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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.

Metabolic labeling with clickable noncanonical amino acids has enabled nascent proteome profiling, which can be performed in a cell-type-specific manner. However, nascent proteomics in an intercellular communication-dependent manner remains challenging. Here we develop communication-activated profiling of protein expression (CAPPEX), which integrates the LuxI/LuxR quorum sensing circuit with the cell-type-specific nascent proteomics method to enable selective click-labeling of newly synthesized proteins in a specific bacterium upon receiving chemical signals from another reporter bacterium. CAPPEX reveals that E. coli competes with Salmonella for tryptophan as the precursor for indole, and the resulting indole suppressed the expression of virulence factors in Salmonella. This tryptophan-indole axis confers attenuation of Salmonella invasion in host cells and living mice. The CAPPEX strategy should be widely applicable for investigating various interbacterial communication processes.

We report here the development of clickable and highly near-infrared (NIR) fluorescent lanthanide (Ln) complexes for bioorthogonal labeling of biomolecules. These azide- or alkyne-functionalized Ln complexes are hydrophilic and fluorogenic, exhibiting a strong increase of NIR fluorescence upon conjugation with biomolecules. Metabolic labeling of biomolecules with azide or alkyne, followed by click labeling with the Ln complexes, enables NIR fluorescence (NIRF) imaging of DNA, RNA, proteins, and glycans in cells. Furthermore, multicolor imaging is performed by combining click-labeling with the Ln complexes and immunostaining. In addition, the Ln complexes is compatible with click-expansion microscopy (click-ExM), which enables high-resolution NIRF imaging of cellular glycoproteins. Finally, the Ln complexes can be used for time-of-flight secondary-ion mass spectrometry (ToF-SIMS) imaging, thus achieving the first example of dual-modal imaging combining NIRF and SIMS microscopies.

Mammalian cell cycle is a central process for tissue growth and maintenance. Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification has been found to occur on several important cell cycle regulators. However, the O-GlcNAcylated proteome has not been extensively profiled during cell cycle progression. Herein, we report a quantitative profiling of protein O-GlcNAcylation sites in cell proliferation, by using an O-GlcNAc chemoproteomic strategy. In HeLa cells, a total of 902, 439, and 872 high-confidence O-GlcNAcylation sites distributed on 414, 265, and 425 proteins are identified in the interphase, early mitosis, and mitotic exit stages, respectively. The identified O-GlcNAcylation events occur on a variety of important regulators, which are involved in the processes of cell division, DNA repair, and cell death. Furthermore, we show that O-GlcNAcylation is dynamically regulated in a cell cycle stage-dependent manner. Our results provide a valuable resource for investigating the functional roles of O-GlcNAc in the mammalian cell cycle.

Expansion microscopy (ExM) allows super-resolution imaging on conventional fluorescence microscopes, but has been limited to proteins and nucleic acids. Here we develop click-ExM, which integrates click labeling into ExM to enable a 'one-stop-shop' method for nanoscale imaging of various types of biomolecule. By click labeling with biotin and staining with fluorescently labeled streptavidin, a large range of biomolecules can be imaged by the standard ExM procedure normally used for proteins. Using 18 clickable labels, we demonstrate click-ExM on lipids, glycans, proteins, DNA, RNA and small molecules. We demonstrate that click-ExM is applicable in cell culture systems and for tissue imaging. We further show that click-ExM is compatible with signal-amplification techniques and two-color imaging. Click-ExM thus provides a convenient and versatile method for super-resolution imaging, which may be routinely used for cell and tissue samples.

Metabolic labeling of glycans with clickable unnatural sugars has enabled glycan analysis in multicellular systems. However, cell-type-specific labeling of glycans in vivo remains challenging. Here we develop genetically encoded metabolic glycan labeling (GeMGL), a cell-type-specific strategy based on a bump-and-hole pair of an unnatural sugar and its matching engineered enzyme. N-pentynylacetylglucosamine (GlcNAl) serves as a bumped analog of N-acetylglucosamine (GlcNAc) that is specifically incorporated into glycans of cells expressing a UDP-GlcNAc pyrophosphorylase mutant, AGX2

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.

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.

Expansion microscopy (ExM) allows super-resolution imaging on conventional fluorescence microscopes, but has been limited to proteins and nucleic acids. Here we develop click-ExM, which integrates click-labeling into ExM to enable a “one-stop-shop” method for nanoscale imaging of various types of biomolecules. Using 18 clickable labels for click-ExM imaging of DNA, RNA, proteins, lipids, glycans and small molecules, we demonstrate its universality, compatibility with signal-amplification techniques, and broad applications in cellular imaging.

With the growth of the Internet and the advancements in modern communication systems, sending data over the Internet or other media is now a commonplace practice. Consequently, it has become an important issue to secure the confidentiality and integrity of data because the data are sent via the Internet as a primary method of communication. This paper proposes a steganographic method based on the vector quantization coding compression algorithm with elastic indicators, which also incorporates a keystream to enhance security. Our experimental results have shown that the proposed method performs more effectively compared to the method proposed by Lee et al. in 2013.

Membrane voltage is an important biophysical signal that underlies intercellular electrical communications. A fluorescent voltage indicator is presented that enables the investigation of electrical signaling at high spatial resolution. The method is built upon the site-specific modification of microbial rhodopsin proteins with organic fluorophores, resulting in a hybrid indicator scaffold that is one of the most sensitive and fastest orange-colored voltage indicators developed to date. We applied this technique to optically map electrical connectivity in cultured cells, which revealed gap junction-mediated long-range coupling that spanned over hundreds of micrometers.

Metabolic labeling of glycans with bioorthogonal reporters has been widely used for glycan imaging and glycoproteomic profiling. One of the intrinsic limitations of metabolic glycan labeling is the lack of cell-type selectivity. The recently developed liposome-assisted bioorthogonal reporter (LABOR) strategy provides a promising means to overcome this limitation, but the mechanism of LABOR has not been investigated in detail. In this work, we performed a mechanistic study on LABOR and explored its multiplexing capability. Our studies support an endocytosis-salvage mechanism. The ligand-targeted liposomes encapsulating azidosugars are internalized into the endosome via the receptor-mediated endocytosis. Unlike the conventional drug delivery, LABOR does not rely on the endosomal escape pathways. Rather, the liposomes are allowed to enter the lysosome, inside which the azidosugars are released from the liposomes. The released azidosugars then intercept the salvage pathways of monosaccharides and get transported into the cytosol by lysosomal sugar transporters. Based on this mechanism, we expanded the scope of LABOR by evaluating a series of ligand-receptor pairs for targeting sialoglycans in various cell types. Different ligand types including small molecules, antibodies, aptamers, and peptides could be easily implemented into LABOR. Finally, we demonstrated that the sialoglycans in two distinct cell populations in a co-cultured system could be selectively labeled with two distinct chemical reporters by performing a multiplexed LABOR labeling.

Expansion microscopy (ExM) allows super-resolution imaging on conventional fluorescence microscopes, but has been limited to proteins and nucleic acids. Here we develop click-ExM, which integrates click labeling into ExM to enable a 'one-stop-shop' method for nanoscale imaging of various types of biomolecule. By click labeling with biotin and staining with fluorescently labeled streptavidin, a large range of biomolecules can be imaged by the standard ExM procedure normally used for proteins. Using 18 clickable labels, we demonstrate click-ExM on lipids, glycans, proteins, DNA, RNA and small molecules. We demonstrate that click-ExM is applicable in cell culture systems and for tissue imaging. We further show that click-ExM is compatible with signal-amplification techniques and two-color imaging. Click-ExM thus provides a convenient and versatile method for super-resolution imaging, which may be routinely used for cell and tissue samples.

With the continued expansion of the Internet and increased means of communication, it is inevitable that we send data via the Internet or other such media. The important issue of maintaining the confidentiality and integrity of such data when using the Internet as a primary mode of communication also continues to grow in scale. In this paper, we therefore propose an information-hiding method based on the vector quantization (VQ) compression algorithm with elastic indicators; further, we present our design for a key stream to enhance security. Our experimental results have shown that our proposed method performs better as compared to the method introduced by Lee et al. in 2013.

An ultrasensitive biosensor for carcinoembryonic antigen (CEA) was constructed based on fluorescence resonance energy transfer (FRET) between upconverting nanoparticles (UCPs) and palladium nanoparticles (PdNPs). PdNPs was synthesized by the addition of a solution of Na2PdCl4 into a mixture of N2H4·H2O as the reducing agent and 11-mercaptoundecanoic acid (MUDA) as the stabilizer. The CEA aptamer (5'-NH2-ATACCAGCTTATTCAATT-3') was conjugated to hexanedioic acid (HDA) modified UCPs (HDA-UCPs) through an EDC-NHS coupling protocol. The coordination interaction between nitrogen functional groups of the CEA aptamer and PdNPs brought UCPs and PdNPs in close proximity, which resulted in the fluorescence quenching of UCPs to an extent of 85%. And the non-specific fluorescence quenching caused by PdNPs towards HDA-UCPs was negligible. After the introduction of CEA into the UCPs-CEA aptamer-PdNPs fluorescence quenching system, the CEA aptamer preferentially combined with CEA accompanied by the conformational change which weakened the coordination interaction between the CEA aptamer and PdNPs. So fluorescence recovery of UCPs was observed and a linear relationship between the fluorescence recovery of UCPs and the concentration of CEA was obtained in the range from 2pg/mL to 100pg/mL in the aqueous buffer with the detection limit of 0.8pg/mL. The ultrasensitive detection of CEA was also realized in diluted human serum with a linear range from 4pg/mL to 100pg/mL and a detection limit of 1.7pg/mL. This biosensor makes the most of the high quenching ability of PdNPs towards UCPs with negligible non-specific fluorescence quenching and has broad application prospects in biochemistry.

An ultrasensitive biosensing platform for DNA and protein detection is constructed based on the luminescence quenching ability of plasmonic palladium nanoparticles (PdNPs). By growing the particles into large sizes (ca. 30 nm), the plasmonic light absorption of PdNPs is broadened and extended to the visible range with extinction coefficients as high as 10(9) L mol(-1) cm(-1) , enabling complete quenching of fluorescent dyes that emit at diverse ranges and that are tagged to bioprobes. Meanwhile the nonspecific quenching of the dyes (not bound to probes) is negligible, leading to extremely low background signal. Utilizing the affinity of PdNPs towards bioprobes, such as single-stranded (ss) DNA and polypeptide molecules, which is mainly assigned to the coordination interaction, nucleic acid assays with a quantification limit of 3 pM target DNA and protein assay are achieved with a simple mix-and-detect strategy based on the luminescence quenching-and-recovery protocol. This is the first demonstration of biosensing employing plasmonic absorption of nanopalladium, which offers pronounced sensing performances and can be reasonably expected for wide applications.

In the title complex, [Co(CHO2)2(C20H14N4)(H2O)2]n, the CoII ion, lying on an inversion center, is six-coordinated by two O atoms from two monodentate formate ligands, two N atoms from two 2,6-bis­(pyridin-4-yl)-4,4′-bipyridine (4-pybpy) ligands and two water mol­ecules, displaying an octa­hedral geometry. The 4-pybpy ligand, having a twofold rotation axis, functions in a bridging coordination mode, connecting the CoII ions into a corrugated chain along [\overline{1}01]. The chains are further linked into a three-dimensional supra­molecular network by O—H⋯O, C—H⋯N and C—H⋯O hydrogen bonds and π–π stacking inter­actions between the pyridine rings [centroid-to-centroid distance = 3.743 (2) A].