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This study presents the combination of electrochemical cell based biosensor and fluorescence microscopy on a single platform to track biomolecular processes contributing to the morphology changes of the cells. Initial experiments demonstrate that using interdigitated electrodes where the gold microfingers were comparable in width and spacing to a single cell have the optimal sensitivity in the final electrochemical cell based-sensing device. This was determined by measuring the cell index, based on the impedance analysis of bare and cell-covered microelectrodes. The fabrication of the electrodes on a glass substrate enabled the capture of high-resolution fluorescence microscopy images of single cells and related intracellular calcium release inside the HeLa cells via a window incorporated into the gold microelectrode design. As an illustration of the enhanced capability of the combined approach over traditional impedance cellular assay, the opto-electric assay was utilized as a functional readout for G protein couple receptor activation. The simultaneous examinations of cells stimulated with histamine demonstrated an association between time courses of changes in cytosolic calcium concentration and reductions in cell-cell adhesions.

Stochastic optical reconstruction microscopy (STORM) allows wide-field imaging with single-molecule resolution by calculating the coordinates of individual fluorophores from the separation of fluorophore emission in both time and space. Such separation is achieved by photoswitching the fluorophores between a long-lived OFF state and an emissive ON state. Although STORM can image single molecules, molecular counting remains challenging due to undercounting errors from photobleached or overlapping dyes and overcounting artefacts from the repetitive random blinking of dyes. Here we show that fluorophores can be electrochemically switched for STORM imaging (EC-STORM), with excellent control over the switching kinetics, duty cycle and recovery yield. Using EC-STORM, we demonstrate molecular counting by using electrochemical potential to control the photophysics of dyes. The random blinking of dyes is suppressed by a negative potential but the switching-ON event can be activated by a short positive-potential pulse, such that the frequency of ON events scales linearly with the number of underlying dyes. We also demonstrate EC-STORM of tubulin in fixed cells with a spatial resolution as low as ~28 nm and counting of single Alexa 647 fluorophores on various DNA nanoruler structures. This control over fluorophore switching will enable EC-STORM to be broadly applicable in super-resolution imaging and molecular counting.

Synthetic hydrogels have been used widely as extracellular matrix (ECM) mimics due to the ability to control and mimic physical and biochemical cues observed in natural ECM proteins such as collagen, laminin, and fibronectin. Most synthetic hydrogels are formed via covalent bonding resulting in slow gelation which is incompatible with drop-on-demand 3D bioprinting of cells and injectable hydrogels for therapeutic delivery. Herein, we developed an electrostatically crosslinked PEG-based hydrogel system for creating high-throughput 3D in vitro models using synthetic hydrogels to mimic the ECM cancer environment. A 3-arm PEG-based polymer backbone was first modified with either permanent cationic charged moieties (2-(methacryloyloxy)ethyl trimethylammonium) or permanent anionic charged moieties (3-sulfopropyl methacrylate potassium salt). The resulting charged polymers can be conjugated further with various amounts of cell adhesive RGD motifs (0, 25, 75, and 98%) to study the influences of RGD motifs on breast cancer (MCF-7) spheroid formation. Formation, stability, and mechanical properties of hydrogels were tested with, and without, RGD to evaluate the cellular response to material parameters in a 3D environment. The hydrogels can be degraded in the presence of salts at room temperature by breaking the interaction of oppositely charged polymer chains. MCF-7 cells could be released with high viability through brief exposure to NaCl solution. Flow cytometry characterization demonstrated that embedded MCF-7 cells proliferate better in a softer (60 Pa) 3D hydrogel environment compared to those that are stiffer (1160 Pa). As the stiffness increases, the RGD motif plays a role in promoting cell proliferation in the stiffer hydrogel. Flow cytometry characterization demonstrated that embedded MCF-7 cells proliferate better in a softer (60 Pa) 3D hydrogel environment compared to those that are stiffer (1160 Pa). As the stiffness increases, the RGD motif plays a role in promot

Herein is explored a dual optical and electrical cell based biosensor that can provide insights into cellular events. The fabrication steps along with the electrical characterization of the microelectrodes is described. Initial experiments show that the ability of indium tin oxide (ITO) to detect biological cells at the electrode-cell layer interface mainly depends on the size of sensing area. Following this, the impact of conductivity was also explored to assess the real time impedance signal during the adhesion of a non-confluent layer of the cells on different substrates. Electrodes with a higher conductivity than ITO gave much higher senstivity of impedance changes which allowed the dynamics of subtle cellular morphology changes to be monitored at densities far lower than a confluent layer of cells. Finally, the capability of ITO and gold microelectrode as a functional readout for G protein couple receptor activation were determined. These set of analyses highlighted the challenges and opportunities of ITO substrate as a dual cell-based assay for quantitative analysis of subtle changes in cell morphology.

Switchable fluorescent proteins, for which fluorescence can be switched ON and OFF, are widely used for molecule tracking and super resolution imaging. However, the robust use of the switchable fluorescent proteins is still limited as either the switching is not repeatable, or such switching requires irradiation with coupled lasers of different wavelengths. Herein, we report an electrochemical approach to reversible fluorescence switching for enhanced green fluorescent proteins (EGFP) on indium tin oxide coated glass. Our results demonstrate that negative and positive electrochemical potentials can efficiently switch the fluorescent proteins between the dim (OFF) and bright (ON) states at the single molecule level. The electrochemical fluorescence switching is fast, reversible, and may be performed up to hundreds of cycles before photobleaching occurs. These findings highlight that this method of electrochemical fluorescence switching can be incorporated into advanced fluorescence microscopy.

Maize (Zea mays) is a major staple crop in Africa, where its yield and the livelihood of millions are compromised by the parasitic witchweed Striga. Germination of Striga is induced by strigolactones exuded from maize roots into the rhizosphere. In a maize germplasm collection, we identified two strigolactones, zealactol and zealactonoic acid, which stimulate less Striga germination than the major maize strigolactone, zealactone. We then showed that a single cytochrome P450, ZmCYP706C37, catalyzes a series of oxidative steps in the maize-strigolactone biosynthetic pathway. Reduction in activity of this enzyme and two others involved in the pathway, ZmMAX1b and ZmCLAMT1, can change strigolactone composition and reduce Striga germination and infection. These results offer prospects for breeding Striga-resistant maize.

We propose a theoretical model for the influence of confinement on biomolecular binding at the single-molecule scale at equilibrium, based on the change of the number of microstates (localization and orientation) upon reaction. Three cases are discussed: DNA sequences shorter and longer than the single strain DNA Kuhn length and spherical proteins, confined into a spherical container (liposome, droplet, etc.). The influence of confinement is found to be highly dependent on the molecular structure and significant for large molecules (relative to container size).

Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Two-photon direct laser writing is capable of nanofabricating arbitrary three-dimensional structures with nanometer accuracy. Here, we explore direct laser writing for high-resolution optical microscopy by fabricating unique 3D optical fiducials for single-molecule tracking and 3D single-molecule localization microscopy. By having control over the position and three-dimensional architecture of the fiducials, we improve axial discrimination and demonstrate isotropic subnanometer 3D focusing (<0.8 nm) over tens of micrometers using a standard inverted microscope. We perform 3D single-molecule acquisitions over cellular volumes, unsupervised data acquisition and live-cell single-particle tracking with nanometer accuracy.

Single molecule experiments have recently attracted enormous interest. Many of these studies involve the encapsulation of a single molecule into nanoscale containers (such as vesicles, droplets and nanowells). In such cases, the single molecule encapsulation efficiency is a key parameter to consider in order to get a statistically significant quantitative information. It has been shown that such encapsulation typically follows a Poisson distribution and such theory of encapsulation has only been applied to the encapsulation of single molecules into perfectly sized monodispersed containers. However, experimentally nanocontainers are usually characterized by a size distribution, and often just a single binding pair (rather than a single molecule) is required to be encapsulated. Here the use of Poisson distribution is extended to predict the encapsulation efficiency of two different molecules in an association equilibrium. The Poisson distribution is coupled with a log-normal distribution in order to consider the effect of the container size distribution, and the effect of adsorption to the container is also considered. This theory will allow experimentalists to determine what single molecule encapsulation efficiency can be expected as a function of the experimental conditions. Two case studies, based on experimental data, are given to support the theoretical predictions.

T cells are highly sensitive to low levels of antigen, but how this sensitivity is achieved is currently unknown. Here, we imaged proximal TCR-CD3 signal propagation with single molecule localization microscopy (SMLM) in T cells activated with nanoscale clusters of TCR stimuli. We observed the formation of large TCR-CD3 clusters that exceeded the area of the ligand clusters, and required multivalent interactions facilitated by TCR-CD3 phosphorylation for assembly. Within these clustered TCR-CD3 domains, TCR-CD3 signaling spread laterally for ∼500 nm, far beyond the activating site, via non-engaged receptors. Local receptor density determined the functional cooperativity between engaged and non-engaged receptors, but lateral signal propagation was not influenced by the genetic deletion of ZAP70. Taken together, our data demonstrates that clustered ligands induced the clustering of non-ligated TCR-CD3 into domains that cooperatively facilitate lateral signal propagation.

MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B-related metabolite antigens from diverse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system.

Macropinocytosis is a consequence of oncogenic alterations of cancer cells while most healthy cells are non-macropinocytic. It is currently unclear whether macropinocytic cancer cells can be targeted rather than healthy cells, by adjusting the shape and size of nanoparticles. Herein, the endocytosis of two differently shaped nanoparticles; nanorods and nanospheres are compared in cancer and healthy cells. The cells are breast epithelial cancer cells (MCF7) and breast epithelial healthy cells (MCF10A) and pancreas cancer cells (PANC-1 cells) and non-tumourogenic patient-derived cancer-associated fibroblasts (CAFs). The endocytosis pathway is quantified by a combination of pair correlation microscopy and endocytosis inhibitors. MCF7 cells use clathrin-mediated endocytosis and macropinocytosis to take up the nanorods while MCF10A cells use predominantly clathrin-mediated endocytosis. Based on the comparison of endocytic behavior of cancer and healthy cells, MCF7 cells can be induced to take up more nanorods and suppress the metabolism and endocytosis of nanorods in MCF10A cells. The nanorods allow targeting to breast cancer MCF7 cells and pancreas cancer cells over the healthy cells. This study opens exciting possibilities for shape to target the cancer cells over healthy cells, by adjusting nanoparticle shape.

Impedance spectroscopy is a widely used technique for monitoring cell-surface interactions and morphological changes, typically based on averaged signals from thousands of cells. However, acquiring impedance data at the single cell level, can potentially reveal cell-to-cell heterogeneity for example in response to chemotherapeutic agents such as doxorubicin. Here, we present a generic platform where light is used to define and localize the electroactive area, thus enabling the impedance measurements for selected single cells. We firstly tested the platform to assess phenotypic changes in breast cancer cells, at the single cell level, using the change in the cell impedance. We next show that changes in electrochemical noise reflects instantaneous responses of the cells to drugs, prior to any phenotypical changes. We used doxorubicin and monensin as model drugs and found that both drug influx and efflux events affect the impedance noise signals. Finally, we show how the electrochemical noise signal can be combined with fluorescence microscopy, to show that the noise provides information on cell susceptibility and resistance to drugs at the single cell level. Together the combination of electrochemical impedance and electrochemical noise with fluorescence microscopy provides a unique approach to understanding the heterogeneity in the response of single cells to stimuli where there is not phenotypic change.

It was recently shown that it is possible to exploit the nanoparticle shape to selectively target endocytosis pathways found in cancer and not healthy cells. It is important to understand and compare the endocytosis pathways of nanoparticles in both cancer and healthy cells to restrict the healthy cells from taking up anticancer drugs to help reduce the side effects for patients. Here, the clathrin-mediated endocytosis inhibitor, hydroxychloroquine, and the anticancer drug, doxorubicin, are loaded into the same mesoporous silica nanorods. The use of nanorods was found to restrict the uptake by healthy cells but allowed cancer cells to take up the nanorods via the macropinocytosis pathway. Furthermore, it is shown that the nanorods can selectively deliver doxorubicin to the nucleus of breast cancer cells and to the cytoplasm of pancreatic cancer cells. The dual-drug-loaded nanorods were able to selectively kill the breast cancer cells in the presence of healthy breast cells. This study opens exciting possibilities of targeting cancer cells based on the material shape rather than targeting antibodies.

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.

A key part of any super-resolution technique involves accurately correcting for mechanical motion of the sample and setup during acquisition. If left uncorrected, drift degrades the resolution of the final reconstructed image and can introduce unwanted artifacts. Here, we describe how to implement active stabilization, thereby reducing drift to ~1 nm across all three dimensions. In this protocol, we show how to implement our method on custom and standard microscopy hardware. We detail the construction of a separate illumination and detection path, dedicated exclusively to acquiring the diffraction pattern of fiducials deposited on the imaging slide. We also show how to focus lock and adjust the focus in arbitrary nanometer step size increments. Our real-time focus locking is based on kHz calculations performed using the graphics processing unit. The fast calculations allow for rapid repositioning of the sample, which reduces drift below the photon-limited localization precision. Our approach allows for a single-molecule and/or super-resolution image acquisition free from movement artifacts and eliminates the need for complex algorithms or hardware installations. The method is also useful for long acquisitions which span over hours or days, such as multicolor super resolution. Installation does not require specialist knowledge and can be implemented in standard biological laboratories. The full protocol can be implemented within ~2 weeks.

FRET has been massively used to see if biomolecules were bounded or not by labelling both biomolecules by one dye of a FRET pair. This should give a digital answer to the question (fluorescence of the acceptor: high FRET efficency: molecules associated, fluorescence of the donor: low FRET efficency: molecules dissociated). This has been used, inter alia, at the single-molecule scale in containers, such as liposomes. One perspective of the field is to reduce the container’s size to study the effect of confinement on binding. The problem is that if the two dyes are encapsulated inside a small liposome, they could have a significant probability to be close one from the other one (and thus to undergo a high FRET efficiency event without binding). This is why we suggest here a theoretical model which gives mean FRET efficiency as a function of liposome radius (the model applies to any spherical container) and Förster radius to help the experimentalist to choose their experimental set-up. Besides, the influence of side effect on mean FRET efficiency has been studied as well. We show here that if this “background FRET” is most of the time non-quantitative, it can remain significant and which makes data analysis trickier. We could show as well that if this background FRET obviously increases when liposome radius decreases, this variation was lower than the one which could be expected because of side effect. We show as well the FRET efficiency function distribution which let the experimentalist know the probability to get one FRET efficiency value.

The data quality of high-resolution imaging can be markedly improved with active stabilization, which is based on feedback loops within the microscope that maintain the sample in the same location throughout the experiment. The purpose is to provide a highly accurate focus lock, therefore eliminating drift and improving localization precision. Here, we describe a step-by-step protocol for building a total internal reflection microscope combined with the feedback loops necessary for sample and detection stabilization, which we routinely use in single-molecule localization microscopy (SMLM). The performance of the final microscope with feedback loops, called feedback SMLM, has previously been described. We demonstrate how to build a replica of our system and include a list of the necessary optical components, tips, and an alignment strategy.

Incorporation of fluorinated moieties in polymeric nanoparticles has been shown in many instances to increase their uptake by living cells and, hence, has proven to be a useful approach to enhancing delivery to cells. However, it remains unclear how incorporation of fluorine affects critical transport processes, such as interactions with membranes, intracellular transport, and tumor penetration. In this study, we investigate the influence of fluorine on transport properties using a series of rationally designed poly(oligo(ethylene glycol) methyl ether acrylate)-block-perfluoropolyether (poly(OEGA)m-PFPE) copolymers. Copolymers with different fluorine contents were prepared and exhibit aggregate in solution in a manner dependent on the fluorine content. Doxorubicin-conjugated poly(OEGA)20-PFPE nanoparticles with lower fluorine content exist in solution as unimers, leading to greater exposure of hydrophobic PFPE segments to the cell surface. This, in turn, results in greater cellular uptake, deeper tumor penetration, as well as enhanced therapeutic efficacy compared to that with the micelle-state nanoaggregates (poly(OEGA)10-PFPE and poly(OEGA)5-PFPE) with higher fluorine content but with less PFPE exposed to the cell membranes. Our results demonstrate that the aggregation behavior of these fluorinated polymers plays a critical role in internalization and transport in living cells and 3D spheroids, providing important design criteria for the preparation of highly effective delivery agents.

Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ∼1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

Biotechnology; Cell Biology; Biomaterials

Malignant melanoma displays a high degree of cellular plasticity during disease progression. Signals in the tumor microenvironment are believed to influence melanoma plasticity through changes in the epigenetic state to guide dynamic differentiation and de-differentiation. Here we uncover a relationship between geometric features at perimeter regions of melanoma aggregates, and reprogramming to a stem cell-like state through histone marks H3K4Me2 and H3K9Ac. Using an in vitro tumor microengineering approach, we find spatial enrichment of these histone modifications with concurrent expression of stemness markers. The epigenetic modifier PRDM14 overlaps with H3K9Ac and shows elevated expression in cells along regions of perimeter curvature. siRNA knockdown of PRDM14 abolishes the MIC phenotype suggesting a role in regulating melanoma heterogeneity. Our results suggest mechanotransduction at the periphery of melanoma aggregates may orchestrate the activity of epigenetic modifiers to regulate histone state, cellular plasticity, and tumorigenicity.

Recently, DNA-PAINT single-molecule localization microscopy (SMLM) has shown great promise for quantitative imaging; however, labelling strategies thus far have relied on multivalent and affinity-based approaches. Here, the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA-docking strands and application of SMLM via DNA-PAINT is demonstrated. tagPAINT is then used for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy. The fluorescence intensity of the Alexa Fluor 647 decreased, or reached background levels, at reducing potentials but returned to normal levels at oxidizing potentials. These electrochemically induced changes in fluorescence were sensitive to pH despite that BSA-Alexa Fluor 647 fluorescence without applied potential is insensitive to pH between values of 4–10. The observed pH dependence indicated the involvement of electron and proton transfer in the fluorescence switching mechanism.

Although F-actin has a large number of binding partners and regulators, the number of phenotypic states available to the actin cytoskeleton is unknown. Here, we quantified 74 features defining filamentous actin (F-actin) and cellular morphology in >25 million cells after treatment with a library of 114,400 structurally diverse compounds. After reducing the dimensionality of these data, only ∼25 recurrent F-actin phenotypes emerged, each defined by distinct quantitative features that could be machine learned. We identified 2,003 unknown compounds as inducers of actin-related phenotypes, including two that directly bind the focal adhesion protein, talin. Moreover, we observed that compounds with distinct molecular mechanisms could induce equivalent phenotypes and that initially divergent cellular responses could converge over time. These findings suggest a conceptual parallel between the actin cytoskeleton and gene regulatory networks, where the theoretical plasticity of interactions is nearly infinite, yet phenotypes in vivo are constrained into a limited subset of practicable configurations.

© 2019 The Royal Society of Chemistry. This article focuses on how nanoparticle shape affects the cellular internalisation and transport of nanoparticles inside cells and what the different analytical methods for determining nanoparticle internalisation by cells tell us. Rod-shaped nanoparticles typically show greater cellular internalisation relative to spherical nanoparticles, although there are studies with contradictory conclusions. The contradiction may be a result of differences in the materials being used in the comparison and/or a result of the analytical method employed. Finally, future opportunities in studying cellular internalisation with 3D cell-culture models and light-sheet microscopy are discussed.

Nanopore sensors detect individual species passing through a nanoscale pore. This experimental paradigm suffers from long analysis times at low analyte concentration and non-specific signals in complex media. These limit effectiveness of nanopore sensors for quantitative analysis. Here, we address these challenges using antibody-modified magnetic nanoparticles ((anti-PSA)-MNPs) that diffuse at zero magnetic field to capture the analyte, prostate-specific antigen (PSA). The (anti-PSA)-MNPs are magnetically driven to block an array of nanopores rather than translocate through the nanopore. Specificity is obtained by modifying nanopores with anti-PSA antibodies such that PSA molecules captured by (anti-PSA)-MNPs form an immunosandwich in the nanopore. Reversing the magnetic field removes (anti-PSA)-MNPs that have not captured PSA, limiting non-specific effects. The combined features allow detecting PSA in whole blood with a 0.8 fM detection limit. Our ‘magnetic nanoparticle, nanopore blockade’ concept points towards a strategy to improving nanopore biosensors for quantitative analysis of various protein and nucleic acid species.

Modern high-throughput biosensors with sensitivity down to a single analyte molecule may be possible with single-molecule localization microscopy (SMLM). Functionalized surfaces can be fabricated with self-assembly monolayer chemistry on indium tin oxide (ITO) substrates but not glass. However, characterizations of SMLM-compatible fluorophores are primarily performed on glass substrates. Here we collect single-molecule kinetics data of isolated Alexa Fluor 647 molecules on bare and functionalized glass and ITO surfaces. Extracting the photophysical dynamics of the fluorophores allows direct comparison of behavior of this dye on these substrates and fitting data to a model that accounts for multiple reversible dark states. All surfaces had sensitivity sufficient to image single fluorophore molecules. Photophysical kinetics observed are similar between the two substrates. The photon yield from individual fluorophores was greatest on bare glass, but functionalized ITO surfaces showed superior yield to functionalized glass surfaces and nearly matched the yield of bare glass. Together these results indicate functionalized ITO as a promising substrate for modern single-molecule biosensors.

To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia-telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.

Nanofabricated and nanopatterned surfaces have revealed the sensitivity of cell adhesion to nanoscale variations in the spacing of adhesive ligands such as the tripeptide arginine-glycine-aspartic acid (RGD). To date, surface characterisation and cell adhesion are often examined in two separate experiments so that the localisation of ligands and adhesion proteins cannot be combined in the same image. Here we developed self-assembled monolayer chemistry for indium tin oxide (ITO) surfaces for single molecule localisation microscopy (SMLM). Cell adhesion and spreading were sensitive to average RGD spacing. At low average RGD spacing, a threshold exists of 0.8 RGD peptides per µm2 that tether cells to the substratum but this does not enable formation of focal adhesions. These findings suggest that cells can sense and engage single adhesive ligands but ligand clustering is required for cell spreading. Thus, our data reveal subtle differences in adhesion biology that may be obscured in ensemble measurements.

Single-molecule localization microscopy (SMLM) has created the opportunity of pushing fluorescence microscopy from being a biological imaging tool to a surface characterization and possibly even a quantitative analytical tool. The latter could be achieved by molecular counting using pointillist SMLM data sets. However, SMLM is especially sensitive to background fluorescent signals, which influences any subsequent analysis. Therefore, fabricating sensing surfaces that resist nonspecific adsorption of proteins, even after multiple modification steps, has become paramount. Herein is reported two different ways to modify surfaces: dichlorodimethylsilane-biotinylated bovine serum albumin-Tween-20 (DbT20) and poly-l-lysine grafted polyethylene glycol (PLL-PEG) mixed with biotinylated PLL-PEG (PLL-PEG/PEGbiotin). The results show that the ability to resist nonspecific adsorption of DbT20 surfaces deteriorates with an increase in the number of modification steps required after the addition of the DbT20, which limits the applicability of this surface for SMLM. As such, a new surface for SMLM that employs PLL-PEG/PEGbiotin was developed that exhibits ultralow amounts of nonspecific protein adsorption even after many modification steps. The utility of the surface was demonstrated for human influenza hemagglutinin-tagged mEos2, which was directly pulled down from cell lysates onto the PLL-PEG/PEGbiotin surface. The results strongly indicated that the PLL-PEG/PEGbiotin surface satisfies the criteria of SMLM imaging of a negligible background signal and negligible nonspecific adsorption.

Introduction: Interest in mesoporous silica nanoparticles for drug delivery has resulted in a good understanding of the impact of size and surface chemistry of these nanoparticles on their performance as drug carriers. Shape has emerged as an additional factor that can have a significant effect on delivery efficacy. Rod-shaped mesoporous silica nanoparticles show improvements in drug delivery relative to spherical mesoporous silica nanoparticles. Areas covered: This review summarises the synthesis methods for producing rod-shaped mesoporous silica nanoparticles for use in nanomedicine. The second part covers recent progress of mesoporous silica nanorods by comparing the impact of sphere and rod-shape on drug delivery efficiency. Expert opinion: As hollow mesoporous silica nanorods are capable of higher drug loads than most other drug delivery vehicles, such particles will reduce the amount of mesoporous silica in the body for efficient therapy. However, the importance of nanoparticle shape on drug delivery efficiency is not well understood for mesoporous silica. Studies that visualize and quantify the uptake pathway of mesoporous silica nanorods in specific cell types and compare the cellular uptake to the well-studied nanospheres should be the focus of research to better understand the role of shape in uptake.

For many normal and aberrant cell behaviours, it is important to understand the origin of cellular heterogeneity. Although powerful methods for studying cell heterogeneity have emerged, they are more suitable for common rather than rare cells. Exploring the heterogeneity of rare single cells is challenging because these rare cells must be first pre-concentrated and undergo analysis prior to classification and expansion. Here, a versatile capture & release platform consisting of an antibody-modified and electrochemically cleavable semiconducting silicon surface for release of individual cells of interest is presented. The captured cells can be interrogated microscopically and tested for drug responsiveness prior to release and recovery. The capture & release strategy was applied to identify rare tumour cells from whole blood, monitor the uptake of, and response to, doxorubicin and subsequently select cells for single-cell gene expression based on their response to the doxorubicin.

Two important challenges in the field of 19F magnetic resonance imaging (MRI) are the maintenance of high fluorine content without compromising imaging performance, and effective targeting of small particles to diseased tissue. To address these challenges, we have developed a series of perfluoropolyether (PFPE)-based hyperbranched (HBPFPE) nanoparticles with attached peptide aptamer as targeting ligands for specific in vivo detection of breast cancer with high 19F MRI sensitivity. A detailed comparison of the HBPFPE nanoparticles (NPs) with the previously reported trifluoroethyl acrylate (TFEA)-based polymers demonstrates that the mobility of fluorinated segments of the HBPFPE nanoparticles is significantly enhanced (19F T2 > 80 ms vs 31 ms), resulting in superior MR imaging sensitivity. Selective targeting was confirmed by auto- and pair correlation analysis of fluorescence microscopy data, in vitro immunofluorescence, in vivo 19F MRI, ex vivo fluorescence and 19F NMR. The results highlight the high efficiency of aptamers for targeting and the excellent sensitivity of the PFPE moieties for 19F MRI. Of relevance to in vivo applications, the PFPE-based polymers exhibit much faster clearance from the body than the previously introduced perfluorocarbon emulsions (t1/2 ∼20 h vs up to months). Moreover, the aptamer-conjugated NPs show significantly higher tumor-penetration, demonstrating the potential of these imaging agents for therapeutic applications. This report of the synthesis of polymeric aptamer-conjugated PFPE-based 19F MRI CAs with high fluorine content (∼10 wt %) demonstrates that these NPs are exciting candidates for detecting diseases with high imaging sensitivity.

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

Membrane charge has a critical role in protein trafficking and signaling. However, quantification of the effective electrostatic potential of cellular membranes has remained challenging. We developed a fluorescence membrane charge sensor (MCS) that reports changes in the membrane charge of live cells via Förster resonance energy transfer (FRET). MCS is permanently attached to the inner leaflet of the plasma membrane and shows a linear, reversible and fast response to changes of the electrostatic potential. The sensor can monitor a wide range of cellular treatments that alter the electrostatic potential, such as incorporation and redistribution of charged lipids and alterations in cytosolic ion concentration. Applying the sensor to T cell biology, we used it to identify charged membrane domains in the immunological synapse. Further, we found that electrostatic interactions prevented spontaneous phosphorylation of the T cell receptor and contributed to the formation of signaling clusters in T cells.

Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis.

Herein is presented a proof-of-concept study of protease sensing that combines nontoxic silicon quantum dots (SiQDs) with Förster resonance energy transfer (FRET). The SiQDs serve as the donor and an organic dye as the acceptor. The dye is covalently attached to the SiQDs using a peptide linker. Enzymatic cleavage of the peptide leads to changes in FRET efficiency. The combination of interfacial design and optical imaging presented in this work opens opportunities for use of nontoxic SiQDs relevant to intracellular sensing and imaging.

Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision.

Modern fluorescence microscopy requires software-controlled illumination sources with high power across a wide range of wavelengths. Diode lasers meet the power requirements and combining multiple units into a single fiber launch expands their capability across the required spectral range. We present the NicoLase, an open-source diode laser combiner, fiber launch, and software sequence controller for fluorescence microscopy and super-resolution microscopy applications. Two configurations are described, giving four or six output wavelengths and one or two single-mode fiber outputs, with all CAD files, machinist drawings, and controller source code openly available.

While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins,and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR) signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR-CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.

Nanoparticle size, surface charge and material composition are known to affect the uptake of nanoparticles by cells. However, whether nanoparticle shape affects transport across various barriers inside the cell remains unclear. Here we used pair correlation microscopy to show that polymeric nanoparticles with different shapes but identical surface chemistries moved across the various cellular barriers at different rates, ultimately defining the site of drug release. We measured how micelles, vesicles, rods and worms entered the cell and whether they escaped from the endosomal system and had access to the nucleus via the nuclear pore complex. Rods and worms, but not micelles and vesicles, entered the nucleus by passive diffusion. Improving nuclear access, for example with a nuclear localization signal, resulted in more doxorubicin release inside the nucleus and correlated with greater cytotoxicity. Our results therefore demonstrate that drug delivery across the major cellular barrier, the nuclear envelope, is important for doxorubicin efficiency and can be achieved with appropriately shaped nanoparticles.