Single-molecule Labeling Research Articles

Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin

We present phalloidin-based points accumulation for imaging in nanoscale topography (phalloidin-PAINT), enabling quantitative superresolution imaging of filamentous actin (F-actin) in the cell body and delicate membrane protrusions. We demonstrate that the intrinsic phalloidin dissociation enables PAINT superresolution microscopy in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-PAINT are its ability to consistently quantify F-actin at the nanoscale throughout the entire cell and its enhanced preservation of fragile cellular structures. In a proof-of-concept study, we employed phalloidin-PAINT to superresolve F-actin structures in U2OS and dendritic cells (DCs). We demonstrate more consistent F-actin quantification in the cell body and structurally delicate membrane protrusions of DCs compared with direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse DCs as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane protrusions on the culture glass surface after lipopolysaccharide exposure. The concept of our work opens new possibilities for quantitative protein-specific PAINT using commercially available reagents.

Single-molecule labeling achieves enhanced nanoscale imaging of F-actin in fragile membrane structures of dendritic cells

Single-molecule labeling achieves enhanced nanoscale imaging of F-actin in fragile membrane structures of dendritic cells

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

Failure to Thrive: Impaired BDNF Transport along the Cortical–Striatal Axis in Mouse Q140 Neurons of Huntington’s Disease

Boosting trophic support to striatal neurons by increasing levels of brain-derived neurotrophic factor (BDNF) has been considered as a target for therapeutic intervention for several neurodegenerative diseases, including Huntington's disease (HD). To aid in the implementation of such a strategy, a thorough understanding of BDNF cortical-striatal transport is critical to help guide its strategic delivery. In this manuscript, we investigate the dynamic behavior of BDNF transport along the cortical-striatal axis in Q140 primary neurons, a mouse model for HD. We examine this by using single-molecule labeling of BDNF conjugated with quantum dots (QD-BDNF) to follow the transport along the cortical-striatal axis in a microfluidic chamber system specifically designed for the co-culture of cortical and striatal primary neurons. Using this approach, we observe a defect of QD-BDNF transport in Q140 neurons. Our study demonstrates that QD-BDNF transport along the cortical-striatal axis involves the impairment of anterograde transport within axons of cortical neurons, and of retrograde transport within dendrites of striatal neurons. One prominent feature we observe is the extended pause time of QD-BDNF retrograde transport within Q140 striatal dendrites. Taken together, these finding support the hypothesis that delinquent spatiotemporal trophic support of BDNF to striatal neurons, driven by impaired transport, may contribute to the pathogenesis of HD, providing us with insight into how a BDNF supplementation therapeutic strategy may best be applied for HD.

Classification of DNA-FRET based single-molecule spectroscopic labels in anti-Brownian electrokinetic (ABEL) trap

Fluorescent tags are usually distinguished by their color, making fluorescence multiplexing at the single-molecule level challenging since only a few tags can be distinguished due to the overlap in their emission spectra and the low signal-to-noise ratios inherent in single-molecule measurements. We have engineered a series of spectroscopically distinguishable single-molecule labels whose emission is characterized by unique combinations of photophysical parameters including brightness, lifetime, and emission spectra.

Advances in Optical Single-Molecule Detection: En Route to Supersensitive Bioaffinity Assays.

The ability to detect low concentrations of analytes and in particular low‐abundance biomarkers is of fundamental importance, e.g., for early‐stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single‐molecule bioaffinity assays. While many review articles have highlighted the potentials of single‐molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real‐world applications as one should expect. This Review provides a theoretical background on single‐molecule—or better digital—assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single‐molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.

Single-Molecule Fluorescence Microscopy for Probing the Electrochemical Interface.

The electrochemical interface is an ultrathin interfacial region between the electrode and solution where electrochemical reactions occur. The study of the electrochemical interface continues to be one of the most exciting directions in modern electrochemistry research. Much of our existing knowledge about the electrochemical interface comes from ensemble measurements and ex situ imaging of the electrode surface. Due to its enormous complexity and highly dynamic nature, however, new imaging tools that can probe the interface in situ with ultrahigh spatial and temporal resolution and single-molecule sensitivity are apparently needed. Single-molecule fluorescence microscopy (SMFM) has emerged as a powerful tool that is uniquely suited for studying the electrochemical interface. In this mini-review, we first give a brief overview of various existing SMFM methods for studying electrochemical problems. We then discuss several exciting research topics involving the use of SMFM methods for studying surface-immobilized molecules, single freely diffusing molecules, single molecules as catalytic reaction indicators, and single-molecule labeling and imaging of interfacial nanobubbles. We anticipate that we will continue to see a rapid increase in publications on stochastic electrochemistry of single molecules and nanoparticles. The increased use of SMFM will likely bring new information to our study of the electrochemical interface.

Single-molecule labeling for studying trafficking of renal transporters.

The ability to detect and track single molecules presents the advantage of visualizing the complex behavior of transmembrane proteins with a time and space resolution that would otherwise be lost with traditional labeling and biochemical techniques. Development of new imaging probes has provided a robust method to study their trafficking and surface dynamics. This mini-review focuses on the current technology available for single-molecule labeling of transmembrane proteins, their advantages, and limitations. We also discuss the application of these techniques to the study of renal transporter trafficking in light of recent research.

Digital Microarrays: Single-Molecule Readout with Interferometric Detection of Plasmonic Nanorod Labels.

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology's Achilles' heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ("digital") regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique's simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.

Live-cell single-molecule labeling and analysis of myosin motors with quantum dots.

Quantum dots (QDs) are a powerful tool for quantitatively analyzing dynamic cellular processes by single-particle tracking. However, tracking of intracellular molecules with QDs is limited by their inability to penetrate the plasma membrane and bind to specific molecules of interest. Although several techniques for overcoming these problems have been proposed, they are either complicated or inconvenient. To address this issue, in this study, we developed a simple, convenient, and nontoxic method for labeling intracellular molecules in cells using HaloTag technology and electroporation. We labeled intracellular myosin motors with this approach and tracked their movement within cells. By simultaneously imaging myosin movement and F-actin architecture, we observed that F-actin serves not only as a rail but also as a barrier for myosin movement. We analyzed the effect of insulin on the movement of several myosin motors, which have been suggested to regulate intracellular trafficking of the insulin-responsive glucose transporter GLUT4, but found no significant enhancement in myosin motor motility as a result of insulin treatment. Our approach expands the repertoire of proteins for which intracellular dynamics can be analyzed at the single-molecule level.

Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology.

BackgroundStructural variants (SVs) are less common than single nucleotide polymorphisms and indels in the population, but collectively account for a significant fraction of genetic polymorphism and diseases. Base pair differences arising from SVs are on a much higher order (>100 fold) than point mutations; however, none of the current detection methods are comprehensive, and currently available methodologies are incapable of providing sufficient resolution and unambiguous information across complex regions in the human genome. To address these challenges, we applied a high-throughput, cost-effective genome mapping technology to comprehensively discover genome-wide SVs and characterize complex regions of the YH genome using long single molecules (>150 kb) in a global fashion.ResultsUtilizing nanochannel-based genome mapping technology, we obtained 708 insertions/deletions and 17 inversions larger than 1 kb. Excluding the 59 SVs (54 insertions/deletions, 5 inversions) that overlap with N-base gaps in the reference assembly hg19, 666 non-gap SVs remained, and 396 of them (60%) were verified by paired-end data from whole-genome sequencing-based re-sequencing or de novo assembly sequence from fosmid data. Of the remaining 270 SVs, 260 are insertions and 213 overlap known SVs in the Database of Genomic Variants. Overall, 609 out of 666 (90%) variants were supported by experimental orthogonal methods or historical evidence in public databases. At the same time, genome mapping also provides valuable information for complex regions with haplotypes in a straightforward fashion. In addition, with long single-molecule labeling patterns, exogenous viral sequences were mapped on a whole-genome scale, and sample heterogeneity was analyzed at a new level.ConclusionOur study highlights genome mapping technology as a comprehensive and cost-effective method for detecting structural variation and studying complex regions in the human genome, as well as deciphering viral integration into the host genome.Electronic supplementary materialThe online version of this article (doi:10.1186/2047-217X-3-34) contains supplementary material, which is available to authorized users.

The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision

The double-helix point spread function microscope encodes the axial (z) position information of single emitters in wide-field (x,y) images, thus enabling localization in three dimensions (3D) inside extended volumes. We experimentally determine the statistical localization precision σ of this approach using single emitters in a cell under typical background conditions, demonstrating σ < 20 nm laterally and <30 nm axially for N ≈ 1180 photons per localization. Combined with light-induced blinking of single-molecule labels, we present proof-of-concept imaging beyond the optical diffraction limit of microtubule network structures in fixed mammalian cells over a large axial range in three dimensions.

Synthesis and Characterization of DNA-Based Micro- and Nanodumbbell Structures

Static barcode labels for individual molecules, based on a sequence of fluorescent segments deterministically encoded in a DNA scaffold, are emerging as sensitive tools for direct digital quantification assays, such as RNA transcript profiling. The next generation of digital barcoding will require dynamic single-molecule labels, which can be controllably translocated, stretched, and read serially by an electronic or optical point detector. Towards that goal, structures consisting of a single magnetic and a single non-magnetic particle connected by a DNA-based linker were synthesized at micron (microdumbbells) and submicron (nanodumbells) scales via successive streptavidin-biotin attachments. Visualization of the structures with fluorescent and atomic force microscopy revealed the desired dumbbell morphology, thus demonstrating the suitability of the synthesis scheme. The stretched DNA-based linker is a convenient template for deterministic, sequence specific information encoding, which indicates the potential of the dumbbell-shaped nanostructures as magnetophoretically driven dynamic barcode labels.

Mapping the Polarization Pattern of Plasmon Modes Reveals Nanoparticle Symmetry

Mapping the Polarization Pattern of Plasmon Modes Reveals Nanoparticle Symmetry

Self-Assembly of Small Gold Colloids with Functionalized Gold Nanorods

We present a general strategy to stabilize gold nanorod suspensions with mono- and bifunctional polyethylene glycol (PEG) and to attach a controlled number of nanoparticles or biomolecules. Characterization by gel electrophoresis, transmission electron microscopy (TEM), and optical dark-field microscopy show the specific binding of functionalized nanorods to their target while avoiding nonspecific binding to substrates, matrices, and other particles. Such nanorods are well suited for self-assembly of nanostructures and single-molecule labeling.

T cell killing does not require the formation of a stable mature immunological synapse.

A notable feature of T lymphocyte recognition on other cell surfaces is the formation of a stable mature immunological synapse. Here we use a single-molecule labeling method to directly measure the number of ligands a cytotoxic T cell engages and track the consequences of that interaction by three-dimensional video microscopy. Like helper T cells, cytotoxic T cells were able to detect even a single foreign antigen but required about ten complexes of peptide-major histocompatibility complex (pMHC) to achieve full calcium increase and to form a mature synapse. Thus, cytotoxic T cells and helper T cells are more uniform in their antigen sensitivities than previously thought. Furthermore, only three pMHC complexes were required for killing, showing that stable synapse formation and complete signaling are not required for cytotoxicity.