Single-molecule Fluorescence Imaging Research Articles

Improving the Photostability of Red- and Green-Emissive Single-Molecule Fluorophores via Ni2+ Mediated Excited Triplet-State Quenching.

Methods to improve the photostability/photon output of fluorophores without compromising their signal stability are of paramount importance in single-molecule fluorescence (SMF) imaging applications. We show herein that Ni2+ provides a suitable photostabilizing agent for three green-emissive (Cy3, ATTO532, Alexa532) and three red-emissive (Cy5, Alexa647, ATTO647N) fluorophores, four of which are regularly utilized in SMF studies. Ni2+ works via photophysical quenching of the triplet excited state eliminating the potential for reactive intermediates being formed. Measurements of survival time, average intensity, and mean number of photons collected for the six fluorophores show that Ni2+ increased their photostability 10- to 45-fold, comparable to photochemically based systems, without compromising the signal intensity or stability. Comparative studies with existing photostabilizing strategies enabled us to score different photochemical and photophysical stabilizing systems, based on their intended application. The realization that Ni2+ allowed achieving a significant increase in photon output both for green- and red-emissive fluorophores positions Ni2+ as a widely applicable tool to mitigate photobleaching, most suitable for multicolor single-molecule fluorescence studies.

A Combination of Diffusion and Active Translocation Localizes Myosin 10 to the Filopodial Tip

Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell, we have used total internal reflection fluorescence microscopy to study the movement of individual full-length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single α-helical and anti-parallel coiled-coil forming regions, which lie between the motor and pleckstrin homology domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia. Deletion of the anti-parallel coiled-coil forming region, but not the EKR-rich region of the single α-helical domain, restored intrafilopodial trafficking, suggesting this region is important in determining myosin 10 motility. We propose a model by which myosin 10 rapidly targets to the filopodial tip via a sequential reduction in dimensionality. Molecules first undergo rapid diffusion within the three-dimensional volume of the cell body. They then exhibit periods of slower two-dimensional diffusion in the plane of the plasma membrane. Finally, they move in a unidimensional, highly directed manner along the polarized actin filament bundle within the filopodium becoming confined to a single point at the tip. Here we have observed directly each phase of the trafficking process using single molecule fluorescence imaging of live cells and have quantified our observations using single particle tracking, autocorrelation analysis, and kymographs.

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level.

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

Nanoscopic Cellular Imaging: Confinement Broadens Understanding.

In recent years, single-molecule fluorescence imaging has been reconciling a fundamental mismatch between optical microscopy and subcellular biophysics. However, the next step in nanoscale imaging in living cells can be accessed only by optical excitation confinement geometries. Here, we review three methods of confinement that can enable nanoscale imaging in living cells: excitation confinement by laser illumination with beam shaping; physical confinement by micron-scale geometries in bacterial cells; and nanoscale confinement by nanophotonics.

In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria.

Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of ∼8 μm2/s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes.

Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli.

Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the ‘proximal' and ‘distal' UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA dissociates from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions.

Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor.

Nanopore technology has become a highly sensitive and powerful tool for single molecule sensing of chemicals and biopolymers. Protein pores have the advantages of size amenability, channel homogeneity, and fabrication reproducibility. But most well-studied protein pores for sensing are too small for passage of peptide analytes that are typically a few nanometers in dimension. The funnel-shaped channel of bacteriophage phi29 DNA packaging motor has previously been inserted into a lipid membrane to serve as a larger pore with a narrowest N-terminal constriction of 3.6 nm and a wider C-terminal end of 6 nm. Here, the utility of phi29 motor channel for fingerprinting of various peptides using single molecule electrophysiological assays is reported. The translocation of peptides is proved unequivocally by single molecule fluorescence imaging. Current blockage percentage and distinctive current signatures are used to distinguish peptides with high confidence. Each peptide generated one or two distinct current blockage peaks, serving as typical fingerprint for each peptide. The oligomeric states of peptides can also be studied in real time at single molecule level. The results demonstrate the potential for further development of phi29 motor channel for detection of disease-associated peptide biomarkers.

Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA–DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA–DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.

Activation of p53 Facilitates the Target Search in DNA by Enhancing the Target Recognition Probability

Tumor suppressor p53 binds to the target in a genome and regulates the expression of downstream genes. p53 searches for the target by combining three-dimensional diffusion and one-dimensional sliding along the DNA. To examine the regulation mechanism of the target binding, we constructed the pseudo-wild type (pseudo-WT), activated (S392E), and inactive (R248Q) mutants of p53 and observed their target binding in long DNA using single-molecule fluorescence imaging. The pseudo-WT sliding along the DNA showed many pass events over the target and possessed target recognition probability (TRP) of 7±2%. The TRP increased to 18±2% for the activated mutant but decreased to 0% for the inactive mutant. Furthermore, the fraction of the target binding by the one-dimensional sliding among the total binding events increased from 63±9% for the pseudo-WT to 87±2% for the activated mutant. Control of TRP upon activation, as demonstrated here for p53, might be a general activation mechanism of transcription factors.

Competitive Assays of Label-Free DNA Hybridization with Single-Molecule Fluorescence Imaging Detection.

Single-molecule imaging of fluorescently labeled biomolecules is a powerful technique for measuring association interactions; however, care must be taken to ensure that the fluorescent labels do not influence the system being probed. Label-free techniques are needed to understand biomolecule interactions free from the influence of an attached label, but these techniques often lack sensitivity and specificity. To solve these challenges, we have developed a competitive assay that uses single-molecule detection to track the population of unlabeled target single-stranded DNA (ssDNA) hybridized with probe DNA immobilized at a glass interface by detecting individual duplexes with a fluorescently labeled "tracer" ssDNA. By labeling a small fraction (<0.2%) of target molecules, the "tracer" DNA tracks the available probe DNA sites without significant competition with the unlabeled target population. Single-molecule fluorescence imaging is a good read-out scheme for competitive assays, as it is sufficiently sensitive to detect tracer DNA on substrates with relatively low densities of probe DNA, ∼10(-3) of a monolayer, so that steric interactions do not hinder DNA hybridization. Competitive assays are used to measure the association constant of complementary strand DNA hybridization of 9- and 10-base pair targets, where the tracer assay predicts the same association constant as a traditional displacement competitive assay. This methodology was used to compare the Ka of hybridization for identical DNA strands differing only by the presence of a fluorescent label tethered to the 5' end of the solution-phase target. The addition of the fluorescent label significantly stabilizes the DNA duplex by 3.6 kJmol(-1), adding more stability than an additional adenine-thymine base-pairing interaction, 2.7 kJmol(-1). This competitive tracer assay could be used to screen a number of labeled and unlabeled target DNA strands to measure the impact of fluorescent labeling on duplex stability. This single-molecule competitive hybridization scheme could be easily adapted into a sensitive assay, where competition between tracer and target oligonucleotides for probe sites could be used to measure concentrations of unlabeled DNA or RNA.

PIF1-Interacting Transcription Factors and Their Binding Sequence Elements Determine the in Vivo Targeting Sites of PIF1.

The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR1 (PIF1) binds G-box elements in vitro and inhibits light-dependent germination in Arabidopsis thaliana A previous genome-wide analysis of PIF1 targeting indicated that PIF1 binds 748 sites in imbibed seeds, only 59% of which possess G-box elements. This suggests the G-box is not the sole determinant of PIF1 targeting. The targeting of PIF1 to specific sites could be stabilized by PIF1-interacting transcription factors (PTFs) that bind other nearby sequence elements. Here, we report PIF1 targeting sites are enriched with not only G-boxes but also with other hexameric sequence elements we named G-box coupling elements (GCEs). One of these GCEs possesses an ACGT core and serves as a binding site for group A bZIP transcription factors, including ABSCISIC ACID INSENSITIVE5 (ABI5), which inhibits seed germination in abscisic acid signaling. PIF1 interacts with ABI5 and other group A bZIP transcription factors and together they target a subset of PIF1 binding sites in vivo. In vitro single-molecule fluorescence imaging confirms that ABI5 facilitates PIF1 binding to DNA fragments possessing multiple G-boxes or the GCE alone. Thus, we show in vivo PIF1 targeting to specific binding sites is determined by its interaction with PTFs and their binding to GCEs.

Direct observation of λ-DNA molecule reversal movement within microfluidic channels under electric field with single molecule imaging technique**Project supported by the National Natural Science Foundation of China (Grant No. 61378083), the International Cooperation Foundation of the National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2011DFA12220), the Major Research Plan of National

The electrodynamic characteristics of single DNA molecules moving within micro-/nano-fluidic channels are important in the design of biomedical chips and bimolecular sensors. In this study, the dynamic properties of λ-DNA molecules transferring along the microchannels driven by the external electrickinetic force were systemically investigated with the single molecule fluorescence imaging technique. The experimental results indicated that the velocity of DNA molecules was strictly dependent on the value of the applied electric field and the diameter of the channel. The larger the external electric field, the larger the velocity, and the more significant deformation of DNA molecules. More meaningfully, it was found that the moving directions of DNA molecules had two completely different directions: (i) along the direction of the external electric field, when the electric field intensity was smaller than a certain threshold value; (ii) opposite to the direction of the external electric field, when the electric field intensity was greater than the threshold electric field intensity. The reversal movement of DNA molecules was mainly determined by the competition between the electrophoresis force and the influence of electro-osmosis flow. These new findings will theoretically guide the practical application of fluidic channel sensors and lab-on-chips for precisely manipulating single DNA molecules.

Moving Kinetics of Nanocars with Hydrophobic Wheels on Solid Surfaces at Ambient Conditions

Motivated by “driving” nanoscopic nanocars on solid substrate surfaces at ambient conditions, we studied the moving kinetics of nanocars on differently modified surfaces. Single molecule fluorescence imaging was used to track the nanocar movement so that the molecules were minimally perturbed. On freshly cleaned, hydroxylated glass surfaces, nanocars with hydrophobic adamantane wheels can diffuse with a relatively large diffusion coefficient of 7.6 × 10–16 m2/s. Both the number of moving molecules and the mobility of the moving molecules decreased over time when the sample was exposed in the air. Similar declinations in movement were observed on a poly(ethylene glycol) (PEG)-modified glass surface, but the declination rate was lowered. The slowing of molecular surface diffusion is correlated to the hydrophobicity of the surface and is likely caused by the adsorption of hydrophobic molecules from the air. A proposed sticky-spots model explains the decreasing apparent diffusion coefficient of the hydrophobi...

Simple calibration of TIR field depth using the supercoiling response of DNA

The combination of single-molecule fluorescence and nanomanipulation techniques into a single experimental platform enables one to carry out correlative analysis of the composition and the activity of complex, multicomponent molecular systems. Here we describe implementation and calibration of such a combined system allowing simultaneous single-molecule force spectroscopy and fluorescence imaging of proteins acting on the DNA using magnetic trapping coupled with fluorescence excitation based on a Total Internal Reflection (TIR), or evanescent, field. We propose a simple and robust in situ method for calibration of the TIR field depth against the mechanical properties of nanomanipulated DNA, and which is made possible by the fact that the magnetic bead used to trap and nanomanipulate DNA and measure its conformation also exhibits autofluorescence in the TIR field. Indeed, the fact that the bead size is on the 1-micron scale does not preclude sensitive probing of an intensity field which decays exponentially on the 0.1micron-scale. We demonstrate the usefulness of this approach by mapping out TIR field depth as a function of the angle of incidence of the illuminating laser at the glass-water interface and showing that one recovers the expected theoretical relationship between field depth and angle of incidence.

Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging.

Interferometric scattering microscopy (iSCAT) is a light scattering-based imaging modality that offers a unique combination of imaging speed and precision for tracking nanoscopic labels and enables label-free optical sensing down to the single-molecule level. In contrast to fluorescence, iSCAT does not suffer from limitations associated with dye photochemistry and photophysics, or the requirement for fluorescent labeling. Here we present a protocol for constructing an iSCAT microscope from commercially available optical components and demonstrate its compatibility with simultaneously operating single-molecule, objective-type, total internal reflection fluorescence microscopy. Given an intermediate level of experience with optics and microscopy, for instance graduate-level familiarity with laser beam steering and optical components, this protocol can be completed in a time frame of 2 weeks.

Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions.

DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2–Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2–Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2–Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2–Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2–Msh3 and Msh2–Msh6 navigate on a crowded genome and suggest how Msh2–Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.

Structure and Dynamics of Filopodia Studied by Electron Cryo-Tomography and Single Molecule Fluorescence Imaging

Filopodia are cellular projections around 100nm in diameter and usually several micrometres in length. They are composed of fascin-bundled actin filaments and the molecular motor myosin‑10 localises at the extreme tip. Filopodia are important in cell migration and in making cell-cell contacts and have been linked with roles in diverse areas of biology including angiogenesis, and path-finding properties of neuronal growth cones. Filopodia can be induced in model cell lines by overexpresion of myosin‑10 and live-cell imaging shows that they undergo stochastic cycles of extension and retraction. Myosin‑10 transports cargo along the actin filament bundle towards the filopodial tip, forming a complex of proteins which are responsible for governing growth, retraction and adhesion. We have used a combination of optical and electron microscopy to study the dynamics and ultra-structure of myosin‑10 induced filopodia. Electron cryo-tomography of flash-frozen vitrified mammalian cells revealed the arrangement of actin filaments within the filopodia. Single molecule imaging by total internal reflection fluorescence microscopy allowed movement of myosin‑10 with the filopodia to be quantified and super-resolution localisation gave structural and mechanical information about the filopodium that could be correlated to our electron tomographic images.

Dynamic DNA Binding Licenses a Eukaryotic Repair Complex to Bypass Protein Roadblocks in Search of DNA Lesions

DNA binding proteins search for specific targets amidst an excess of non-target DNA largely through facilitated diffusion. While DNA is crowded with proteins in vivo, little is known about how protein roadblocks affect diffusion. Here, we use single-molecule fluorescence imaging and DNA curtains generated by nano-fabricated chromium arrays to investigate how Msh2-Msh3, a eukaryotic mismatch repair (MMR) complex, navigates on crowded DNA. We discovered that Msh2-Msh3, in contrast to the homologous protein complex Msh2-Msh6, diffuses on DNA by hopping. Furthermore, hopping allows Msh2-Msh3 to bypass protein roadblocks, including nucleosomes. Despite hopping over roadblocks, we observed that Msh2-Msh3 maintains sufficient contact with DNA to bind single DNA lesions. Remarkably, the primary Msh3 DNA binding domain, the mispair binding domain (MBD), translates the ability to hop to a Msh2-Msh6(3-MBD) chimera. This work provides a model for how Msh2-Msh3 locates lesions outside of replication-coupled MMR, provides insight into the structural pre-requisites for protein hopping, and shows how hopping may accelerate target search in the context of chromatin.

Probing Single-Molecule Ion Channel Conformational Dynamics in Living Cells

Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels that are crucial for cell functions, neuronal signaling, and brain functions. We have developed a new combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging for probing ion channel conformational changes simultaneously with the electrical single channel recording. By combining real-time single-molecule fluorescence imaging measurements with real-time single-channel electric current measurements in artificail lipid bilayers and in living cell membranes, we were able to probe single ion-channel-protein conformational changes simultaneously, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level. We will focus our discussion on the new development and results of real-time imaging of the dynamics of gramicidin, colicin, and NMDA receptor ion channels in lipid bilayers and living cells. We have probed NMDA (N-Methyl-D-Aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open-close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. Based on our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics. Our results shed light on new perspectives of the intrinsic interplay of lipid membrane dynamics, solvation dynamics, and the ion channel functions.