RESUMO
High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were â¼10 PE/â¼80 nm EV diameter for CytoFLEX and â¼10 PEs/â¼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.
Assuntos
Vesículas Extracelulares , Citometria de Fluxo , Citometria de Fluxo/métodos , Citometria de Fluxo/instrumentação , Humanos , Vesículas Extracelulares/metabolismo , Neoplasias Colorretais/diagnóstico , Linhagem Celular Tumoral , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/instrumentaçãoRESUMO
By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.
Assuntos
Imagem Individual de Molécula , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/instrumentação , Humanos , Animais , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/instrumentaçãoRESUMO
Optical microscopy has vastly expanded the frontiers of structural and functional biology, due to the non-invasive probing of dynamic volumes in vivo. However, traditional widefield microscopy illuminating the entire field of view (FOV) is adversely affected by out-of-focus light scatter. Consequently, standard upright or inverted microscopes are inept in sampling diffraction-limited volumes smaller than the optical system's point spread function (PSF). Over the last few decades, several planar and structured (sinusoidal) illumination modalities have offered unprecedented access to sub-cellular organelles and 4D (3D + time) image acquisition. Furthermore, these optical sectioning systems remain unaffected by the size of biological samples, providing high signal-to-noise (SNR) ratios for objective lenses (OLs) with long working distances (WDs). This review aims to guide biologists regarding planar illumination strategies, capable of harnessing sub-micron spatial resolution with a millimeter depth of penetration.
Assuntos
Imageamento Tridimensional/instrumentação , Imagem Individual de Molécula/instrumentação , Imagem com Lapso de Tempo/instrumentação , Iluminação , Microscopia de Fluorescência , Razão Sinal-RuídoRESUMO
Amplification-free genome analysis can revolutionize biology and medicine by uncovering genetic variations among individuals. Here, the authors report on a 3D-integrated nanopore for electrolysis to in situ detection of single-molecule DNA in a cell by ionic current measurements. It consists of a SiO2 multipore sheet and a SiNx nanopore membrane stacked vertically on a Si wafer. Single cell lysis is demonstrated by 106 V m-1 -level electrostatic field focused at the multinanopore. The intracellular molecules are then directly detected as they move through a sensing zone, wherein the authors find telegraphic current signatures reflecting folding degrees of freedom of the millimeter-long polynucleotides threaded through the SiNx nanopore. The present device concept may enable on-chip single-molecule sequencing to multi-omics analyses at a single-cell level.
Assuntos
DNA/análise , Imagem Individual de Molécula/instrumentação , Técnicas Biossensoriais , Humanos , Nanoporos , Dióxido de Silício/química , Imagem Individual de Molécula/métodos , Eletricidade EstáticaRESUMO
Recent advances in light and electron microscopy are uncovering viral lifecycle events with a level of detail never before seen [...].
Assuntos
Interações entre Hospedeiro e Microrganismos , Interpretação de Imagem Assistida por Computador/métodos , Imagem Individual de Molécula/métodos , Humanos , Interpretação de Imagem Assistida por Computador/instrumentação , Microscopia Eletrônica/métodos , Imagem Individual de Molécula/instrumentação , Replicação ViralRESUMO
FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 µm and various well depths ranging from 300 nm to 1 µm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 µm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.
Assuntos
Resinas Acrílicas/química , Separação Celular/métodos , Hidrocarbonetos Fluorados/química , Imunoensaio/métodos , Imagem Individual de Molécula/métodos , Anticorpos/análise , Anticorpos/imunologia , Separação Celular/instrumentação , Escherichia coli , Interações Hidrofóbicas e Hidrofílicas , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Imunoensaio/instrumentação , Imagem Individual de Molécula/instrumentação , Proteínas tau/química , Proteínas tau/imunologiaRESUMO
In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture-conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/µg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.
Assuntos
Técnicas de Química Analítica/métodos , Vesículas Extracelulares , Proteínas/isolamento & purificação , Linhagem Celular , Meios de Cultivo Condicionados , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Humanos , Leucemia Linfocítica Crônica de Células B , Microscopia Eletrônica/métodos , Nanopartículas , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodosRESUMO
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/imunologia , Imagem Individual de Molécula/métodos , Proteínas Virais/genética , Anticorpos Antivirais/sangue , Sequência de Bases , COVID-19/sangue , COVID-19/imunologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/normas , Teste Sorológico para COVID-19/normas , Ensaio de Imunoadsorção Enzimática , Humanos , Soros Imunes/química , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Nasofaringe/virologia , Poliproteínas/sangue , Poliproteínas/genética , RNA Viral/sangue , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade , Imagem Individual de Molécula/instrumentação , Proteínas Virais/sangueRESUMO
Single-Molecule Localization Microscopy (SMLM) provides the ability to determine molecular organizations in cells at nanoscale resolution, but in complex biological tissues, where sample-induced aberrations hamper detection and localization, its application remains a challenge. Various adaptive optics approaches have been proposed to overcome these issues, but the exact performance of these methods has not been consistently established. Here we systematically compare the performance of existing methods using both simulations and experiments with standardized samples and find that they often provide limited correction or even introduce additional errors. Careful analysis of the reasons that underlie this limited success enabled us to develop an improved method, termed REALM (Robust and Effective Adaptive Optics in Localization Microscopy), which corrects aberrations of up to 1 rad RMS using 297 frames of blinking molecules to improve single-molecule localization. After its quantitative validation, we demonstrate that REALM enables to resolve the periodic organization of cytoskeletal spectrin of the axon initial segment even at 50 µm depth in brain tissue.
Assuntos
Encéfalo/patologia , Óptica e Fotônica/métodos , Imagem Individual de Molécula/métodos , Algoritmos , Animais , Células COS , Chlorocebus aethiops , Microscopia de Fluorescência/instrumentação , Ratos , Imagem Individual de Molécula/instrumentação , SoftwareRESUMO
Single-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental modalities often resort to molecule immobilization for long observation times and do not always approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with ultrahigh resolving power in FRET efficiency. Importantly, single-molecule diffusivity is used to provide additional size and shape information for hydrodynamic profiling of individual molecules, which, together with the concurrently measured intramolecular conformation through FRET, enables a holistic and dynamic view of biomolecules and their complexes.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Dano ao DNA , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Transferência Ressonante de Energia de Fluorescência/instrumentação , Hidrodinâmica , Dispositivos Lab-On-A-Chip , Conformação Molecular , Ácidos Nucleicos Heteroduplexes/química , Fótons , Imagem Individual de Molécula/instrumentaçãoRESUMO
Non-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and ß2-spectrin in neurons. ASTER's sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.
Assuntos
Iluminação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Algoritmos , Animais , Células COS , Chlorocebus aethiops , Lasers , Luz , Microtúbulos , Reprodutibilidade dos TestesRESUMO
Cell-extracellular matrix sensing plays a crucial role in cellular behavior and leads to the formation of a macromolecular protein complex called the focal adhesion. Despite their importance in cellular decision making, relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here, we combine cRGD-ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling. We uncover that integrin receptor clustering is governed by a non-random organization with complexes spaced at 20-30 nm distances. The DNA-based tension sensor and analysis framework provide powerful tools to study a multitude of receptor-ligand interactions where forces are involved in ligand-receptor binding.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , DNA/metabolismo , Nanotecnologia/métodos , Imagem Individual de Molécula/métodos , Actinas/química , Actinas/ultraestrutura , Adesão Celular , Análise por Conglomerados , Citoesqueleto/ultraestrutura , DNA/química , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Humanos , Ligantes , Ligação Proteica , Imagem Individual de Molécula/instrumentação , Propriedades de Superfície , Talina/genética , Talina/metabolismoRESUMO
Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Antígenos de Histocompatibilidade/química , Receptores de Antígenos de Linfócitos T/química , Imagem Individual de Molécula/métodos , Anticorpos de Cadeia Única/química , Animais , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/imunologia , Citocromos c/química , Transferência Ressonante de Energia de Fluorescência/instrumentação , Antígenos de Histocompatibilidade/imunologia , Cinética , Ligantes , Bicamadas Lipídicas/química , Camundongos , Peptídeos/química , Receptores de Antígenos de Linfócitos T/imunologia , Imagem Individual de Molécula/instrumentação , Anticorpos de Cadeia Única/imunologia , Análise Espaço-TemporalRESUMO
This article presents answers to the questions on superresolution and structured illumination microscopy (SIM) as raised in the editorial of this collection of articles (https://doi.org/10.1098/rsta.2020.0143). These answers are based on my personal views on superresolution in light microscopy, supported by reasoning. Discussed are the definition of superresolution, Abbe's resolution limit and the classification of superresolution methods into nonlinear-, prior knowledge- and near-field-based superresolution. A further focus is put on the capabilities and technical aspects of present and future SIM methods. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.
Assuntos
Microscopia de Fluorescência/métodos , Algoritmos , Animais , Análise de Fourier , Humanos , Processamento de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Luz , Aprendizado de Máquina , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/estatística & dados numéricos , Dinâmica não Linear , Fenômenos Ópticos , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/estatística & dados numéricosRESUMO
We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope set-up and a mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localization microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as 'blinking'), detected and localized. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that stimulated emission depletion and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.
Assuntos
Microscopia de Fluorescência/instrumentação , Imagem Individual de Molécula/instrumentação , Anfíbios , Animais , Cromossomos/química , Cromossomos/ultraestrutura , Desenho de Equipamento , Corantes Fluorescentes , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Compostos Orgânicos , Estudo de Prova de Conceito , Imagem Individual de Molécula/métodos , Complexo Sinaptonêmico/química , Complexo Sinaptonêmico/ultraestrutura , XantenosRESUMO
Polysaccharides are important biomacromolecules existing in all plants, most of which are integrated into a fibrillar structure called the cell wall. In the absence of an effective methodology for polysaccharide analysis that arises from compositional heterogeneity and structural flexibility, our knowledge of cell wall architecture and function is greatly constrained. Here, we develop a single-molecule approach for identifying plant polysaccharides with acetylated modification levels. We designed a solid-state nanopore sensor supported by a free-standing SiN x membrane in fluidic cells. This device was able to detect cell wall polysaccharide xylans at concentrations as low as 5 ng/µL and discriminate xylans with hyperacetylated and unacetylated modifications. We further demonstrated the capability of this method in distinguishing arabinoxylan and glucuronoxylan in monocot and dicot plants. Combining the data for categorizing polysaccharide mixtures, our study establishes a single-molecule platform for polysaccharide analysis, opening a new avenue for understanding cell wall structures, and expanding polysaccharide applications.
Assuntos
Botânica/métodos , Nanoporos , Oryza/metabolismo , Polissacarídeos/análise , Imagem Individual de Molécula/métodos , Botânica/instrumentação , Imagem Individual de Molécula/instrumentaçãoRESUMO
The ability of magnetic tweezers to apply forces and measure molecular displacements has resulted in its extensive use to study the activity of enzymes involved in various aspects of nucleic acid metabolism. These studies have led to the discovery of key aspects of protein-protein and protein-nucleic acid interaction, uncovering dynamic heterogeneities that are lost to ensemble averaging in bulk experiments. The versatility of magnetic tweezers lies in the possibility and ease of tracking multiple parallel single-molecule events to yield statistically relevant single-molecule data. Moreover, they allow tracking both fast millisecond dynamics and slow processes (spanning several hours). In this chapter, we present the protocols used to study the interaction between E. coli SSB, single-stranded DNA (ssDNA), and E. coli RecQ helicase using magnetic tweezers. In particular, we propose constant force and force modulation assays to investigate SSB binding to DNA, as well as to characterize various facets of RecQ helicase activity stimulation by SSB.
Assuntos
DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , RecQ Helicases/metabolismo , Imagem Individual de Molécula/instrumentação , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Fenômenos Magnéticos , Ligação Proteica , Fatores de TempoRESUMO
G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.
Assuntos
DNA/química , Quadruplex G , Guanina/química , Proteínas Proto-Oncogênicas c-kit/química , Imagem Individual de Molécula/métodos , DNA Super-Helicoidal/química , Cinética , Desnaturação de Ácido Nucleico , Oncogenes , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Imagem Individual de Molécula/instrumentaçãoRESUMO
Atomic force microscopy (AFM) is one of the most versatile tools currently used in nanoscience. AFM allows for performing nondestructive imaging of almost any sample in either air or liquid, regardless whether the specimen is insulating, conductive, transparent, or opaque. It also allows for measuring interaction forces between a sharp probe and a sample surface, therefore allowing to probe nanomechanical properties of the specimen by either applying a controlled force or pulling the sample. It can provide topography, mechanical, magnetic, and conductive maps for very different type of samples. Transferred to the field of biology, today, AFM is the only microscopy technique able to produce images from biomolecules to bacteria and cells with nanometric resolution in aqueous media. Here, we will focus on the biological applications of AFM to flavoproteins. Despite references in the literature are scarce in this particular field, here it is described how imaging with AFM can contribute to describe catalysis mechanisms of some flavoenzymes, how oxidation states or binding of relevant ligands influence the association state of molecules, the dynamics of functional quaternary assemblies, and even visualize structural differences of individual protein molecules. Furthermore, we will show how force spectroscopy can be used to obtain the kinetic parameters, the dissociation landscape and the mechanical forces that maintain flavoprotein complexes, including the possibility to specifically detect particular flavoproteins on a sample.
Assuntos
Flavoproteínas/metabolismo , Imagem Individual de Molécula/métodos , Ligantes , Microscopia de Força Atômica , Oxirredução , Ligação Proteica , Imagem Individual de Molécula/instrumentaçãoRESUMO
Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.