Nanoscale imaging of quantum dot dimers using time-resolved super-resolution microscopy combined with scanning electron microscopy.

Time-resolved super-resolution microscopy was used in conjunction with scanning electron microscopy to image individual colloidal CdSe/CdS semiconductor quantum dots (QD) and QD dimers. The photoluminescence (PL) lifetimes, intensities, and structural parameters were acquired with nanometer scale spatial resolution and sub-nanosecond time resolution. The combination of these two techniques was more powerful than either alone, enabling us to resolve the PL properties of individual QDs within QD dimers as they blinked on and off, measure interparticle distances, and identify QDs that may be participating in energy transfer. The localization precision of our optical imaging technique was ∼3 nm, low enough that the emission from individual QDs within the dimers could be spatially resolved. While the majority of QDs within dimers acted as independent emitters, at least one pair of QDs in our study exhibited lifetime and intensity behaviors consistent with resonance energy transfer from a shorter lifetime and lower intensity donor QD to a longer lifetime and higher intensity acceptor QD. For this case, we demonstrate how the combined super-resolution optical imaging and scanning electron microscopy data can be used to characterize the energy transfer rate.

Increased Mortality in Mice following Immunoprophylaxis Therapy with High Dosage of Nicotinamide in Burkholderia Persistent Infections.

Bacterial persistence, known as noninherited antibacterial resistance, is a factor contributing to the establishment of long-lasting chronic bacterial infections. In this study, we examined the ability of nicotinamide (NA) to potentiate the activity of different classes of antibiotics against Burkholderia thailandensis persister cells. Here we demonstrate that addition of NA in in vitro models of B. thailandensis infection resulted in a significant depletion of the persister population in response to various classes of antibiotics. We applied microfluidic bioreactors with a continuous medium flow to study the effect of supplementation with an NA gradient on the recovery of B. thailandensis persister populations. A coculture of human neutrophils preactivated with 50 µM NA and B. thailandensis resulted in the most efficient reduction in the persister population. Applying single-cell RNA fluorescence in situ hybridization analysis and quantitative PCR, we found that NA inhibited gene expression of the stringent response regulator relA, implicated in the regulation of the persister metabolic state. We also demonstrate that a therapeutic dose of NA (250 mg/kg of body weight), previously applied as immunoprophylaxis against antibiotic-resistant bacterial species, produced adverse effects in an in vivo murine model of infection with the highly pathogenic bacterium Burkholderia pseudomallei, indicating that therapeutic dose and metabolite effects have to be carefully evaluated and tailored for every case of potential clinical application.

DNA Templated Metal Nanoclusters: From Emergent Properties to Unique Applications.

Metal nanoclusters containing a few to several hundred atoms with sizes ranging from sub-nanometer to ∼2 nm occupy an intermediate size regime that bridges larger plasmonic nanoparticles and smaller metal complexes. With strong quantum confinement, metal nanoclusters exhibit molecule-like properties. This Account focuses on noble metal nanoclusters that are synthesized within a single stranded DNA template. Compared to other ligand protected metal nanoclusters, DNA-templated metal nanoclusters manifest intriguing physical and chemical properties that are heavily influenced by the design of DNA templates. For example, DNA-templated silver nanoclusters can show bright fluorescence, tunable emission colors, and enhanced stability by tuning the sequence of the encapsulating DNA template. DNA-templated gold nanoclusters can also serve as excellent cocatalysts, which are integratable with other biocatalysts such as enzymes. In this Account, DNA-templated silver and gold nanoclusters are selected as paradigm systems to showcase their emergent properties and unique applications. We first discuss the DNA-templated silver nanoclusters with a focus on the creation of a complementary palette of emission colors, which has potential applications for multiplex assays. The importance of the DNA template toward enhanced stability of silver nanoclusters is also demonstrated. We then introduce a special class of activable fluorescence probes that are based on the fluorescence turn-on phenomena of DNA-templated silver nanoclusters, which are named nanocluster beacons (NCBs). NCBs have distinct advantages over molecular beacons for nucleic acid detection, and their emission mechanisms are also discussed in detail. We then discuss a universal method of creating novel DNA-silver nanocluster aptamers for protein detection with high specificity. The remainder of the Account is devoted to the DNA-templated gold nanoclusters. We demonstrate that DNA-gold nanoclusters can serve as enhancers for enzymatic reduction of oxygen, which is one of the most important reactions in biofuel cells. Although DNA-templated metal nanoclusters are still in their infancy, we anticipate they will emerge as a new type of functional nanomaterial with wide applications in biology and energy science. Future research will focus on the synthesis of size selected DNA-metal nanoclusters with atomic monodispersity, structural determination of different sized DNA-metal nanoclusters, and establishment of structure-property correlations. Some long-standing mysteries, such as the origin of fluorescence and mechanism for emission color tunability, constitute the central questions regarding the photophysical properties of DNA-metal nanoclusters. On the application side, more studies are required to understand the interaction between nanocluster and biological systems. In the foreseeable future, one can expect that new biosensors, catalysts, and functional devices will be invented based on the intriguing properties of well-designed DNA-metal nanoclusters and their composites. Overall, DNA-metal nanoclusters can add additional spotlights into the highly vibrant field of ligand protected, quantum sized metal nanoclusters.

Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements.

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of "burst" confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering it within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope's multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope's capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.

3-Dimensional Tracking of Non-blinking 'Giant' Quantum Dots in Live Cells.

While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or 'blinking,' which causes ambiguity in particle trajectory analysis and limits tracking duration. Here, non-blinking 'giant' quantum dots (gQDs) are exploited to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3D SPT microscope. There is a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to commercially available QDs. A time-gated photon-pair correlation analysis is implemented to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows the observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which are quantified using a time-dependent diffusion coefficient and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, especially in cases where changes in cellular dynamics are occurring on the time scale of several minutes.

Counting small RNA in pathogenic bacteria.

Here, we present a modification to single-molecule fluorescence in situ hybridization that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (~200 nucleotide) nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 0 and 10 transcripts. The numbers of these RNA are both increased (by a factor of 2.5× for YSR35 and 3.5× for YSP8) upon a temperature shift from 25 to 37 °C, suggesting they play a role in pathogenesis. The copy number distribution of sRNAs from bacteria-to-bacteria are well-fit with a bursting model of gene transcription. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in cellular regulatory networks.

Disorder limited exciton transport in colloidal single-wall carbon nanotubes.

We present measurements of S(1) exciton transport in (6,5) carbon nanotubes at room temperature in a colloidal environment. Exciton diffusion lengths associated with end quenching paired with photoluminescence lifetimes provide a direct basis for determining a median diffusion constant of approximately 7.5 cm(2)s(-1). Our experimental results are compared to model diffusion constants calculated using a realistic exciton dispersion accounting for a logarithmic correction due to the exchange self-energy and a nonequilibrium distribution between bright and dark excitons. The intrinsic diffusion constant associated with acoustic phonon scattering is too large to explain the observed diffusion length, and as such, we attribute the observed transport to disorder-limited diffusional transport associated with the dynamics of the colloidal interface. In this model an effective surface potential limits the exciton mean free path to the same size as that of the exciton wave function, defined by the strength of the electron-hole Coulomb interaction.

A fluorescence light-up Ag nanocluster probe that discriminates single-nucleotide variants by emission color.

Rapid and precise screening of small genetic variations, such as single-nucleotide polymorphisms (SNPs), among an individual's genome is still an unmet challenge at point-of-care settings. One crucial step toward this goal is the development of discrimination probes that require no enzymatic reaction and are easy to use. Here we report a new type of fluorescent molecular probe, termed a chameleon NanoCluster Beacon (cNCB), that lights up into different colors upon binding SNP targets. NanoCluster Beacons (NCBs) are collections of a small number of Ag atoms templated on single-stranded DNA that fluoresce strongly when placed in proximity to particular DNA sequences, termed enhancers. Here we show the fluorescence emission color of a NCB can change substantially (a shift of 60-70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. Chameleon NCBs exploit this color shift to directly detect SNPs, based on the fact that different SNPs produce a different alignment between the Ag nanocluster and the enhancer. This SNP detection method has been validated on all single-nucleotide substitution scenarios in three synthetic DNA targets, in six disease-related SNP targets, and in two clinical samples taken from patients with ovarian serous borderline tumors. Samples with single-nucleotide variations can be easily identified by the naked eye under UV excitation, making this method a reliable and low-cost assay with a simple readout format.

Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria.

Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial-fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence in situ hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial-fungal relationships.

A DNA--silver nanocluster probe that fluoresces upon hybridization.

DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that "lights up" upon target binding. Since NCBs do not rely on Forster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers.

Time-resolved three-dimensional molecular tracking in live cells.

We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 ms to follow three-dimensional molecular motion. This method has substantial advantages over three-dimensional molecular tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 µm demonstrated here), substantially lower excitation powers (15 µW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon antibunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye-labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatiotemporal dynamics of live cells, we follow individual QD-labeled IgE-FcεRI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell.

A gain series method for accurate EMCCD calibration.

Calibration of the gain and digital conversion factor of an EMCCD is necessary for accurate photon counting. We present a new method to quickly calibrate multiple gain settings of an EMCCD camera. Acquiring gain-series calibration data and analyzing the resulting images with the EMCCD noise model more accurately estimates the gain response of the camera. Furthermore, we develop a method to compare the results from different calibration approaches. Gain-series calibration outperforms all other methods in this self-consistency test.

Widespread bacterial diversity within the bacteriome of fungi.

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

3D particle transport in multichannel microfluidic networks with rough surfaces.

The transport of particles and fluids through multichannel microfluidic networks is influenced by details of the channels. Because channels have micro-scale textures and macro-scale geometries, this transport can differ from the case of ideally smooth channels. Surfaces of real channels have irregular boundary conditions to which streamlines adapt and with which particle interact. In low-Reynolds number flows, particles may experience inertial forces that result in trans-streamline movement and the reorganization of particle distributions. Such transport is intrinsically 3D and an accurate measurement must capture movement in all directions. To measure the effects of non-ideal surface textures on particle transport through complex networks, we developed an extended field-of-view 3D macroscope for high-resolution tracking across large volumes ([Formula: see text]) and investigated a model multichannel microfluidic network. A topographical profile of the microfluidic surfaces provided lattice Boltzmann simulations with a detailed feature map to precisely reconstruct the experimental environment. Particle distributions from simulations closely reproduced those observed experimentally and both measurements were sensitive to the effects of surface roughness. Under the conditions studied, inertial focusing organized large particles into an annular distribution that limited their transport throughout the network while small particles were transported uniformly to all regions.

Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation.

The heterogeneity of mRNA and protein expression at the single-cell level can reveal fundamental information about cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of genes. Here we describe a fully automated system to digitally count the intron, mRNA, and protein content of up to five genes of interest simultaneously in single-cells. Full system automation of 3D microscope scans and custom image analysis routines allows hundreds of individual cells to be automatically segmented and the mRNA-protein content to be digitally counted. Single-molecule intron and mRNA content is measured by single-molecule fluorescence in-situ hybridization (smFISH), while protein content is quantified though the use of antibody probes. To mimic immune response to bacterial infection, human monocytic leukemia cells (THP-1) were stimulated with lipopolysaccharide (LPS), and the expression of two inflammatory genes, IL1ß (interleukin 1ß) and TNF-α (tumor necrosis factor α), were simultaneously quantified by monitoring the intron, mRNA, and protein levels over time. The simultaneous labeling of cellular content allowed for a series of correlations at the single-cell level to be explored, both in the progressive maturation of a single gene (intron-mRNA-protein) and comparative analysis between the two immune response genes. In the absence of LPS stimulation, mRNA expression of IL1ß and TNF-α were uncorrelated. Following LPS stimulation, mRNA expression of the two genes became more correlated, consistent with a model in which IL1ß and TNF-α upregulation occurs in parallel through independent mechanistic pathways. This smFISH methodology can be applied to different complex biological systems to provide valuable insight into highly dynamic gene mechanisms that determine cell plasticity and heterogeneity of cellular response.

Confocal, three-dimensional tracking of individual quantum dots in high-background environments.

We demonstrate a custom confocal fluorescence-microscope that is capable of tracking individual quantum dots undergoing three-dimensional Brownian motion (diffusion coefficient approximately 0.5 microm(2)/s) in environments with a signal-to-background ratio as low as 2:1, significantly worse than observed in a typical cellular environment. By utilizing a pulsed excitation source and time-correlated single photon counting, the time-resolved photon stream can be used to determine changes in the emission lifetime as a function of position and positively identify single quantum dots via photon-pair correlations. These results indicate that this microscope will be capable of following protein and RNA transport throughout the full three-dimensional volume of a live cell for durations up to 15 s.

Measuring an antibody affinity distribution molecule by molecule.

Single molecule fluorescence microscopy was used to observe the binding and unbinding of hapten decorated quantum dots to individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

Probing dimerization and intraprotein fluorescence resonance energy transfer in a far-red fluorescent protein from the sea anemone Heteractis crispa.

Proteins from Anthozoa species are homologous to the green fluorescent protein (GFP) from Aequorea victoria but with absorption/emission properties extended to longer wavelengths. HcRed is a far-red fluorescent protein originating from the sea anemone Heteractis crispa with absorption and emission maxima at 590 and 650 nm, respectively. We use ultrasensitive fluorescence spectroscopic methods to demonstrate that HcRed occurs as a dimer in solution and to explore the interaction between chromophores within such a dimer. We show that red chromophores within a dimer interact through a Forster-type fluorescence resonance energy transfer (FRET) mechanism. We present spectroscopic evidence for the presence of a yellow chromophore, an immature form of HcRed. This yellow chromophore is involved in directional FRET with the red chromophore when both types of chromophores are part of one dimer. We show that by combining ensemble and single molecule methods in the investigation of HcRed, we are able to sort out subpopulations of chromophores with different photophysical properties and to understand the mechanism of interaction between such chromophores. This study will help in future quantitative microscopy investigations that use HcRed as a fluorescent marker.

The creation of a novel fluorescent protein by guided consensus engineering.

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.

Site-specific dimensions across a highly denatured protein; a single molecule study.

Do highly denatured proteins adopt random coil configurations? Here, we address this question by measuring residue-to-residue separations across the denatured FynSH3 domain. Using single-molecule Forster resonance energy transfer techniques, we have collected transfer efficiency probability distributions for dye-labeled, denatured protein. Applying maximum likelihood analysis to the interpretation of these distributions, we have determined the through-space distance between five residue pairs in the protein's guanidine hydrochloride-unfolded and trifluoroethanol-unfolded states. We find that, while the dimensions of the guanidine hydrochloride -unfolded molecule generally coincide with the dimensions predicted for a random coil ensemble, potentially statistically significant deviations from random coil behavior are also evident. These small, site-specific deviations may provide a means of reconciling earlier, scattering-based evidence for the random coil nature of the unfolded state with more site-specific spectroscopic evidence suggesting residual structure. We have also studied the unfolded ensemble populated in 50% trifluoroethanol, a denaturant that induces a highly helical unfolded state. We find that the size and shape of the unfolded ensemble under these conditions is effectively indistinguishable from that populated in guanidinium hydrochloride solutions, suggesting that the gross structure of the denatured state is, perhaps surprisingly, independent of the chemistry of the cosolvent.