNA detection and visualization. It is almost nonfluorescent in PBS buffer and exhibits 182-fold enhancement in fluorescence within 20 s after the application of mtDNA in the solution, with a detection limit of 55.1 μg/L. Using dual-color Hessian-structured illumination microscopy, we have demonstrated that CNQ-labeled mtDNA structures are distinct from those labeled by TFAM-EGFP. Finally, we have used two-photon confocal scanning microscopy (λex = 850 nm) to monitor the nondestructive doxorubicin-induced mtDNA damage in live cells.

To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM. Hessian-SIM enables spatiotemporal resolution of 88 nm and 188 Hz, and hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.

Total internal reflection fluorescence microscopy (TIRF microscopy) uses a rapid decay of evanescent waves to excite fluorophores within several hundred nanometers (nm) beneath the plasma membrane, which can effectively suppress excitation of fluorescence signals in the deep layers. From image stacks obtained with a plurality of different incident angles, a three-dimensional spatial structure of the observed sample can be reconstructed by a Multi-Angle-TIRF (MA-TIRF) algorithm that provides an axial resolution of ~50 nm. Taking into account the point spread function (PSF) of the TIRF microscopes, we further increase its lateral resolution by introducing a fast deconvolution algorithm into the reconstruction of MA-TIRF data (DMA-TIRF), which is approached in just one step of minimizing the reconstruction function. We also introduce a TV regularization term in the deconvolution algorithm to suppress artifacts induced by the excessive noise. Therefore, based on the hardware of existing MA-TIRF microscopes, the proposed DMA-TIRF algorithm has achieved lateral and axial resolutions of ~200 and ~50 nm, respectively.

The crystal-field splitting levels in YPO4 Eu3+ micro-crystals are investigated by the Zeeman splitting in photoluminescence spectrum. X-ray diffraction pattern and microscopic images indicate small tetragonal nano-particles attaching on the surfaces of the mainly hexagonal micro-crystals. The peaks correspond to the transitions from 5D0 excited state to 7F1 and 7F2 ground states are split in pulsed high magnetic fields, crystal-field splitting levels associated with different magnetic moments are clarified, which are completely different from that was supposed by the X-ray, microscopy, and zero-field photoluminescence results.

We have synthesized compounds of Ca3Co2−xFexO6 (x = 0–1.5) by sol–gel reaction method. X-ray diffraction data show that the solubility limit of Fe in the spin-chain compound Ca3Co2O6 is about x = 0.7. For the rhombohedral ( R 3 ¯ c ) samples with x ≤ 0.7, the upward deviation from the paramagnetic Curie–Weiss law in the inverse magnetic susceptibility (H/M) tends to be suppressed at x = 0.7. Correlatively, the paramagnetic Curie temperature θP approaches zero at this concentration. The M(H) curves exhibit a well-defined ferrimagnetic state, which becomes smeared out with increasing Fe concentration and nearly disappears at x = 0.7. For the triclinic ( P 1 ¯ ) samples with x = 1.1, the magnetization is much small and its ground state is antiferromagnetic. The H/M curve measured up to 300 K does not follow the Curie–Weiss law. Finally, ESR spectra composed of eight hyperfine splittings due to Co nuclear spin are observed for x = 0.4.

The electrical transport and magnetization measurements have been carried out on Al-doped polycrystalline intermetallic compounds Dy 50− x Al x Ag 50 ( x = 0, 0.3, 0.6, 1.2, 1.8) in a pulsed high magnetic field, in which multi-step magnetization is observed. Partial substitute of non-magnetic Al 3+ for Dy 3+ ions in the compounds increases the critical magnetic fields and the relative area of the magnetic hysteresis loop, which result from the pinning effect, lattice distortion, the change of coupling strength and dilution effect related to the Al 3+ doping.The experimental results indicate that non-magnetic Al 3+ ions and induced amorphous phase can pin the rotation and/or growth of magnetic domains, thus, the critical magnetic field can be enhanced by doping non-magnetic ions in the magnetic materials, especially in the permanent magnet materials.

The magnetization properties of (Tb0.2Pr0.8)(Fe0.4Co0.6)1.9−x C x ( $x=0$ –0.5) were investigated in a pulsed high magnetic field. Obvious unsymmetry magnetic hysteresis loops were observed, in which the magnetization measured in the second(negative) magnetic field route was larger than that measured in the first(positive) one. Meanwhile, the unsymmetry of the hysteresis loops was C doping levels, magnetic fields and temperatures dependent. The experimental results suggest that the unusual hysteresis phenomenon result mainly from the collapse of the pinning center and the formation of ferromagnetic(FM) pseudo-domain.

We describe and analyze the inverse Gaussian apodization among the three co-directional guided modes using the supermodes theory and the transfer matrix method. The propagation condition is related to the two phase matching distribution induced by two long period co-spatial gratings. It is seen that one input mode can be converted into another two modes with each mode having two peaks at its transmission spectrum. Through adopting the inverse Gaussian apodization, the input mode A turns to having two peaks while the converted mode B has six peaks and mode C has three peaks around the central frequency in the transmission spectrum. Of particular interest is when adjusting the maximum coupling apodization strength from 100 to 1500, the frequency space in mode A changes nearly linearly from 340 GHz to 5100 GHz which is twice of the mode B at each coupling strength, while the mode C can be turned nearly linearly from 350 GHz to 5200 GHz. Additionally, the power at the central frequency of input mode A will almost totally move to the converted mode C. This kind of design can form the basis for a new generation of multi-wavelength-tunable bandpass optical filtering mode converting, add/drop multiplexing and optical switching.

[1] The heterogeneous oxidation of SO2 by O3 on NaCl particles has been studied using diffuse reflectance infrared Fourier transform spectroscopy. The formation of sulfite and sulfate onthe surface wasidentified, andthe rolesofO3andwater inthe oxidation processes were determined. The results showed that in the presence of O3 ,S O2 could be oxidized to sulfate on the surface of NaCl particles. The reaction is first order in O3 and zero order in SO2. The initial reactive uptake coefficient for SO2 [(0.6–9.8)� 10 14 molecule cm � 3 ] oxidation by O3 [(1.2–12)� 10 14 molecule cm � 3 ] was determined to be (4.8–0.7)� 10 � 8 using the Brunauer-Emmett-Teller area as the reactive area and (9.8–1.4)� 10 � 5 using the geometric area at 40% relative humidity. A three-stage mechanism that involves the adsorption of O3 results in an alkalescent surface, the adsorption of SO2 followed by O3 oxidation is proposed, and the adsorption of O3 on the NaCl surface is the rate-determining step. The proposed mechanism can well explain the experiment results. Furthermore, the surface oxidation on mixtures of NaCl with other components such as CaCO3 ,A l2O3 ,T iO2, MgCl2 6H2O, MgO, elemental carbon, and soot were studied. The reactivity of mixtures can be predicted from the reactivity of the single component with each component weighted by its abundance in the mixture. The catalytic and basic additives could enhance the production of sulfate on the NaCl surface.

The mitigation of intra-channel nonlinearities in 10 Gbaud coherent NRZ-QPSK systems via a nonlinear electrical equaliser based on Volterra theory is experimentally demonstrated. Without the requirement of prior knowledge of the fibre link, the equaliser significantly improves the system performance compared to digital dispersion compensation only.