We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.

The combination of specific probes and advanced optical microscopy now allows quantitative probing of biochemical reactions in living cells. On selected systems, one can detect and track a particular protein with single-molecule sensitivity, nanometer spatial precision, and millisecond time resolution. Metabolites, usually difficult to detect, can be imaged and monitored in living cells with coherent anti-Stokes Raman scattering microscopy. Here, we describe the application of these techniques in studying gene expression, active transport, and lipid metabolism.

SummaryDormant bacterial spores are extraordinarily resistant to environmental insults and are vectors of various illnesses. However, spores cannot cause disease unless they germinate and become vegetative cells. The molecular details of initiation of germination are not understood, but proteins essential in early stages of germination, such as nutrient germinant receptors (GRs) and GerD, are located in the spore inner membrane. In this study, we examine how these germination proteins are organized in dormant Bacillus subtilis spores by expressing fluorescent protein fusions that were at least partially functional and observing spores by fluorescence microscopy. We show that GRs and GerD colocalize primarily to a single cluster in dormant spores, reminiscent of the organization of chemoreceptor signalling complexes in Escherichia coli. GRs require all their subunits as well as GerD for clustering, and also require diacylglycerol addition to GerD and GRs' C protein subunits. However, different GRs cluster independently of each other, and GerD forms clusters in the absence of all the GRs. We predict that the clusters represent a functional germination unit or 'germinosome' in the spore inner membrane that is necessary for rapid and cooperative response to nutrients, as conditions known to block nutrient germination also disrupt the protein clusters.

Fluorescence imaging in the second near-infrared window (NIR-II) is a new technique that permits visualization of deep anatomical features with unprecedented spatial resolution. Although attractive, effectively suppressing the interference signal of the background is still an enormous challenge for obtaining target-specific NIR-II imaging in the complex and dynamic physiological environment. Herein, dual-pathological-parameter cooperatively activatable NIR-II fluorescence nanoprobes (HISSNPs) are developed whereby hyaluronic acid chains and disulfide bonds act as the "double locks" to lock the fluorescence-quenched aggregation state of the NIR-II fluorescence dyes for performing ultrahigh specific imaging of tumors in vivo. The fluorescence can be lit up only when the "double locks" are opened by reacting with the "dual smart keys" (overexpressed hyaluronidase and thiols in tumor) simultaneously. In vivo NIR-II imaging shows that they reduce nonspecific activitation and achieve ultralow background fluorescence, which is 10.6-fold lower than single-parameter activatable probes (HINPs) in the liver at 15 h postinjection. Consequently, these "dual lock-and-key"-controlled HISSNPs exhibit fivefold higher tumor-to-normal tissue ratio than "single lock-and-key"-controlled HINPs at 24 h postinjection, attractively realizing ultrahigh specificity of tumor imaging. This is thought to be the first attempt at implementing ultralow background interference with the participation of multiple pathological parameters in NIR-II fluorescence imaging.

Using photoactivatable fluorescent protein as an intracellular protein label for single-molecule tracking offers several advantages over the traditional methods. Here we demonstrate the technique of photoactivation single-molecule tracking by investigating the mobility dynamics of intracellular FtsZ protein molecules in live Escherichia coli cells. FtsZ is a prokaryotic cytoskeleton protein (a homolog of tubulin) and plays important roles in cytokinesis. We demonstrate two heterogeneous subpopulations of FtsZ molecules with distinct diffusional dynamics. The FtsZ molecules forming the Z-rings near the center of the cell were mostly stationary, consistent with the assumption that they are within polymeric filamentous structures. The rest of the FtsZ molecules, on the other hand, undergo Brownian motion spanning the whole cell length. Surprisingly, the diffusion of FtsZ is spatially restricted to helical-shaped regions, implying an energy barrier for free diffusion. Consistently, the measured mean-square displacements of FtsZ showed anomalous diffusion characteristics. These results demonstrated the feasibility and advantages of photoactivation single-molecule tracking, and suggested new levels of complexity in the prokaryotic membrane organization.

Radicals, organic molecules with unpaired electrons, are applied across different scientific disciplines such as electronics, energy storage and biochemistry.

The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore's DPA once rapid DPA release had begun (⌬T release ) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for ⌬T release were generally 0.5 to 3 min at 25 to 37°C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination >15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T lag , between mixing spores with nutrient germinants and the beginning of ⌬T release . (iv) A number of factors decrease spores' T lag values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.Spores of various Bacillus species are metabolically dormant and can survive for years in this state (30). However, spores constantly sense their environment, and if appropriate small molecules termed germinants are present, spores can rapidly return to life in the process of germination followed by outgrowth (25,29,30). The germinants that most likely trigger spore germination in the environment are low-molecularweight nutrient molecules, the identities of which are strain and species specific, including amino acids, sugars, and purine nucleosides. Metabolism of these nutrient germinants is not needed for the triggering of spore germination. Rather, these germinants are recognized by germinant receptors (GRs) located in the spore's inner membrane that recognize their cognate germinants in a stereospecific manner (17,24,25,29). Spores have a number of such GRs, with three functional GRs in Bacillus subtilis spores and even more in Bacillus anthracis, Bacillus cereus, and Bacillus megaterium spores (6,29,30).Binding of nutrient germinants to some single GRs is sufficient to trigger spore germination, for example the triggering of B...

Our exploiting versatile multimodal theranostic agent aims to integrate the complementary superiorities of photoacoustic imaging (PAI), second near-infrared (NIR-II, 1000-1700) fluorescence and T 1 -weighted magnetic resonance imaging (MRI) with an ultimate objective of perfecting cancer diagnosis, thus improving cancer therapy efficacy. Herein, we engineered and prepared a water-soluble gadolinium-chelated conjugated polymer-based theranostic nanomedicine (PFTQ-PEG-Gd NPs) for in vivo tri-mode PA/MR/NIR-II imaging-guided tumor photothermal therapy (PTT). Methods : We firstly constructed a semiconducting polymer composed of low-bandgap donor-acceptor (D-A) which afforded the strong NIR absorption for PAI/PTT and long fluorescence emission to NIR-II region for in vivo imaging. Then, the remaining carboxyl groups of the polymeric NPs could effectively chelate with Gd 3+ ions for MRI. The in vitro characteristics of the PFTQ-PEG-Gd NPs were studied and the in vivo multimode imaging as well as anti-tumor efficacy of the NPs was evaluated using 4T1 tumor-bearing mice. Results : The obtained theranostic agent showed excellent chemical and optical stability as well as low biotoxicity. After 24 h of systemic administration using PQTF-PEG-Gd NPs, the tumor sites of living mice exhibited obvious enhancement in PA, NIR-II fluorescence and positive MR signal intensities. Better still, a conspicuous tumor growth restraint was detected under NIR light irradiation after administration of PQTF-PEG-Gd NPs, indicating the efficient photothermal potency of the nano-agent. Conclusion : we triumphantly designed and synthesized a novel and omnipotent semiconducting polymer nanoparticles-based theranostic platform for PAI, NIR-II fluorescence imaging as well as positive MRI-guided tumor PTT in living mice. We expect that such a novel organic nano-platform manifests a great promise for high spatial resolution and deep penetration cancer theranostics.