Single-particle Fluorescence Imaging Research Articles

The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Highly enhanced photoluminescence and suppressed blinking of N-doped carbon dots by targeted passivation of amine group for mechanistic insights and Vis-NIR excitation bioimaging application

Carbon dots (CDs) have attracted extensive attention owing to their attractive optical properties, small size, low cost, and good biocompatibility. However, there are still challenges in unequivocally unraveling the photoluminescence (PL) origin and the structure-function relationship associated with the surface groups of CDs. Herein, we have successfully enhanced the PL efficiency of GA/N-CDs by targeted passivation of the amine groups of N-doped CDs (N-CDs) with glutaraldehyde (GA), to facilitate their bioimaging applications under one-photon and two-photon excitations. In particular, we have systematically investigated the regulation effects of this surface modification on the optical properties, exciton dynamics, and blinking behavior using steady-state and time-resolved spectroscopies, and single-particle fluorescence imaging microscopes. The effective passivation of the amine surface group of N-CDs not only regulates the energy levels and properties of surface states, but importantly activates an additional emissive center via an aldehyde-amine condensation reaction. Meanwhile, the hydrogen bonding interactions between GA and the related surface groups of N-CDs further restrain the non-radiative exciton relaxations. This synergetic effect greatly enhances the PL efficiency of two separate emission centers and the photostability of GA/N-CDs. Moreover, the photoblinking of N-CDs is significantly suppressed and the two-photon excitation PL property is also markedly increased after the surface modification, endowing GA/N-CDs the outstanding performances for fluorescence bioimaging under Vis-NIR excitations. Our findings contribute to mechanistic insights into the correlation between photophysical properties and surface states/groups, as well as demonstrate an effective post-modification strategy to improve the PL efficiency and stability of carbon-based dots.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Shell-dependent blinking and ultrafast carrier dynamics in CdxZn1-xSeyS1-y@ZnS core/shell quantum dots

Fluorescent semiconductor quantum dots (QDs) are promising nanocrystals with excellent optical properties for applications in many fields. However, the severe blinking of QDs limits their practical applications that rely on single-dot emission. In this study, the shell-dependent blinking behavior and ultrafast carrier dynamics in CdxZn1-xSeyS1-y@ZnS core/shell QDs with smooth interface were investigated by using single particle fluorescence imaging and time-resolved spectroscopy. Our observations indicate that the on-time fraction of emission blinking for CdxZn1-xSeyS1-y QDs can be effectively increased with capping the ZnS shell. Meanwhile, the ultrafast dynamics including the hot carrier relaxation within hundreds of femtoseconds, carriers capture by defect states in tens of picoseconds, and electron-hole nonradiative recombination with several nanoseconds have been clearly identified for the as-prepared QDs with different shell thickness. We found that above carrier dynamics demonstrate an obvious dependence of ZnS shell thickness due to the effective isolation of the internal carriers in the CdxZn1-xSeyS1-y core from the decreased interfacial defects. These findings suggest that the capping shell can effectively regulate the carrier dynamics and blinking behavior, and improve the optical properties of CdxZn1-xSeyS1-y @ZnS QDs, which are significant for understanding the correlation between the structure and optical properties of fluorescent QDs.

Luminescence-enhanced conjugated polymer dots through thermal treatment for cell imaging.

Conjugated polymer dots (Pdots) are often used as excellent fluorescent probes in the biomedical field. In the process of preparing Pdots, the rapid change of the solvent polarity will result in a messy and defective stacking of the polymer chains in the particle, and these stacking defects of the polymer chains may weaken its luminescence properties. Here, we try to optimize the stacking of the conjugated polymer chains by the thermal annealing treatment. After the low temperature thermal treatment, the fluorescence intensity of Pdots can be enhanced by about 11%-29%, and Pdots maintain their original stability and biosafety. We used transmission electron microscopy (TEM) and single particle fluorescence imaging to reveal the possible mechanism of the chain stacking optimization process, that is, the thermal annealing process of Pdots is the competition between internal chain rearrangement in the particle and particle aggregation. The luminescence-enhanced Pdots exhibit good cellular imaging performance. These results prove that it is feasible to extend the thermal annealing treatment from planar polymer devices to polymer nanoparticles. It provides the possibility to realize stable and complex biological imaging applications using Pdots with a simple molecular structure, and a mature improvement scheme for the mass preparation of Pdots.

Addressing Particle Compositional Heterogeneities in Super-Resolution-Enhanced Live-Cell Ratiometric pH Sensing with Ultrasmall Fluorescent Core-Shell Aluminosilicate Nanoparticles.

The interrogation of metabolic parameters like pH in live-cell experiments using optical super-resolution microscopy (SRM) remains challenging. This is due to a paucity of appropriate metabolic probes enabling live-cell SRM-based sensing. Here we introduce ultrasmall fluorescent core-shell aluminosilicate nanoparticle sensors (FAM-ATTO647N aC' dots) that covalently encapsulate a reference dye (ATTO647N) in the core and a pH-sensing moiety (FAM) in the shell. Only the reference dye exhibits optical blinking enabling live-cell stochastic optical reconstruction microscopy (STORM). Using data from cells incubated for 60 minutes with FAM-ATTO647N aC' dots, pixelated information from total internal reflection fluorescence (TIRF) microscopy-based ratiometric sensing can be combined with that from STORM-based localizations via the blinking reference dye in order to enhance the resolution of ratiometric pH sensor maps beyond the optical diffraction limit. A nearest-neighbor interpolation methodology is developed to quantitatively address particle compositional heterogeneity as determined by separate single-particle fluorescence imaging methods. When combined with STORM-based estimates of the number of particles per vesicle, vesicle size, and vesicular motion as a whole, this analysis provides detailed live-cell spatial and functional information, paving the way to a comprehensive mapping and understanding of the spatiotemporal evolution of nanoparticle processing by cells important, e.g. for applications in nanomedicine.

Real-time molecular-level visualization of mass flow during patterned photopolymerization of liquid-crystalline monomers

Single-particle fluorescence imaging is used to monitor dynamic processes that occur during patterned photopolymerization of liquid-crystalline monomers. A spatial gradient of chemical potential can be created at the border of bright and dark regions by structured illumination in the photopolymerization process, leading to mutual diffusion of polymers and monomers. Analysis of the fluorescence from single quantum dots doped into the monomers at minute concentrations enables visualization of highly directional flow from the illuminated region where the photopolymerization proceeds toward a masked unpolymerized region. This directional mass flow causes flow-induced orientation of the polymers that is subsequently fixed by completion of the polymerization reaction, resulting in a mesoscopic aligned area of the polymer film.

Enhancing porphyrin photostability when locked in metal-organic frameworks.

Porphyrins have been widely used in many optical devices given their unique photochemical properties. Their poor photostability has, however, limited their wide applications in bioimaging and biosensing schemes. Herein, we report the remarkable photostability enhancement of the porphyrin, carboxyphenyl porphyrin (TCPP-H2) when locked in a zirconium based metal-organic framework (MOF-525). Steady-state ensemble fluorescence spectroscopy experiments showed minimal changes (2%) in the recorded signal when MOF-525 was continuously illuminated as compared to a 16% decrease for free porphyrins. Single particle fluorescence imaging revealed bright microparticles with exceptional photostability and no-blinking within the experiment window. This study highlights the use of metal-organic frameworks for preparing photostable microstructures by leveraging on their unique self-assembly properties.

Phycobiliproteins: A Novel Green Tool from Marine Origin Blue-Green Algae and Red Algae.

Marine species are comprising about a half of the whole global biodiversity; the sea offers an enormous resource for novel bioactive compounds. Several of the marine origin species show multifunctional bioactivities and characteristics that are useful for a discovery and/or reinvention of biologically active compounds. For millennia, marine species that includes cyanobacteria (blue-green algae) and red algae have been targeted to explore their enormous potential candidature status along with a wider spectrum of novel applications in bio- and non-bio sectors of the modern world. Among them, cyanobacteria are photosynthetic prokaryotes, phylogenetically a primitive group of Gramnegative prokaryotes, ranging from Arctic to Antarctic regions, capable of carrying out photosynthesis and nitrogen fixation. In the recent decade, a great deal of research attention has been paid on the pronouncement of bio-functional proteins along with novel peptides, vitamins, fine chemicals, renewable fuel and bioactive compounds, e.g., phycobiliproteins from marine species, cyanobacteria and red algae. Interestingly, they are extensively commercialized for natural colorants in food and cosmetics, antimicrobial, antioxidant, anti-inflammatory, neuroprotective, hepatoprotective agents and fluorescent neo-glycoproteins as probes for single particle fluorescence imaging fluorescent applications in clinical and immunological analysis. However, a comprehensive knowledge and technological base for augmenting their commercial utilities are lacking. Therefore, this paper will provide an overview of the phycobiliproteins-based research literature from marine cyanobacteria and red algae. This review is also focused towards analyzing global and commercial activities with application oriented-based research. Towards the end, the information is also given on the potential biotechnological and biomedical applications of phycobiliproteins.

Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres.

Telomeric repeat-containing RNA (TERRA) controls the structure and length of telomeres through interactions with numerous telomere-binding proteins. However, little is known about the mechanism by which TERRA regulates the accessibility of the proteins to telomeres, mainly because of the lack of spatiotemporal information of TERRA and its-interacting proteins. We developed a fluorescent probe to visualize endogenous TERRA to investigate its dynamics in living cells. Single-particle fluorescence imaging revealed that TERRA accumulated in a telomere-neighboring region and trapped diffusive heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), thereby inhibiting hnRNPA1 localization to the telomere. These results suggest that TERRA regulates binding of hnRNPA1 to the telomere in a region surrounding the telomere, leading to a deeper understanding of the mechanism of TERRA function.

Functionalization of emissive conjugated polymer nanoparticles by coprecipitation: consequences for particle photophysics and colloidal properties

The functionalization of polyfluorene (PFO) nanoparticles by coprecipitation of the conjugated polymer with an amphiphilic comb polymer, consisting of a hydrophobic polystyrene backbone with hydrophilic, carboxylic acid-terminated polyethylene oxide side-chains (PS-PEG-COOH), is investigated. The comb polymer affects the properties of the formed hybrid nanoparticles. Non-functionalized particles are typically larger (28 nm) than functionalized ones (20 nm); peak molar extinction coefficients are found to differ in a similar trend. Zeta potentials are negative, consistent with negative surface charge on PFO particles due to chemical defect formation, with additional charge on functionalized particles due to the pendant carboxylic acid groups. Emission quantum yields of functionalized particles are typically larger, consistent with lower efficiency of energy transfer to quenchers in smaller particles and weaker PFO interchain interactions due to chain dilution. The trend in per-particle fluorescence brightness values, as confirmed by single particle fluorescence imaging, reflects the nanoparticle extinction coefficients. Photostability studies on aqueous dispersions of hybrid particles indicate mild photobrightening under continuous illumination while PFO particles exhibit slow exponential emission decay. Functionalized particles are also resistant to aggregation during exposure to adenocarcinoma cells. Generally, the hybrid particles exhibit more favorable time-, pH- and medium-dependent stabilities, likely due to steric and electrostatic stabilization by PEG-carboxylic acid functionalities. Overall, the functionalized particles exhibit attractive properties: Reasonably small size, tight size distribution, high absorption cross section, radiative rate and emission quantum yield, excellent brightness and photostability, and good colloidal stability.

Distinguishing Direct and Indirect Photoelectrocatalytic Oxidation Mechanisms Using Quantitative Single-Molecule Reaction Imaging and Photocurrent Measurements

Light-driven semiconductor-catalyzed oxidation reactions are of fundamental importance in photocatalysis and photoelectrocatalysis for removing organic contaminants in wastewater, solar energy conversion, and fine chemical synthesis. The underlying reaction mechanism is often unclear because it is difficult to measure directly and specifically the semiconductor-catalyzed reaction rates. For example, an organic molecule could be oxidized “directly” by photogenerated holes that are transported from the semiconductor interior to the semiconductor–electrolyte interface or “indirectly” by photogenerated intermediates (e.g., hydroxyl radical, superoxide anion, or hydrogen peroxide) that are produced at the semiconductor surface in aqueous solution. New experimental approaches that can distinguish these pathways are thus desirable. Here we introduce quantitative single-molecule, single-particle fluorescence imaging to measure the photoelectrocatalytic oxidation rate of a model organic substrate, amplex red, on t...

Brightness calibrates particle size in single particle fluorescence imaging.

This Letter provides a novel approach to quantify the particle sizes of highly bright semiconductor polymer dots (Pdots) for single-particle imaging and photobleaching studies. A quadratic dependence of single-particle brightness on particle size was determined by single-particle fluorescence imaging and intensity statistics. In terms of the same imaging conditions, the particle diameter can be quantified by comparing the individual brightness intensity with associated calibration curve. Based on this sizing method, photobleaching trajectories and overall photon counts emitted by single particles were analyzed. It is found that photobleaching rate constants of different sized Pdots are not strongly dependent on particle diameter except the sparsely occurring fluorescence blinking in certain dim particles and the rapid photobleaching component in some bright particles. The overall photon counts increase with increasing particle diameter. However, those larger than 30nm deviate away from the increasing tendency. These results reveal the significance of selecting appropriate Pdots (≤30 nm) for single-particle imaging and tracking applications.

Discovery of New Nitridosilicate Phosphors for Solid State Lighting by the Single-Particle-Diagnosis Approach

Discovery of novel luminescent materials is of fundamental importance in the advancement of solid state lighting and flat panel display technologies. In this work, we report a single-particle-diagnosis method for the discovery of new phosphors by just characterizing a luminescent crystalline particle as small as 10 μm in diameter. We explored single-particle fluorescence imaging and spectroscopy techniques to evaluate the photoluminescence of a phosphor particle distinguished from a complex powder mixture and applied a high-resolution single-crystal X-ray diffractometer to determine its crystal structure. The approach enabled us to discover two new phosphors in the Ba3N2–Si3N4–AlN ternary system: Ba5Si11Al7N25:Eu2+ and BaSi4Al3N9:Eu2+. Ba5Si11Al7N25:Eu2+ crystallizes in the space group of Pnnm (no. 58) with a = 9.5923(2), b = 21.3991(5), c = 5.8889 (2) Å and Z = 2, while BaSi4Al3N9:Eu2+ in the space group of P21/C (no.14) with a = 5.8465(4), b = 26.7255(18), c = 5.8386(4) Å, β = 118.897° and Z = 4. The single-particle photoluminescence of Ba5Si11Al7N25:Eu2+ shows yellow emission (λem = 568 nm, fwhm = 98 nm) and a quantum efficiency of 36% under the 405 nm excitation. BaSi4Al3N9:Eu2+ shows blue emission (λem = 500 nm, fwhm = 67 nm) upon the 365 nm excitation.

Size-dependent property and cell labeling of semiconducting polymer dots.

Semiconducting polymer dots (Pdots) represent a new class of fluorescent nanoparticles for biological applications. In this study, we investigated their size-dependent fluorescence and cellular labeling properties. We demonstrate that the polymer conformation in solution phase largely affects the polymer folding and packing during the nanoparticle preparation process, resulting in solution-phase control over the fluorescence properties of semiconducting polymer nanoparticles. The resulting Pdots exhibit apparent size dependent absorption and emission, a characteristic feature of different chain packing behaviors due to the preparation conditions. Single-particle fluorescence imaging was employed to perform a side-by-side comparison on the Pdot brightness, indicating a quadratic dependence of single-particle brightness on particle size. Upon introducing a positively charged dye Nile blue, all the three type of Pdots were quenched very efficiently (Ksv > 1 × 10(7) M(-1)) in an applied quenching process at low dye concentrations, but exhibit apparent difference in quenching efficiency with increasing dye concentration. Furthermore, Pdots of different sizes were used for cell uptake and cellular labeling involving biotin-streptavidin interactions. Fluorescence imaging together with flow cytometry studies clearly showed size dependent labeling brightness. Small-sized Pdots appear to be more effective for immunolabeling of cell surface, whereas medium-sized Pdots exhibit the highest uptake efficiency. This study provides a concrete guidance for selecting appropriate particle size for biological imaging and sensing applications.

Key Interactions for Clathrin Coat Stability

Clathrin-coated vesicles are major carriers of vesicular traffic in eukaryotic cells. This endocytic pathway relies on cycles of clathrin coat assembly and Hsc70-mediated disassembly. Here we identify histidine residues as major determinants of lattice assembly and stability. They are located at the invariant interface between the proximal and distal segments of clathrin heavy chains, in triskelions centered on two adjacent vertices of the coated-vesicle lattice. Mutation of these histidine residues to glutamine alters the pH dependence of coat stability. We then describe single-particle fluorescence imaging experiments in which we follow the effect of these histidine mutations on susceptibility to Hsc70-dependent uncoating. Coats destabilized by these mutations require fewer Hsc70 molecules to initiate disassembly, as predicted by a model in which Hsc70 traps conformational distortions during the auxilin- and Hsc70:ATP-mediated uncoating reaction.

Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization

Ammonium is a preferred source of nitrogen for plants but is toxic at high levels. Plant ammonium transporters (AMTs) play an essential role in NH4(+) uptake, but the mechanism by which AMTs are regulated remains unclear. To study how AMTs are regulated in the presence of ammonium, we used variable-angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1;3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1;3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress, however, AMT1;3-EGFPs were found to amass into clusters, which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1;2 background. Single-particle analysis of AMT1;3-EGFPs in the clathrin heavy chain 2 mutant (chc2 mutant) and Flotllin1 artificial microRNA (Flot1 amiRNA) backgrounds, together with chemical inhibitor treatments, demonstrated that the endocytosis of AMT1;3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways, but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1;3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1;3 from the plasma membrane.

High-intensity near-IR fluorescence in semiconducting polymer dots achieved by cascade FRET strategy.

Near-IR (NIR) emitting semiconducting polymer dots (Pdots) with ultrabright fluorescence have been prepared for specific cellular targeting. A series of π-conjugated polymers were synthesized to form water dispersible multicomponent Pdots by an ultrasonication-assisted co-precipitation method. By optimizing cascade energy transfer in Pdots, high-intensity NIR fluorescence (φ = 0.32) with tunable excitations, large absorption-emission separation (up to 330 nm), and narrow emission bands (FWHM = 44 nm) have been achieved. Single-particle fluorescence imaging show that the as-prepared NIR Pdots were more than three times brighter than the commercially available Qdot705 with comparable sizes under identical conditions of excitation and detection. Because of the covalent introduction of carboxylic acid groups into polymer side chains, the bioconjugation between NIR-emitting Pdots and streptavidins can be readily completed via these functional groups on the surface of Pdots. Furthermore, through flow cytometry and confocal fluorescence microscopy the NIR-emitting Pdot-streptavidin conjugates proved that they could effectively label EpCAM receptors on the surface of MCF-7 cells, via specific binding between streptavidin and biotin.

Quantitative 3D Tracing of Gene-delivery Viral Vectors in Human Cells and Animal Tissues

Trafficking through a variety of cellular structures and organelles is essential for the interaction between gene-delivery vectors (i.e., adeno-associated virus (AAV) and liposomes) and host cells/tissues. Here, we present a method of computer-assisted quantitative 3D biodistribution microscopy that samples the whole population of fluorescently-labeled vectors and document their trafficking routes. Using AAV as a working model, we first experimentally defined numerical parameters for the singularity of Cy5-labeled particles by combining confocal microscopy and atomic force microscopy (AFM). We then developed a robust approach that integrates single-particle fluorescence imaging with 3D deconvolution and isosurface rendering to quantitate viral distribution and trafficking in human cells as well as animal tissues at the single-particle level. Using this quantitative method, we uncovered an as yet uncharacterized rate-limiting step during viral cell entry, while delineating nuclear accumulation of virions during the first 8 hours postinfection. Further, our studies revealed for the first time that following intramuscular injection, AAV spread progressively across muscle tissues through endomysium between myofibers instead of traversing through target cells. Such 3D resolution and quantitative dissection of vector-host interactions at the subcellular level should significantly improve our ability to resolve trafficking mechanisms of gene-delivery particles and facilitate the development of enhanced viral vectors.

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating

Heat shock cognate protein 70 (Hsc70) supports remodeling of protein complexes -- for example, disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by electron cryomicroscopy, in the vicinity of each occupied site and that accumulation of local strains destabilises the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.