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1.
Nat Methods ; 21(6): 1082-1093, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38831208

ABSTRACT

The point spread function (PSF) of a microscope describes the image of a point emitter. Knowing the accurate PSF model is essential for various imaging tasks, including single-molecule localization, aberration correction and deconvolution. Here we present universal inverse modeling of point spread functions (uiPSF), a toolbox to infer accurate PSF models from microscopy data, using either image stacks of fluorescent beads or directly images of blinking fluorophores, the raw data in single-molecule localization microscopy (SMLM). Our modular framework is applicable to a variety of microscope modalities and the PSF model incorporates system- or sample-specific characteristics, for example, the bead size, field- and depth- dependent aberrations, and transformations among channels. We demonstrate its application in single or multiple channels or large field-of-view SMLM systems, 4Pi-SMLM, and lattice light-sheet microscopes using either bead data or single-molecule blinking data.


Subject(s)
Microscopy, Fluorescence , Single Molecule Imaging , Single Molecule Imaging/methods , Microscopy, Fluorescence/methods , Algorithms , Image Processing, Computer-Assisted/methods , Fluorescent Dyes/chemistry , Models, Theoretical
2.
Nat Methods ; 21(6): 928, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38866985
3.
J Phys Chem B ; 128(23): 5576-5589, 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38833567

ABSTRACT

Single-molecule free diffusion experiments enable accurate quantification of coexisting species or states. However, unequal brightness and diffusivity introduce a burst selection bias and affect the interpretation of experimental results. We address this issue with a photon-by-photon maximum likelihood method, burstML, which explicitly considers burst selection criteria. BurstML accurately estimates parameters, including photon count rates, diffusion times, Förster resonance energy transfer (FRET) efficiencies, and population, even in cases where species are poorly distinguished in FRET efficiency histograms. We develop a quantitative theory that determines the fraction of photon bursts corresponding to each species and thus obtain accurate species populations from the measured burst fractions. In addition, we provide a simple approximate formula for burst fractions and establish the range of parameters where unequal brightness and diffusivity can significantly affect the results obtained by conventional methods. The performance of the burstML method is compared with that of a maximum likelihood method that assumes equal species brightness and diffusivity, as well as standard Gaussian fitting of FRET efficiency histograms, using both simulated and real single-molecule data for cold-shock protein, protein L, and protein G. The burstML method enhances the accuracy of parameter estimation in single-molecule fluorescence studies.


Subject(s)
Fluorescence Resonance Energy Transfer , Diffusion , Photons , Likelihood Functions , Single Molecule Imaging/methods
4.
Nat Commun ; 15(1): 5113, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38879529

ABSTRACT

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.


Subject(s)
Adenosine Triphosphate , DNA Helicases , RNA Polymerase II , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Single Molecule Imaging , Transcription Termination, Genetic , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , RNA Polymerase II/metabolism , Adenosine Triphosphate/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , Single Molecule Imaging/methods , RNA Helicases/metabolism , RNA Helicases/genetics , Transcription, Genetic , RNA, Fungal/metabolism , RNA, Fungal/genetics , DNA, Fungal/metabolism , DNA, Fungal/genetics , Hydrolysis
5.
Nature ; 629(8014): 1062-1068, 2024 May.
Article in English | MEDLINE | ID: mdl-38720082

ABSTRACT

Most chemistry and biology occurs in solution, in which conformational dynamics and complexation underlie behaviour and function. Single-molecule techniques1 are uniquely suited to resolving molecular diversity and new label-free approaches are reshaping the power of single-molecule measurements. A label-free single-molecule method2-16 capable of revealing details of molecular conformation in solution17,18 would allow a new microscopic perspective of unprecedented detail. Here we use the enhanced light-molecule interactions in high-finesse fibre-based Fabry-Pérot microcavities19-21 to detect individual biomolecules as small as 1.2 kDa, a ten-amino-acid peptide, with signal-to-noise ratios (SNRs) >100, even as the molecules are unlabelled and freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of subpopulations in mixed samples. Notably, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight and composition but different conformation can also be resolved. Detection is based on the creation of a new molecular velocity filter window and a dynamic thermal priming mechanism that make use of the interplay between optical and thermal dynamics22,23 and Pound-Drever-Hall (PDH) cavity locking24 to reveal molecular motion even while suppressing environmental noise. New in vitro ways of revealing molecular conformation, diversity and dynamics can find broad potential for applications in the life and chemical sciences.


Subject(s)
Peptides , Single Molecule Imaging , Diffusion , Isomerism , Light , Peptides/analysis , Peptides/chemistry , Peptides/radiation effects , Signal-To-Noise Ratio , Single Molecule Imaging/methods , Solutions , Protein Conformation , Molecular Weight , Motion
6.
Curr Opin Struct Biol ; 86: 102825, 2024 06.
Article in English | MEDLINE | ID: mdl-38723560

ABSTRACT

Knowledge of the structure and dynamics of biomolecules is key to understanding the mechanisms underlying their biological functions. Single-particle cryo-electron microscopy (cryo-EM) is a powerful structural biology technique to characterize complex biomolecular systems. Here, we review recent advances of how Molecular Dynamics (MD) simulations are being used to increase and enhance the information extracted from cryo-EM experiments. We will particularly focus on the physics underlying these experiments, how MD facilitates structure refinement, in particular for heterogeneous and non-isotropic resolution, and how thermodynamic and kinetic information can be extracted from cryo-EM data.


Subject(s)
Cryoelectron Microscopy , Molecular Dynamics Simulation , Cryoelectron Microscopy/methods , Thermodynamics , Kinetics , Single Molecule Imaging/methods
7.
Int J Mol Sci ; 25(9)2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38731890

ABSTRACT

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.


Subject(s)
Tumor Suppressor Protein p53 , Tumor Suppressor p53-Binding Protein 1 , Tumor Suppressor Protein p53/metabolism , Humans , Tumor Suppressor p53-Binding Protein 1/metabolism , Single Molecule Imaging/methods , Microscopy, Fluorescence/methods , Protein Binding , Cell Line, Tumor , Mitochondria/metabolism
8.
Nanotechnology ; 35(33)2024 May 31.
Article in English | MEDLINE | ID: mdl-38772350

ABSTRACT

Single-molecule detection technology is a technique capable of detecting molecules at the single-molecule level, characterized by high sensitivity, high resolution, and high specificity. Nanopore technology, as one of the single-molecule detection tools, is widely used to study the structure and function of biomolecules. In this study, we constructed a small-sized nanopore with a pore-cavity-pore structure, which can achieve a higher reverse capture rate. Through simulation, we investigated the electrical potential distribution of the nanopore with a pore-cavity-pore structure and analyzed the influence of pore size on the potential distribution. Accordingly, different pore sizes can be designed based on the radius of gyration of the target biomolecules, restricting their escape paths inside the chamber. In the future, nanopores with a pore-cavity-pore structure based on two-dimensional thin film materials are expected to be applied in single-molecule detection research, which provides new insights for various detection needs.


Subject(s)
DNA , Nanopores , DNA/chemistry , Nanotechnology/methods , Single Molecule Imaging/methods
9.
Methods Mol Biol ; 2799: 225-242, 2024.
Article in English | MEDLINE | ID: mdl-38727910

ABSTRACT

Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies.


Subject(s)
Fluorescence Resonance Energy Transfer , Receptors, N-Methyl-D-Aspartate , Single Molecule Imaging , Fluorescence Resonance Energy Transfer/methods , Receptors, N-Methyl-D-Aspartate/metabolism , Receptors, N-Methyl-D-Aspartate/chemistry , Single Molecule Imaging/methods , Protein Conformation , Kinetics , Fluorescent Dyes/chemistry , Humans , Protein Binding
11.
Ultramicroscopy ; 263: 113986, 2024 Sep.
Article in English | MEDLINE | ID: mdl-38762964

ABSTRACT

Nucleolin is overexpressed on the surface of pancreatic cancer cells and are regarded as the remarkable therapeutic target. Aptamers are capable of binding the external domain of nucleolin on the cell surface with high affinity and specificity. But nucleolin has not been localized on pancreatic cancer cells at very high spatial resolution, and the interactions between nucleolin and aptamers have not been investigated at very high force resolution level. In this work, nucleolin was localized on pancreatic cancer and normal cells by aptamers (9FU-AS1411-NH2, AS1411-NH2 and CRONH2) in Single Molecule Recognition Imaging mode of Atomic Force Microscopy. There are plenty of nucleolin on the surfaces of pancreatic cancer cells (area percentage about 5 %), while there are little nucleolin on the surfaces of normal cells. The interactions between three types of aptamers and nucleolins on the surfaces of pancreatic cancer cells were investigated by Single Molecule Force Spectroscopy. The unbinding forces of nucleolins-(9FU-AS1411-NH2) are larger than nucleolins-(AS1411-NH2). The dissociation activation energy on nucleolin-(9FU-AS1411-NH2) is higher than nucleolin-(AS1411-NH2), which indicates that the former complex is more stable and harder to dissociate than the later complex. There are no unbinding forces between nucleolin and CRONH2. All these demonstrate that nucleolin was localized on pancreatic cancer and normal cells at single molecule level quantitatively, and the interactions (unbinding forces and kinetics) between nucleolin and aptamers were studied at picoNewton level. The approaches and results of this work will pave new ways in the investigations of nucleolin and aptamers, and will also be useful in the studies on other proteins and their corresponding aptamers.


Subject(s)
Aptamers, Nucleotide , Microscopy, Atomic Force , Nucleolin , Pancreatic Neoplasms , Phosphoproteins , RNA-Binding Proteins , RNA-Binding Proteins/metabolism , Phosphoproteins/metabolism , Humans , Pancreatic Neoplasms/metabolism , Microscopy, Atomic Force/methods , Cell Line, Tumor , Protein Binding , Single Molecule Imaging/methods
12.
Proc Natl Acad Sci U S A ; 121(22): e2403013121, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38781207

ABSTRACT

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.


Subject(s)
Avidin , Biomolecular Condensates , Biotin , Poloxamer , Biomolecular Condensates/chemistry , Biomolecular Condensates/metabolism , Poloxamer/chemistry , Biotin/chemistry , Biotin/metabolism , Avidin/chemistry , Avidin/metabolism , Fluorescence Recovery After Photobleaching/methods , Surface Properties , Surface-Active Agents/chemistry , Surface-Active Agents/metabolism , Single Molecule Imaging/methods
13.
Methods Mol Biol ; 2807: 61-76, 2024.
Article in English | MEDLINE | ID: mdl-38743221

ABSTRACT

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.


Subject(s)
Cell Membrane , HIV-1 , Microscopy, Fluorescence , Single Molecule Imaging , T-Lymphocytes , Virus Assembly , gag Gene Products, Human Immunodeficiency Virus , HIV-1/physiology , Humans , Cell Membrane/metabolism , Cell Membrane/virology , Single Molecule Imaging/methods , T-Lymphocytes/virology , T-Lymphocytes/metabolism , Microscopy, Fluorescence/methods , gag Gene Products, Human Immunodeficiency Virus/metabolism
14.
Methods Mol Biol ; 2807: 45-59, 2024.
Article in English | MEDLINE | ID: mdl-38743220

ABSTRACT

Latent HIV-1 reservoirs are a major obstacle to the eradication of HIV-1. Several cure strategies have been proposed to eliminate latent reservoirs. One of the key strategies involves the reactivation of latent HIV-1 from cells using latency-reversing agents. However, currently it is unclear whether any of the latency-reversing agents are able to completely reactivate HIV-1 provirus transcription in all latent cells. An understanding of the reactivation of HIV-1 provirus at single-cell single-molecule level is necessary to fully comprehend the reactivation of HIV-1 in the reservoirs. Furthermore, since reactivable viruses in the pool of latent reservoirs are rare, combining single-cell imaging techniques with the ability to visualize a large number of reactivated single cells that express both viral RNA and proteins in a pool of uninfected and non-reactivated cells will provide unprecedented information about cell-to-cell variability in reactivation. Here, we describe the single-cell single-molecule RNA-FISH (smRNA-FISH) method to visualize HIV-1 gag RNA combined with the immunofluorescence (IF) method to detect Gag protein to characterize the reactivated cells. This method allows the visualization of subcellular localization of RNA and proteins before and after reactivation and facilitates absolute quantitation of the number of transcripts per cell using FISH-quant. In addition, we describe a high-speed and high-resolution scanning (HSHRS) fluorescence microscopy imaging method to visualize rare and reactivated cells in a pool of non-reactivated cells with high efficiency.


Subject(s)
Fluorescent Antibody Technique , HIV-1 , In Situ Hybridization, Fluorescence , RNA, Viral , Single Molecule Imaging , Single-Cell Analysis , Virus Activation , Virus Latency , HIV-1/physiology , HIV-1/genetics , Humans , In Situ Hybridization, Fluorescence/methods , RNA, Viral/genetics , Single-Cell Analysis/methods , Single Molecule Imaging/methods , Fluorescent Antibody Technique/methods , HIV Infections/virology , Proviruses/genetics
15.
Nat Methods ; 21(5): 749, 2024 May.
Article in English | MEDLINE | ID: mdl-38745078
16.
Nat Commun ; 15(1): 4403, 2024 May 23.
Article in English | MEDLINE | ID: mdl-38782907

ABSTRACT

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.


Subject(s)
DNA , Single Molecule Imaging , Humans , Single Molecule Imaging/methods , DNA/metabolism , DNA/chemistry , Animals , Nanotechnology/methods , Proteins/metabolism , Proteins/chemistry , Macromolecular Substances/metabolism , Macromolecular Substances/chemistry , Signal-To-Noise Ratio
17.
J Biomed Opt ; 29(6): 066501, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38799979

ABSTRACT

Significance: Spectroscopic single-molecule localization microscopy (sSMLM) takes advantage of nanoscopy and spectroscopy, enabling sub-10 nm resolution as well as simultaneous multicolor imaging of multi-labeled samples. Reconstruction of raw sSMLM data using deep learning is a promising approach for visualizing the subcellular structures at the nanoscale. Aim: Develop a novel computational approach leveraging deep learning to reconstruct both label-free and fluorescence-labeled sSMLM imaging data. Approach: We developed a two-network-model based deep learning algorithm, termed DsSMLM, to reconstruct sSMLM data. The effectiveness of DsSMLM was assessed by conducting imaging experiments on diverse samples, including label-free single-stranded DNA (ssDNA) fiber, fluorescence-labeled histone markers on COS-7 and U2OS cells, and simultaneous multicolor imaging of synthetic DNA origami nanoruler. Results: For label-free imaging, a spatial resolution of 6.22 nm was achieved on ssDNA fiber; for fluorescence-labeled imaging, DsSMLM revealed the distribution of chromatin-rich and chromatin-poor regions defined by histone markers on the cell nucleus and also offered simultaneous multicolor imaging of nanoruler samples, distinguishing two dyes labeled in three emitting points with a separation distance of 40 nm. With DsSMLM, we observed enhanced spectral profiles with 8.8% higher localization detection for single-color imaging and up to 5.05% higher localization detection for simultaneous two-color imaging. Conclusions: We demonstrate the feasibility of deep learning-based reconstruction for sSMLM imaging applicable to label-free and fluorescence-labeled sSMLM imaging data. We anticipate our technique will be a valuable tool for high-quality super-resolution imaging for a deeper understanding of DNA molecules' photophysics and will facilitate the investigation of multiple nanoscopic cellular structures and their interactions.


Subject(s)
Deep Learning , Single Molecule Imaging , Animals , Single Molecule Imaging/methods , Humans , Chlorocebus aethiops , COS Cells , Microscopy, Fluorescence/methods , Image Processing, Computer-Assisted/methods , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/analysis , Algorithms , Histones/chemistry , Histones/analysis
18.
Cell Syst ; 15(5): 475-482.e6, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38754367

ABSTRACT

Image-based spatial transcriptomics methods enable transcriptome-scale gene expression measurements with spatial information but require complex, manually tuned analysis pipelines. We present Polaris, an analysis pipeline for image-based spatial transcriptomics that combines deep-learning models for cell segmentation and spot detection with a probabilistic gene decoder to quantify single-cell gene expression accurately. Polaris offers a unifying, turnkey solution for analyzing spatial transcriptomics data from multiplexed error-robust FISH (MERFISH), sequential fluorescence in situ hybridization (seqFISH), or in situ RNA sequencing (ISS) experiments. Polaris is available through the DeepCell software library (https://github.com/vanvalenlab/deepcell-spots) and https://www.deepcell.org.


Subject(s)
Deep Learning , Gene Expression Profiling , In Situ Hybridization, Fluorescence , Transcriptome , In Situ Hybridization, Fluorescence/methods , Transcriptome/genetics , Gene Expression Profiling/methods , Software , Humans , Single-Cell Analysis/methods , Image Processing, Computer-Assisted/methods , Single Molecule Imaging/methods , Animals , Supervised Machine Learning
19.
Nat Commun ; 15(1): 4178, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38755200

ABSTRACT

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments.


Subject(s)
Chromatin , Nucleosomes , Single Molecule Imaging , Nucleosomes/metabolism , Chromatin/metabolism , Chromatin/chemistry , Humans , Single Molecule Imaging/methods , Cell Nucleus/metabolism , Histones/metabolism , HeLa Cells , Diffusion
20.
Int J Mol Sci ; 25(9)2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38732092

ABSTRACT

In this work, we apply single-molecule fluorescence microscopy and spectroscopy to probe plasmon-enhanced fluorescence and Förster resonance energy transfer in a nanoscale assemblies. The structure where the interplay between these two processes was present consists of photoactive proteins conjugated with silver nanowires and deposited on a monolayer graphene. By comparing the results of continuous-wave and time-resolved fluorescence microscopy acquired for this structure with those obtained for the reference samples, where proteins were coupled with either a graphene monolayer or silver nanowires, we find clear indications of the interplay between plasmonic enhancement and the energy transfer to graphene. Namely, fluorescence intensities calculated for the structure, where proteins were coupled to graphene only, are less than for the structure playing the central role in this study, containing both silver nanowires and graphene. Conversely, decay times extracted for the latter are shorter compared to a protein-silver nanowire conjugate, pointing towards emergence of the energy transfer. Overall, the results show that monitoring the optical properties of single emitters in a precisely designed hybrid nanostructure provides an elegant way to probe even complex combination of interactions at the nanoscale.


Subject(s)
Fluorescence Resonance Energy Transfer , Graphite , Nanowires , Silver , Silver/chemistry , Nanowires/chemistry , Graphite/chemistry , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/chemistry , Proteins/chemistry , Microscopy, Fluorescence/methods , Single Molecule Imaging/methods
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