Direct Imaging of Single Plasmonic Metal Nanoparticles in Capillary with Laser Light-Sheet Scattering Imaging.

Understanding the heterogeneous distribution of the physical and chemical properties of plasmonic metal nanoparticles is fundamentally important to their basic and applied research. Traditionally, they are obtained either indirectly via bulk spectroscopic measurements plus electron microscopic characterizations or through single molecule/particle imaging of nanoparticles immobilized on planar substrates. In this study, by using light-sheet scattering microscopy with a supercontinuum white laser, highly sensitive imaging of individual metal nanoparticles (MNPs) flowing inside a capillary, driven by either pressure or electric field, was achieved for the first time. We demonstrate that single plasmonic nanoparticles with different size or chemical modification could be differentiated through their electrophoretic mobility in a few minutes. This technique could potentially be applied to high throughput characterization and evaluation of single metal nanoparticles as well as their dynamic interactions with various local environments.

Sequence-Modulated Interactions between Single Multivalent DNA-Conjugated Gold Nanoparticles.

DNA-conjugated gold nanoparticle (AuNP) is an attractive building block to construct elegant plasmonic nanomaterials by self-assembly but the complicated interaction between multivalent nanoconjugates governing the assembly process and the properties of assembled structures remains poorly understood. Herein, with an in situ kinetic single-particle imaging method, we report the dynamic interaction between single multivalent DNA-conjugated AuNPs quantitatively depends on the nucleic acid sequence in nanoconjugates. From the binding dynamics analysis, it was found that the binding of nanoconjugates with DNA length longer than nine bases is kinetically irreversible and the binding rate is dependent on both the sequence length and GC content, enabling us to predict the rational modulation of binding rates of individual building blocks for stepwise assembly. Moreover, the reversibility for the multivalent interaction between single nanoconjugates at constant temperature can be reinstated by adopting the DNA sequence with single-nucleotide mismatch and the lifetime for nanoconjugates at bound state can be tailored by changing the mismatch positions in DNA strands, providing new opportunity to create active nanostructures with controlled dynamic properties. All these findings provide new insights for understanding the multivalent interaction during the assembly process at the single-nanoconjugate level and predicting the programmable self-assembly of engineered nanoconjugates for the fabrication of dynamic nanomaterials.

Single Particle Tracking of Peptides-Modified Nanocargo on Lipid Membrane Revealing Bulk-Mediated Diffusion.

Understanding the detailed diffusion behavior of the nanocargo on lipid membrane can afford deep insight into the surface chemistry controlled translocation mechanism for the rational design of an efficient delivery system. By tracking the diffusion trajectory of transacting activator of transcription (TAT, a cell penetrating peptide) peptides-modified nanocargo on lipid membrane, bulk-mediated (intermittent hopping) diffusion was observed for the first time after a blended modification of TAT peptides and polyethylene glycol (PEG) molecules onto the nanoparticle surface. In contrast to random walk or confined diffusion, the nanoparticles could be temporarily confined for random waiting times between surface displacements produced by excursions through the bulk fluid, which was not noted before. Non-Gaussian distributed step length (with a stretched power law like tail) was observed, making large displacements much more probable than one would predict for regular Gaussian decay. This kind of larger displacement would therefore significantly facilitate a kinetically controlled surface searching process like heterogeneous penetration site recognition on a fluidic membrane with suitable spatial orientation.

Fluorescent gold nanodots based sensor array for proteins discrimination.

A series of dual-ligand cofunctionalized fluorescent gold nanodots with similar fluorescence and diverse surface properties has been designed and synthesized to build a protein sensing array. Using this "chemical nose/tongue" strategy, fluorescence response patterns can be obtained on the array and identified via linear discriminant analysis (LDA). Eight proteins have been well distinguished at low concentration (A280 = 0.005) based on this sensor array. The practicability of this sensor array was further validated by high accuracy (100%) examination of 48 unknown protein samples.

Selective Colorimetric Detection of Hydrogen Sulfide Based on Primary Amine-Active Ester Cross-Linking of Gold Nanoparticles.

Hydrogen sulfide (H2S) is a highly toxic environmental pollutant and also an important gaseous transmitter. Therefore, selective detection of H2S is very important, and visual detection of it with the naked eye is preferred in practical applications. In this study, thiolated azido derivates and active esters functionalized gold nanoparticles (AE-AuNPs)-based nanosensors have been successfully prepared for H2S perception. The sensing principle consists of two steps: first, H2S reduces the azide group to a primary amine; second, a cross-linking reaction between the primary amine and active ester induces the aggregation of AuNPs. The AE-AuNPs-based nanosensors show high selectivity toward H2S over other anions and thiols due to the specific azide-H2S chemistry. Under optimal conditions, 0.2 µM H2S is detectable using a UV-vis spectrophotometer, and 4 µM H2S can be easily detected by the naked eye. In addition, the practical application of the designed nanosensors was evaluated with lake water samples.

Optical analysis of biological hydrogen sulphide: an overview of recent advancements.

Determining the levels of endogenous hydrogen sulphide in real time has become increasingly crucial because of its important biological roles in various physiological and pathological processes. Optical methods allowing sensitive, multiplex and dynamic analysis in a non-invasive manner have attracted much attention in biological and biomedical analysis. This review provides an overview of recent advancements in optical analysis of biological hydrogen sulphide, with a focus on fluorescence and non-fluorescence optical strategies for sensing and imaging subcellular hydrogen sulphide in living biosystems.

Direct imaging of transmembrane dynamics of single nanoparticles with darkfield microscopy: improved orientation tracking at cell sidewall.

Investigation of the cellular internalization processes of individual nanoparticles (NPs) is of great scientific interest with implications to drug delivery and NP biosafety. Herein, by using dual-channel polarization darkfield microcopy (DFM) and single gold nanorods (AuNRs) as orientation probes, we developed a method that is capable of monitoring AuNR orientation dynamics during its transmembrane process. With annular oblique illumination and a birefringent prism to split AuNR plasmonic scattering into two channels of orthogonal polarizations, the AuNR azimuth and polar angles are obtained from their intensity difference and intensity sum. By placing the focal plane of the microscope objective at the elevated cell sidewall rather than at the flat cell top, interference from cellular background is reduced and the signal-to-noise ratio of AuNR orientation sensing is improved significantly, especially for AuNRs inserting into the membrane at a large out-of-plane angle. As a result, we were able to capture the complete membrane-crossing dynamics of single AuNRs. This powerful method could be utilized to obtain valuable insights on NP endocytosis mechanisms of various cells.

Sensitive and selective detection of copper ions with highly stable polyethyleneimine-protected silver nanoclusters.

Copper is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, sensitive Cu(2+) detection is very important to prevent over-ingestion, and visual detection using unaugmented vision is preferred for practical applications. In this study, hyperbranched polyethyleneimine-protected silver nanoclusters (hPEI-AgNCs) were successfully synthesized using a facile, one-pot reaction under mild conditions. The hPEI-AgNCs were very stable against extreme pH, ionic strength, temperature, and photoillumination and could act as sensitive and selective Cu(2+) sensing nanoprobes in aqueous solutions with a 10 nM limit of detection. In addition, hPEI-AgNCs-doped agarose hydrogels were developed as an instrument-free and regenerable platform for visual Cu(2+) and water quality monitoring.

Color difference amplification between gold nanoparticles in colorimetric analysis with actively controlled multiband illumination.

Spectral chemical sensing with digital color analysis by using consumer imaging devices could potentially revolutionize personalized healthcare. However, samples with small spectral variations often cannot be differentiated in color due to the nonlinearity of color appearance. In this study, we address this problem by exploiting the color image formation mechanism in digital photography. A close examination of the color image processing pipeline emphasizes that although the color can be represented digitally, it is still a reproducible subjective perception rather than a measurable physical property. That makes it possible to physically manage the color appearance of a nonradiative specimen through engineered illumination. By using scattering light imaging of gold nanoparticles (GNPs) as a model system, we demonstrated via simulation that enlarged color difference between spectrally close samples could be achieved with actively controlled illumination of multiple narrow-band light sources. Experimentally, darkfield imaging results indicate that color separation of single GNPs with various sizes can be significantly improved and the detection limit of GNP aggregation-based colorimetric assays can be much reduced when the conventional spectrally continuous white light was replaced with three independently intensity-controlled laser beams, even though the laser lines were uncorrelated with the LSPR maxima of the GNPs. With low-cost narrow-band light sources widely available today, this actively controlled illumination strategy could be utilized to replace the spectrometer in many spectral sensing applications.

High-throughput sulfide sensing with colorimetric analysis of single Au-Ag core-shell nanoparticles.

We present a high-throughput strategy for sensitive detection of H2S by using individual spherical Au-Ag core-shell plasmonic nanoparticles (PNPs) as molecular probes. This method is based on quantification of color variation of the single PNPs resulting from formation of Ag2S on the particle surface. The spectral response range of the 51 nm PNP was specifically designed to match the most sensitive region of color cameras. A high density of immobilized PNPs and rapid color RGB (red/green/blue) analysis allow a large number of individual PNPs to be monitored simultaneously, leading to reliable quantification of color change of the PNPs. A linear logarithmic dependence on sulfide concentrations from 50 nM to 100 µM was demonstrated by using this colorimetric assay. By designing PNPs with various surface chemistries, similar strategies could be developed to detect other chemically or biologically important molecules.

Subdiffraction-limited plasmonic imaging with anisotropic metal nanoparticles.

We have developed a high-resolution nonfluorescent imaging method based on superlocalization of gold nanorods (AuNRs). By taking advantage of their anisotropic optical property of the plasmonic scattering of AuNRs, selective imaging of only a fraction of AuNRs can be achieved by rotating the sample relative to the linear polarized illumination under cross-polarization microscopy with a high NA objective. The AuNR positions obtained from a series of images could then be used to reconstruct the overall image. Two AuNRs with center-to-center distances of 80 nm were successfully resolved. This simple but deterministic super-resolution imaging technique can potentially be used to fingerprint optically anisotropic metal nanoparticles and their assemblies for labeling, sensing, and encryption applications.

Pericellular matrix plays an active role in retention and cellular uptake of large-sized nanoparticles.

As the outmost coating of cells, the pericellular matrix (PCM) involved in various cellular functions has been exploited previously to be able to accumulate 120 nm Au nanoparticles (NPs), adjust their diffusion coefficient similar to that of membrane receptors, and enhance their uptake efficiency. In this study, the interactions between PCM and NPs with different sizes and materials were systematically investigated. We found that PCM can selectively enhance the retention and cellular uptake of NPs with diameters from 50 to 180 nm, but has no enhancement effect for 20 nm NPs. Identical behaviors of PCM was observed for both Au NPs and polystyrene NPs, indicating that this unique phenomenon is more related to the dimensions of the NPs. The study of single-particle tracking of 50-180 nm NPs on the surface of thick PCM cells revealed that PCM actively adjusts the diffusion coefficient of NPs to ∼0.1 µm(2)/s regardless of their sizes. By blocking the receptor-mediated endocytosis (RME) pathway with four different inhibitors, this active role of PCM can be effectively suppressed, further confirming that the trapping and retention of NPs by PCM is an inherent biological function. These findings provided new insights for better understanding of the RME pathway and may have promising NP-based applications for controlled drug delivery and therapy in biomedicine.

Direct observation of the orientation dynamics of single protein-coated nanoparticles at liquid/solid interfaces.

We have observed the rotational dynamics of single protein-coated gold nanorods (AuNRs) on C18-modified silica surfaces in real time by dual-channel polarization dark-field microscopy. Four different rotational states were identified, depending on the apparent strength of interactions between the AuNRs and the surface. The distributions of the states could be regulated by adjusting the salt concentration, and the state transitions were verified by monitoring the entire desorption process of a single AuNR. Our study provides insight into the interfacial orientation and dynamics of nanoparticles and could be useful for in vitro biophysics and the separation of proteins.

Use of mass spectrometry for imaging metabolites in plants.

We discuss and illustrate recent advances that have been made to image the distribution of metabolites among cells and tissues of plants using different mass spectrometry technologies. These technologies include matrix-assisted laser desorption ionization, desorption electrospray ionization, and secondary ion mass spectrometry. These are relatively new technological applications of mass spectrometry and they are providing highly spatially resolved data concerning the cellular distribution of metabolites. We discuss the advantages and limitations of each of these mass spectrometric methods, and provide a description of the technical barriers that are currently limiting the technology to the level of single-cell resolution. However, we anticipate that advances in the next few years will increase the resolving power of the technology to provide unprecedented data on the distribution of metabolites at the subcellular level, which will increase our ability to decipher new knowledge concerning the spatial organization of metabolic processes in plants.

Can protein conformers be fractionated by crystallization?

Molecular crystallization typically singles out a specific conformation, or a set of conformations that are identical over large parts and may show some flexibility, from a mixture of equilibrating conformations in solution. To critically evaluate the selectivity of this process, human lactate dehydrogenase isozyme 1 (LDH-1) microcrystals were separately dissolved and subsequently assayed inside capillaries with electrophoretically mediated microanalysis (EMMA) at both the ensemble and the single-molecule level. While fragments from the same crystal exhibited identical enzyme activities, different crystals, even when grown from the same drop of mother liquor, showed markedly different activities. Activities of individual molecules from a crystal were found to be essentially identical, whereas molecules obtained directly from solution showed a 4-fold variation in activity. Furthermore, after storage at 37 °C, the distribution of single-molecule LDH activities from solutions of individual crystals broadened and approached that of LDH obtained from the original solution. X-ray crystallography also showed distinct conformations for single microcrystals and confirms that crystallization properly selects even small conformational variants of proteins and that the slow equilibration to multiple stable conformations in solution is responsible for the observed single-molecule heterogeneity.

Frozen translational and rotational motion of human immunodeficiency virus transacting activator of transcription peptide-modified nanocargo on neutral lipid bilayer.

With time-resolved high-precision single-particle tracking methodologies, we explored the adsorption and thermal motion of transacting activator of transcription (TAT) peptide-modified nanocargo on a model lipid bilayer in the nonelectrostatic domain. We found that the lateral and rotational motion of TAT peptide-modified nanocargo could be effectively suppressed on the surface of neutral lipid membrane, a feature that cannot be explained by existing hypotheses. A semiquantitative association activation energy analysis revealed that multiple weak bonds were required for the initial adsorption process. As a result, the localized multiple TAT peptides on the surface of the nanocargo can provide a pathway for the creation of a net of peptide-lipid complexes (e.g., lipid domain). The dragging forces caused by these complexes effectively confined the thermal motion of the nanocargo on the fluid membrane that cannot be achieved by individual peptides with random spatial and conformational distributions. These interesting findings could provide insightful information for the better understanding of the intracellular internalization mechanism of TAT peptide-modified nanocargo and might shed new light on the development of highly efficient intracellular carriers for site-specific delivery of drugs and genes.

Probing single-molecule fluorescence spectral modulation within individual hotspots with subdiffraction-limit image resolution.

The enhancement of the electromagnetic field on the rough metallic nanostructure has been extensively applied to obtain chemical or biological information about molecules with high sensitivity and has received much attention due to its potential applications in new types of devices based on nanoelectronics and nanophotonics. The typical size of the field enhancement area, the so-called hotspot, is approximately 1 order of magnitude smaller than the optical diffraction limit. In the present study, an optical super-resolution microscopic and spectroscopic approach is introduced to explore single-molecule fluorescence within a hotspot where nonhomogeneous spectral modulation is resolved beyond the optical diffraction limit for the first time. Distinct Stokes shifts from individual dyes were directly observed within single hotspots, which were found to be independent of the local electromagnetic field strength. The method reported here provides a robust tool to probe the optical properties of nanoresonantors with high temporal and spatial resolution.

Photobleaching of quantum dots by non-resonant light.

Core-shell quantum dots suffer from photobleaching by light at wavelengths longer than their emission wavelengths. That is, QD photobleaching can be triggered by photons with low energies that are insufficient to pump electrons into the conduction band. The most probable reason is that electrons are pumped into a surface state and then nonradiatively decayed as in conventional photobleaching.

Pericellular matrix enhances retention and cellular uptake of nanoparticles.

A hydrated gel-like pericellular matrix (PCM) covers the surface of all eukaryotic cells and plays a key role in many cellular events, but its effect on nanoparticle internalization has not been studied. Here, using cells with various PCM thicknesses and gold nanoparticles as probes, we demonstrate that, rather than being a barrier to all foreign objects, the PCM can entrap and accumulate NPs, restrict and slow down their diffusion, and enhance their cellular uptake efficiency. Moreover, this newly discovered PCM function consumes energy and seems to be an integral part of the receptor-mediated endocytosis process. These findings are important in understanding the delivery mechanisms of nanocarriers for biomedical applications.

Superlocalization spectral imaging microscopy of a multicolor quantum dot complex.

The key factor of realizing super-resolution optical microscopy at the single-molecule level is to separately position two adjacent molecules. An opportunity to independently localize target molecules is provided by the intermittency (blinking) in fluorescence of a quantum dot (QD) under the condition that the blinking of each emitter can be recorded and identified. Herein we develop a spectral imaging based color nanoscopy which is capable of determining which QD is blinking in the multicolor QD complex through tracking the first-order spectrum, and thus, the distance at tens of nanometers between two QDs is measured. Three complementary oligonucleotides with lengths of 15, 30, and 45 bp are constructed as calibration rulers. QD585 and QD655 are each linked at one end. The measured average distances are in good agreement with the calculated lengths with a precision of 6 nm, and the intracellular dual-color QDs within a diffraction-limited spot are distinguished.