Nanoscopic Characterization of Cell Migration under Flow Using Optical and Electron Microscopy.

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that takes place through essential interactions with adhesion molecules on an endothelial cell layer. The homing process of HSPCs begins with the tethering and rolling of the cells on the endothelial layer, which is achieved by the interaction between selectins on the endothelium to the ligands on HSPC/leukemic cells under shear stress of the blood flow. Although many studies have been based on in vitro conditions of the cells rolling over recombinant proteins, significant challenges remain when imaging HSPC/leukemic cells on the endothelium, a necessity when considering characterizing cell-to-cell interaction and rolling dynamics during cell migration. Here, we report a new methodology that enables imaging of stem-cell-intrinsic spatiotemporal details during its migration on an endothelium-like cell monolayer. We developed optimized protocols that preserve transiently appearing structures on HSPCs/leukemic cells during its rolling under shear stress for fluorescence and scanning electron microscopy characterization. Our new experimental platform is closer to in vivo conditions and will contribute to indepth understanding of stem-cell behavior during its migration and cell-to-cell interaction during the process of homing.

Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization.

BACKGROUND: Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 µm and a width of 30-250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. RESULTS: We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol-maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin-biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. CONCLUSION: The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion.

Organic J-Aggregate Nanodots with Enhanced Light Absorption and Near-Unity Fluorescence Quantum Yield.

Development of biocompatible fluorophores with small size, bright fluorescence, and narrow spectrum translate directly into major advances in fluorescence imaging and related techniques. Here, we discover that a small donor-acceptor-donor-type organic molecule consisting of a carbazole (Cz) donor and benzothiazole (BT) acceptor (CzBTCz) assembles into quasi-crystalline J-aggregates upon a formation of ultrasmall nanoparticles. The 3.5 nm CzBTCz Jdots show a narrow absorption spectrum (fwhm = 27 nm), near-unity fluorescence quantum yield (ϕfl = 0.95), and enhanced peak molar extinction coefficient. The superior spectroscopic characteristics of the CzBTCz Jdots result in two orders of magnitude brighter photoluminescence of the Jdots compared with semiconductor quantum dots, which enables continuous single-Jdots imaging over a 1 h period. Comparison with structurally similar CzBT nanoparticles demonstrates a critical role played by the shape of CzBTCz on the formation of the Jdots. Our findings open an avenue for the development of a new class of fluorescent nanoparticles based on J-aggregates.

Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain.

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

Millimeter-Deep Detection of Single Shortwave-Infrared-Emitting Polymer Dots through Turbid Media.

Fluorescence imaging at longer wavelengths, especially in the shortwave-infrared (SWIR: 1000-1700 nm) region, leads to a substantial decrease in light attenuation, scattering, and background autofluorescence, thereby enabling enhanced penetration into biological tissues. The limited selection of fluorescent probes is a major bottleneck in SWIR fluorescence imaging. Here, we develop SWIR-emitting nanoparticles composed of donor-acceptor-type conjugated polymers. The bright SWIR fluorescence of the polymer dots (primarily attributable to their large absorption cross-section and high fluorescence saturation intensity (as high as 113 kW·cm-2)) enables the unprecedented detection of single particles as small as 14 nm through millimeter-thick turbid media. Unlike most SWIR-emitting nanomaterials, which have an excited-state lifetime in the range of microseconds to milliseconds, our polymer dots exhibit a subnanosecond excited-state lifetime. These characteristics enable us to demonstrate new time-gated single-particle imaging with a high signal-to-background ratio. These findings expand the range of potential applications of single-particle deep-tissue imaging.

Uncoupling of sister replisomes during eukaryotic DNA replication.

The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes. Some data imply that they travel away from one another and thus function independently. Alternatively, sister replisomes may form a stationary, functional unit that draws parental DNA toward itself. If this "double replisome" model is correct, a constrained DNA molecule should not undergo replication. To test this prediction, lambda DNA was stretched and immobilized at both ends within a microfluidic flow cell. Upon exposure to Xenopus egg extracts, this DNA underwent extensive replication by a single pair of diverging replisomes. The data show that there is no obligatory coupling between sister replisomes and, together with other studies, imply that genome duplication involves autonomously functioning replisomes.

A general approach to break the concentration barrier in single-molecule imaging.

Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.

Shielding Effects Provide a Dominant Mechanism in J-Aggregation-Induced Photoluminescence Enhancement of Carbon Nanotubes.

The unique photophysical properties of single-walled carbon nanotubes (SWCNTs) exhibit great potential for bioimaging applications. This led to extensive exploration of photosensitization methods to improve their faint shortwave infrared (SWIR) photoluminescence. Here, we report the mechanisms of SWCNT-assisted J-aggregation of cyanine dyes and the associated photoluminescence enhancement of SWCNTs in the SWIR spectral region. Surprisingly, we found that excitation energy transfer between the cyanine dyes and SWCNTs makes a negligible contribution to the overall photoluminescence enhancement. Instead, the shielding of SWCNTs from the surrounding water molecules through hydrogen bond-assisted macromolecular reorganization of ionic surfactants triggered by counterions and the physisorption of the dye molecules on the side walls of SWCNTs play a primary role in the photoluminescence enhancement of SWCNTs. We observed 2 orders of magnitude photoluminescence enhancement of SWCNTs by optimizing these factors. Our findings suggest that the proper shielding of SWCNTs is the critical factor for their photoluminescence enhancement, which has important implications for their application as imaging agents in biological settings.

α1,3-fucosylation treatment improves cord blood CD34 negative hematopoietic stem cell navigation.

For almost two decades, clinicians have overlooked the diagnostic potential of CD34neg hematopoietic stem cells because of their limited homing capacity relative to CD34posHSCs when injected intravenously. This has contributed to the lack of appeal of using umbilical cord blood in HSC transplantation because its stem cell count is lower than bone marrow. The present study reveals that the homing and engraftment of CD34negHSCs can be improved by adding the Sialyl Lewis X molecule via α1,3-fucosylation. This unlocks the potential for using this more primitive stem cell to treat blood disorders because our findings show CD34negHSCs have the capacity to regenerate cells in the bone marrow of mice for several months. Furthermore, our RNA sequencing analysis revealed that CD34negHSCs have unique adhesion pathways, downregulated in CD34posHSCs, that facilitate interaction with the bone marrow niche. Our findings suggest that CD34neg cells will best thrive when the HSC resides in its microenvironment.

Single-molecule study on polymer diffusion in a melt state: effect of chain topology.

We report a new methodology for studying diffusion of individual polymer chains in a melt state, with special emphasis on the effect of chain topology. A perylene diimide fluorophore was incorporated into the linear and cyclic poly(THF)s, and real-time diffusion behavior of individual chains in a melt of linear poly(THF) was measured by means of a single-molecule fluorescence imaging technique. The combination of mean squared displacement (MSD) and cumulative distribution function (CDF) analysis demonstrated the broad distribution of diffusion coefficient of both the linear and cyclic polymer chains in the melt state. This indicates the presence of spatiotemporal heterogeneity of the polymer diffusion which occurs at much larger time and length scales than those expected from the current polymer physics theory. We further demonstrated that the cyclic chains showed marginally slower diffusion in comparison with the linear counterparts, to suggest the effective suppression of the translocation through the threading-entanglement with the linear matrix chains. This coincides with the higher activation energy for the diffusion of the cyclic chains than of the linear chains. These results suggest that the single-molecule imaging technique provides a powerful tool to analyze complicated polymer dynamics and contributes to the molecular level understanding of the chain interaction.

Spatiotemporal visualization of subcellular dynamics of carbon nanotubes.

To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of postuptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards.

A single-molecule study on polymer fluid dynamics in porous media.

Understanding the dynamic behavior of polymeric fluids in porous media is essential for vast geoscience applications, particularly enhanced oil recovery and polymer-enhanced soil washing, to clean up soil contamination. During the past decades, the behavior of polymeric fluids in microscopic space has only been investigated using ensemble-averaged experimental methods in which a bulk phase behavior of the fluids characterizes flow mechanisms. Multiple flow mechanisms have been proposed based on ensemble-averaged data; however, microscale characterization of the interactions between polymers and solid surfaces and the mechanisms governing polymer retention and permeability reduction as well as the reversibility of polymer retention are lacking, resulting in a limited understanding of the flow mechanisms. Here we report direct visualization and multi-scale characterization of the dynamic behavior of polymer molecules in a representative porous medium by integrating microfluidics with single-molecule imaging. We demonstrate that the polymers' adsorption, entrapment and hydrodynamic retention contribute to their overall retention in porous media. Our study illustrates how microfluidics can help in understanding the dynamic behavior of polymers, their interactions with the solid/fluid interface and their effects on flow properties. Additionally, it demonstrates the role of microfluidic platforms in providing a more representative and accurate model for polymer retention and permeability reduction in porous media. The obtained insights encourage the development of improved models that better capture the behavior of complex fluids in confined environments and have significant implications for a wide range of applications in geoscience, materials science, and rheology.

Experimental and numerical investigation of polymer pore-clogging in micromodels.

Polymers have been used effectively in the Oil & Gas Industry for a variety of field applications, such as enhanced oil recovery (EOR), well conformance, mobility control, and others. Polymer intermolecular interactions with the porous rock, in particular, formation clogging and the associated alterations to permeability, is a common problem in the industry. In this work, fluorescent polymers and single-molecule imaging are presented for the first time to assess the dynamic interaction and transport behavior of polymer molecules utilizing a microfluidic device. Pore-scale simulations are performed to replicate the experimental observations. The microfluidic chip, also known as a "Reservoir-on-a-Chip" functions as a 2D surrogate to evaluate the flow processes that take place at the pore-scale. The pore-throat sizes of an oil-bearing reservoir rock, which range from 2 to 10 nm, are taken into consideration while designing the microfluidic chip. Using soft lithography, we created the micromodel from polydimethylsiloxane (PDMS). The conventional use of tracers to monitor polymers has a restriction due to the tendency of polymer and tracer molecules to segregate. For the first time, we develop a novel microscopy method to observe the dynamic behavior of polymer pore-clogging and unclogging processes. We provide direct dynamic observations of polymer molecules during their transport within the aqueous phase and their clustering and accumulations. Pore-scale simulations were carried out to simulate the phenomena using a finite-element simulation tool. The simulations revealed a decline in flow conductivity over time within the flow channels that experienced polymer accumulation and retention, which is consistent with the experimental observation of polymer retention. The performed single-phase flow simulations allowed us to assess the flow behavior of the tagged polymer molecules within the aqueous phase. Additionally, both experimental observation and numerical simulations are used to evaluate the retention mechanisms that emerge during flow and how they affect apparent permeability. This work provides new insights to assessing the mechanisms of polymer retention in porous media.

CD34+ HSPCs-derived exosomes contain dynamic cargo and promote their migration through functional binding with the homing receptor E-selectin.

Exosomes are tiny vesicles released by cells that carry communications to local and distant locations. Emerging research has revealed the role played by integrins found on the surface of exosomes in delivering information once they reach their destination. But until now, little has been known on the initial upstream steps of the migration process. Using biochemical and imaging approaches, we show here that exosomes isolated from both leukemic and healthy hematopoietic stem/progenitor cells can navigate their way from the cell of origin due to the presence of sialyl Lewis X modifications surface glycoproteins. This, in turn, allows binding to E-selectin at distant sites so the exosomes can deliver their messages. We show that when leukemic exosomes were injected into NSG mice, they traveled to the spleen and spine, sites typical of leukemic cell engraftment. This process, however, was inhibited in mice pre-treated with blocking E-selectin antibodies. Significantly, our proteomic analysis found that among the proteins contained within exosomes are signaling proteins, suggesting that exosomes are trying to deliver active cues to recipient cells that potentially alter their physiology. Intriguingly, the work outlined here also suggests that protein cargo can dynamically change upon exosome binding to receptors such as E-selectin, which thereby could alter the impact it has to regulate the physiology of the recipient cells. Furthermore, as an example of how miRNAs contained in exosomes can influence RNA expression in recipient cells, our analysis showed that miRNAs found in KG1a-derived exosomes target tumor suppressing proteins such as PTEN.

The Pursuit of Shortwave Infrared-Emitting Nanoparticles with Bright Fluorescence through Molecular Design and Excited-State Engineering of Molecular Aggregates.

Shortwave infrared (SWIR) fluorescence detection gradually becomes a pivotal real-time imaging modality, allowing one to elucidate biological complexity in deep tissues with subcellular resolution. The key challenge for the further growth of this imaging modality is the design of new brighter biocompatible fluorescent probes. This review summarizes the recent progress in the development of organic-based nanomaterials with an emphasis on new strategies that extend the fluorescence wavelength from the near-infrared to the SWIR spectral range and amplify the fluorescence brightness. We first introduce the most representative molecular design strategies to obtain near-infrared-SWIR wavelength fluorescence emission from small organic molecules. We then discuss how the formation of nanoparticles based on small organic molecules contributes to the improvement of fluorescence brightness and the shift of fluorescence to SWIR, with a special emphasis on the excited-state engineering of molecular probes in an aggregate state and spatial packing of the molecules in nanoparticles. We build our discussion based on a historical perspective on the photophysics of molecular aggregates. We extend this discussion to nanoparticles made of conjugated polymers and discuss how fluorescence characteristics could be improved by molecular design and chain conformation of the polymer molecules in nanoparticles. We conclude the article with future directions necessary to expand this imaging modality to wider bioimaging applications including single-particle deep tissue imaging. Issues related to the characterization of SWIR fluorophores, including fluorescence quantum yield unification, are also mentioned.

Photostable polymorphic organic cages for targeted live cell imaging.

Fluorescent microscopy is a powerful tool for studying the cellular dynamics of biological systems. Small-molecule organic fluorophores are the most commonly used for live cell imaging; however, they often suffer from low solubility, limited photostability and variable targetability. Herein, we demonstrate that a tautomeric organic cage, OC1, has high cell permeability, photostability and selectivity towards the mitochondria. We further performed a structure-activity study to investigate the role of the keto-enol tautomerization, which affords strong and consistent fluorescence in dilute solutions through supramolecular self-assembly. Significantly, OC1 can passively diffuse through the cell membrane directly targeting the mitochondria without going through the endosomes or the lysosomes. We envisage that designing highly stable and biocompatible self-assembled fluorophores that can passively diffuse through the cell membrane while selectively targeting specific organelles will push the boundaries of fluorescent microscopy to visualize intricate cellular processes at the single molecule level in live samples.

Absorption linear dichroism measured directly on a single light-harvesting system: the role of disorder in chlorosomes of green photosynthetic bacteria.

Chlorosomes are light-harvesting antennae of photosynthetic bacteria containing large numbers of self-aggregated bacteriochlorophyll (BChl) molecules. They have developed unique photophysical properties that enable them to absorb light and transfer the excitation energy with very high efficiency. However, the molecular-level organization, that produces the photophysical properties of BChl molecules in the aggregates, is still not fully understood. One of the reasons is heterogeneity in the chlorosome structure which gives rise to a hierarchy of structural and energy disorder. In this report, we for the first time directly measure absorption linear dichroism (LD) on individual, isolated chlorosomes. Together with fluorescence-detected three-dimensional LD, these experiments reveal a large amount of disorder on the single-chlorosome level in the form of distributions of LD observables in chlorosomes from wild-type bacterium Chlorobaculum tepidum . Fluorescence spectral parameters, such as peak wavelength and bandwidth, are measures of the aggregate excitonic properties. These parameters obtained on individual chlorosomes are uncorrelated with the observed LD distributions and indicate that the observed disorder is due to inner structural disorder along the chlorosome long axis. The excitonic disorder that is also present is not manifested in the LD distributions. Limiting values of the LD parameter distributions, which are relatively free of the effect of structural disorder, define a range of angles at which the excitonic dipole moment is oriented with respect to the surface of the two-dimensional aggregate of BChl molecules. Experiments on chlorosomes of a triple mutant of Chlorobaculum tepidum show that the mutant chlorosomes have significantly less inner structural disorder and higher symmetry, compatible with a model of well-ordered concentric cylinders. Different values of the transition dipole moment orientations are consistent with a different molecular level organization of BChl's in the mutant and wild-type chlorosomes.

Multi-beam single-molecule defocused fluorescence imaging reveals local anisotropic nature of polymer thin films.

We developed a method to determine full three-dimensional orientation distribution of individual molecules based on wide-field defocused fluorescence imaging. Excitation efficiencies of out-of-plane oriented molecules were improved dramatically by illuminating molecules with multiple laser beams. Our high throughput approach allowed us to obtain unbiased statistical distributions of orientations of doped molecules in spin-coated polymer thin films. We found thickness- and glass transition temperature-dependent distributions of the molecular orientations which reflect local chain orientations and relaxation in the polymer thin films.

Molecular weight dependence of emission intensity and emitting sites distribution within single conjugated polymer molecules.

We investigated exciton migration, trapping and emission processes occurring within a single conjugated polymer molecule by means of superresolution fluorescence localization microscopy. This methodology allowed us to locate the spatial distribution of emitting sites within single chains with nanometre precision. The study was done on individual poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) molecules with average molecular weights ranging from 215,000 to 1,440,000 and with narrow weight distributions. We found that the mean emission intensity increases proportionally to the polymer molecular weight. The localization experiments suggest that the emitting sites are distributed nearly uniformly within a single chain and that the sites are on average 10 nm apart, irrespective of the molecular weight of the polymer. Furthermore, spatial contours formed by all the combined emitting sites within one chain show elongated shapes, in agreement with a rod-like structure of MEH-PPV in a collapsed state.

Single-molecule imaging and microfluidic platform reveal molecular mechanisms of leukemic cell rolling.

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that occurs through key interactions between adhesion molecules. Tethering and rolling of the cells on endothelium, the crucial initial step of the adhesion cascade, is mediated by interactions between selectins expressed on endothelium to their ligands expressed on HSPCs/leukemic cells in flow. Although multiple factors that affect the rolling behavior of the cells have been identified, molecular mechanisms that enable the essential slow and stable cell rolling remain elusive. Here, using a microfluidics-based single-molecule live cell fluorescence imaging, we reveal that unique spatiotemporal dynamics of selectin ligands on the membrane tethers and slings, which are distinct from that on the cell body, play an essential role in the rolling of the cell. Our results suggest that the spatial confinement of the selectin ligands to the tethers and slings together with the rapid scanning of a large area by the selectin ligands, increases the efficiency of selectin-ligand interactions during cell rolling, resulting in slow and stable rolling of the cell on the selectins. Our findings provide novel insights and contribute significantly to the molecular-level understanding of the initial and essential step of the homing process.