Measurement science has been converging to smaller and smaller samples, such that it is now possible to detect single molecules. This Review focuses on the next generation of analytical tools that ...combine single‐molecule detection with the ability to measure many single molecules simultaneously and/or process larger and more complex samples. Such single‐molecule sensors constitute a new type of quantitative analytical tool, as they perform analysis by molecular counting and thus potentially capture the heterogeneity of the sample. This Review outlines the advantages and potential of these new, quantitative single‐molecule sensors, the measurement challenges in making single‐molecule devices suitable for analysis, the inspiration biology provides for overcoming these challenges, and some of the solutions currently being explored.
More than the sum of its parts: Advances in measurement science have seen a progressive reduction in sample size to the point that single‐molecule measurements are today commonplace. A new generation of sensors is expected that perform quantitative analysis by measuring many single‐molecule events. This Review discusses the challenges, opportunities, and recent developments in quantitative single‐molecule sensors.
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.
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.
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.
It is now recognized that lipids and proteins in cellular membranes are not homogenously distributed. A high degree of membrane order is the biophysical hallmark of cholesterol-enriched lipid rafts, ...which may induce the lateral sorting of proteins within the membrane. Here we describe a quantitative fluorescence microscopy technique for imaging localized lipid environments and measuring membrane lipid order in live and fixed cells, as well as in intact tissues. The method is based on the spectral ratiometric imaging of the polarity-sensitive membrane dyes Laurdan and di-4-ANEPPDHQ. Laurdan typically requires multiphoton excitation, making it suitable for the imaging of tissues such as whole, living zebrafish embryos, whereas di-4-ANEPPDHQ imaging can be achieved with standard confocal microscopes. This approach, which takes around 4 h, directly examines the organization of cellular membranes and is distinct from alternative approaches that infer membrane order by measuring probe partitioning or dynamics.
Clustering of the T-cell receptor (TCR) is thought to initiate downstream signalling. However, the detection of protein clustering with high spatial and temporal resolution remains challenging. Here ...we establish a Förster resonance energy transfer (FRET) sensor, named CliF, which reports intermolecular associations of neighbouring proteins in live cells. A key advantage of the single-chain FRET sensor is that it can be combined with image correlation spectroscopy (ICS), single-particle tracking (SPT) and fluorescence lifetime imaging microscopy (FLIM). We test the sensor with a light-sensitive actuator that induces protein aggregation upon radiation with blue light. When applied to T cells, the sensor reveals that TCR triggering increases the number of dense TCR-CD3 clusters. Further, we find a correlation between cluster movement within the immunological synapse and cluster density. In conclusion, we develop a sensor that allows us to map the dynamics of protein clustering in live T cells.
Lipid microdomains are postulated to regulate many membrane-associated processes but have remained highly controversial. Here we provide the first direct evidence that the plasma membrane of intact, ...live cells is comprised of a sub-resolution mixture of approximately 76% ordered and 24% disordered lipid domains, which correspond to liquid-ordered and -disordered model membranes. These measurements were based on the unmixing of fluorescence lifetime decays (phasor analysis) obtained from environmentally sensitive membrane dyes that report the degree of lipid packing. Using the transmembrane protein Linker for Activation of T cells (LAT) as an example, we demonstrate that association with ordered domains retarded LAT diffusion and decreased clustering in meso-scaled protein domains as analysed by super-resolution microscopy. Our data therefore propose a membrane model in which the majority of the plasma membrane is covered by cholesterol-dependent, ordered lipid domains that contribute to the non-random distribution and diffusion of membrane constituents.
For the fabrication of perovskite solar cells (PSCs) using a solution process, it is essential to understand the characteristics of the perovskite precursor solution to achieve high performance and ...reproducibility. The colloids (iodoplumbates) in the perovskite precursors under various conditions were investigated by UV–visible absorption, dynamic light scattering, photoluminescence, and total internal reflection fluorescence microscopy techniques. Their local structure was examined by in situ X-ray absorption fine structure studies. Perovskite thin films on a substrate with precursor solutions were characterized by transmission electron microscopy, X-ray diffraction analysis, space-charge-limited current, and Kelvin probe force microscopy. The colloidal properties of the perovskite precursor solutions were found to be directly correlated with the defect concentration and crystallinity of the perovskite film. This work provides guidelines for controlling perovskite films by varying the precursor solution, making it possible to use colloid-engineered lead halide perovskite layers to fabricate efficient PSCs.
Telomeres regulate DNA damage response (DDR) and DNA repair activity at chromosome ends. How telomere macromolecular structure contributes to ATM regulation and its potential dissociation from ...control over non-homologous end joining (NHEJ)-dependent telomere fusion is of central importance to telomere-dependent cell aging and tumor suppression. Using super-resolution microscopy, we identify that ATM activation at mammalian telomeres with reduced TRF2 or at human telomeres during mitotic arrest occurs specifically with a structural change from telomere loops (t-loops) to linearized telomeres. Additionally, we find the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that ATM activation and telomere linearity occur separately from telomere fusion via NHEJ and that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1 arrest, when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that specifically regulate ATM activation independent of telomere mechanisms to inhibit NHEJ.
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•The TRFH domain of TRF2 regulates t-loop formation•ATM is suppressed at chromosome ends when telomeres are in a t-loop conformation•ATM is activated when telomeres are linearized by altered TRF2 or mitotic arrest•Linearized DDR-positive telomeres can remain NHEJ resistant even during G1 arrest
Van Ly et al. identify that telomere loops (t-loops) function as conformational switches that regulate ATM activity at chromosome ends. They find ATM activity is suppressed when telomeres adopt a t-loop conformation and that ATM is activated with linearized chromosome ends.
Recently developed super‐resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can ...drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been hampered by the resolution limit of optical microscopy, making the definition of their properties problematic and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state‐of‐the art model.
Editor's suggested further reading in BioEssays: Super‐resolution imaging prompts re‐thinking of cell biology mechanisms and Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair‐correlation analysis
We review how new super‐resolution fluorescence microscopy methods such as photoactivated localisation microscopy (PALM), stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) are being used to study membrane organisation. We especially focus on ordered‐phase lipid microdomains (lipid rafts) and their role in regulating protein distribution and diffusion.