Single-molecule Fluorescence Detection Research Articles

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is, however, a growing gap between the state of the art and what is accessible to biologists, biochemists, medical researchers, or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed microspectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy, time-correlated single-photon counting and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines, are provided, to allow non-optics specialists to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and open-source Brick-MIC platform will be a valuable tool for many laboratories worldwide.

Single-molecule digital sizing of proteins in solution

The physical characterization of proteins in terms of their sizes, interactions, and assembly states is key to understanding their biological function and dysfunction. However, this has remained a difficult task because proteins are often highly polydisperse and present as multicomponent mixtures. Here, we address this challenge by introducing single-molecule microfluidic diffusional sizing (smMDS). This approach measures the hydrodynamic radius of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. smMDS allows for ultrasensitive sizing of proteins down to femtomolar concentrations and enables affinity profiling of protein interactions at the single-molecule level. We show that smMDS is effective in resolving the assembly states of protein oligomers and in characterizing the size of protein species within complex mixtures, including fibrillar protein aggregates and nanoscale condensate clusters. Overall, smMDS is a highly sensitive method for the analysis of proteins in solution, with wide-ranging applications in drug discovery, diagnostics, and nanobiotechnology.

Hexagonal Plasmonic Arrays for High-Throughput Multicolor Single-Molecule Studies.

Nanophotonic biosensors offer exceptional sensitivity in the presence of strong background signals by enhancing and confining light in subwavelength volumes. In the field of nanophotonic biosensors, antenna-in-box (AiB) designs consisting of a nanoantenna within a nanoaperture have demonstrated remarkable single-molecule fluorescence detection sensitivities under physiologically relevant conditions. However, their full potential has not yet been exploited as current designs prohibit insightful correlative multicolor single-molecule studies and are limited in terms of throughput. Here, we overcome these constraints by introducing aluminum-based hexagonal close-packed AiB (HCP-AiB) arrays. Our approach enables the parallel readout of over 1000 HCP-AiBs with multicolor single-molecule sensitivity up to micromolar concentrations using an alternating three-color excitation scheme and epi-fluorescence detection. Notably, the high-density HCP-AiB arrays not only enable high-throughput studies at micromolar concentrations but also offer high single-molecule detection probabilities in the nanomolar range. We demonstrate that robust and alignment-free correlative multicolor studies are possible using optical fiducial markers even when imaging in the low millisecond range. These advancements pave the way for the use of HCP-AiB arrays as biosensor architectures for high-throughput multicolor studies on single-molecule dynamics.

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and opensource Brick-MIC platform will be a valuable tool for many laboratories worldwide.

Technologies for investigating single-molecule chemical reactions

Abstract Single molecules, the smallest independently stable units in the material world, serve as the fundamental building blocks of matters. Among different branches of single-molecule sciences, single-molecule chemical reactions, by revealing the behavior and properties of individual molecules at the molecular scale, are particularly attractive because they can advance the understanding of chemical reaction mechanisms and help to address key scientific problems in broad fields such as physics, chemistry, biology, and materials science. This review provides a timely, comprehensive overview of single-molecule chemical reactions based on various technical platforms such as scanning probe microscopy, single-molecule junction, single nanostructure, single-molecule fluorescence detection, and crossed molecular beam. We present multidimensional analyses of single-molecule chemical reactions, offering new perspectives for research in different areas like photocatalysis/electrocatalysis, organic reactions, surface reactions, and biological reactions. Finally, we discuss the opportunities and challenges in this thriving field of single-molecule chemical reaction.

An optofluidic antenna for enhancing the sensitivity of single-emitter measurements

Many single-molecule investigations are performed in fluidic environments, for example, to avoid unwanted consequences of contact with surfaces. Diffusion of molecules in this arrangement limits the observation time and the number of collected photons, thus, compromising studies of processes with fast or slow dynamics. Here, we introduce a planar optofluidic antenna (OFA), which enhances the fluorescence signal from molecules by about 5 times per passage, leads to about 7-fold more frequent returns to the observation volume, and significantly lengthens the diffusion time within one passage. We use single-molecule multi-parameter fluorescence detection (sm-MFD), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) measurements to characterize our OFAs. The antenna advantages are showcased by examining both the slow (ms) and fast (50 μs) dynamics of DNA four-way (Holliday) junctions with real-time resolution. The FRET trajectories provide evidence for the absence of an intermediate conformational state and introduce an upper bound for its lifetime. The ease of implementation and compatibility with various microscopy modalities make OFAs broadly applicable to a diverse range of studies.

Simultaneous electrical and single-molecule fluorescence detection in windowed microelectrode cavity arrays

Simultaneous electrical and single-molecule fluorescence detection in windowed microelectrode cavity arrays

Single primer/self-template-powered isothermal amplification for single-molecule quantification of multiple DNA glycosylases

DNA glycosylase assumes a pivotal role in the preservation of genome integrity by recognizing and excising damaged bases. Herein, we develop a single primer/self-template-powered cascade rolling circle amplification (RCA) and apurinic/apyrimidinic endonuclease (APE1)-mediated signal amplification for simultaneous detection of human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG). The uniqueness of this approach lies in the use of custom-designed hairpin probes to establish a prerequisite for RCA: the conformational change of hairpin probe to circular template, which effectively suppress undesired non-specific amplification. We design two hairpin probes featuring elongated stem structure and 3′-phosphate (3′-PO4) modifications. The stems of hairpin probes are modified with glycosylase substrates and BER process can cleave hairpin probes, resulting in the generation of single-stranded DNAs (ssDNAs) with 3′-hydroxyl (3′-OH) ends. These ssDNAs can function as padlock probes to form circular template upon ligation by ligase. Ultimately, the self-template-initiated RCA and APE1-mediated signal amplification cascade occurs, generating a substantial number of free Cy3 and Cy5 fluorophores that can be simply visualized by single-molecule fluorescence detection. This method demonstrates excellent specificity and sensitivity, with a limit of detection (LOD) of 3.42 × 10-12 U/μL for hAAG and 2.41 × 10-12 U/μL for UDG. This method also can be used for screening of potential inhibitor and kinetic parameter measurement. Most importantly, this method enables accurate profiling of glycosylases expression levels in various cancer cell lines and detection of glycosylase activities at the single-cell level, offering a bright future in cancer detection, medical research, and precision medicine.

Single-molecule fluorescence methods for protein biomarker analysis.

Proteins have been considered key building blocks of life. In particular, the protein content of an organism and a cell offers significant information for the in-depth understanding of the disease and biological processes. Single-molecule protein detection/sequencing tools will revolutionize clinical (proteomics) research, offering ultrasensitivity for low-abundance biomarker (protein) detection, which is important for the realization of early-stage disease diagnosis and single-cell proteomics. This improved detection/measurement capability delivers new sets of techniques to explore new frontiers and address important challenges in various interdisciplinary areas including nanostructured materials, molecular medicine, molecular biology, and chemistry. Importantly, fluorescence-based methods have emerged as indispensable tools for single protein detection/sequencing studies, providing a higher signal-to-noise ratio (SNR). Improvements in fluorescent dyes/probes and detector capabilities coupled with advanced (image) analysis strategies have fueled current developments for single protein biomarker detections. For example, in comparison to conventional ELISA (i.e., based on ensembled measurements), single-molecule fluorescence detection is more sensitive, faster, and more accurate with reduced background, high-throughput, and so on. In comparison to MS sequencing, fluorescence-based single-molecule protein sequencing can achieve the sequencing of peptides themselves with higher sensitivity. This review summarizes various typical single-molecule detection technologies including their methodology (modes of operation), detection limits, advantages and drawbacks, and current challenges with recent examples. We describe the fluorescence-based single-molecule protein sequencing/detection based on five kinds of technologies such as fluorosequencing, N-terminal amino acid binder, nanopore light sensing, and DNA nanotechnology. Finally, we present our perspective for developing high-performance fluorescence-based sequencing/detection techniques.

Pulse-shaped broadband multiphoton excitation for single-molecule fluorescence detection in the far field

Multiphoton excitation of fluorescence has many potential advantages over resonant (one-photon) excitation, but the method has not found widespread use for ultrasensitive applications. We recently described an approach to the multiphoton excitation of single molecules that uses a pulse shaper to compress and tailor pulses from an ultrafast broadband laser in order to optimise the brightness and signal-to-background ratio following non-linear excitation. Here we provide a detailed description of the setup and illustrate its use and potential by optimising two-photon fluorescence of a common fluorophore, rhodamine 110, at the single-molecule level. We also show that a DNA oligonucleotide labelled with a fluorescent nucleobase analogue, tC, can be detected using two-photon FCS, whereas one-photon excitation causes rapid photobleaching. The ability to improve the signal-to-background ratio and to reduce the incident power required to attain a given brightness can be applied to the multiphoton excitation of any fluorescent species, from small molecules with low multiphoton cross sections to the brightest nanoparticles.

Entropy-Driven Self-Assembly of Single Quantum Dot Sensor for Catalytic Imaging of Telomerase in Living Cells

Telomerase is a highly valuable cancer diagnosis biomarker and a promising cancer therapy target. So far, most telomerase assays are limited by the involvement of tedious procedures, multiple enzymes, and complicated reaction schemes. Sensitive monitoring of low-abundant telomerase in living cells remains a challenge. Herein, we demonstrate an entropy-driven catalytic assembly of quantum dot (QD) sensors for accurate detection and imaging of telomerase activity in living cells. In this sensor, target telomerase specifically catalyzes extension of telomerase primer, and the extended primer subsequently acts as a catalyst to continuously initiate entropy-driven catalytic reaction, generating a large number of fluorophore- and biotin-labeled DNAs that can be self-assembled on the QD surface to induce an efficient Föster resonance energy transfer signal. The proposed sensor requires a single step for both recognition and amplification of the telomerase signal, eliminating the use of either protein enzymes or laborious procedures. Taking advantage of the inherent superiority of single-molecule fluorescence detection and high amplification efficiency of the entropy-driven reaction, this sensor demonstrates single-cell sensitivity for the in vitro assay. Moreover, it is capable of screening the telomerase inhibitor, discriminating different tumor cells from normal ones, and even real-time imaging telomerase in living cells, providing a novel platform for telomerase-associated cancer diagnosis and drug screening.

A dark intermediate in the fluorogenic reaction between tetrazine fluorophores and trans-cyclooctene

Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, trans-cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy.

Label-Free Optical Analysis of Biomolecules in Solid-State Nanopores: Toward Single-Molecule Protein Sequencing.

Sequence identification of peptides and proteins is central to proteomics. Protein sequencing is mainly conducted by insensitive mass spectroscopy because proteins cannot be amplified, which hampers applications such as single-cell proteomics and precision medicine. The commercial success of portable nanopore sequencers for single DNA molecules has inspired extensive research and development of single-molecule techniques for protein sequencing. Among them, three challenges remain: (1) discrimination of the 20 amino acids as building blocks of proteins; (2) unfolding proteins; and (3) controlling the motion of proteins with nonuniformly charged sequences. In this context, the emergence of label-free optical analysis techniques for single amino acids and peptides by solid-state nanopores shows promise for addressing the first challenge. In this Perspective, we first discuss the current challenges of single-molecule fluorescence detection and nanopore resistive pulse sensing in a protein sequencing. Then, label-free optical methods are described to show how they address the single-amino-acid identification within single peptides. They include localized surface plasmon resonance detection and surface-enhanced Raman spectroscopy on plasmonic nanopores. Notably, we report new data to show the ability of plasmon-enhanced Raman scattering to record and discriminate the 20 amino acids at a single-molecule level. In addition, we discuss briefly the manipulation of molecule translocation and liquid flow in plasmonic nanopores for controlling molecule movement to allow high-resolution reading of protein sequences. We envision that a combination of Raman spectroscopy with plasmonic nanopores can succeed in single-molecule protein sequencing in a label-free way.

Improving single molecule fluorescence detection with zero-mode waveguide nanoapertures

Zero-mode waveguides (ZMW) are nanoapertures milled in opaque metal films with subwavelength diameters. Thanks to their strong light confinement, ZMWs have been reported to enable single molecule detection in concentrated solutions of several micromolars with enhanced fluorescence brightness. In this talk, I will describe recent applications of the ZMW concept:

Broadband single-molecule fluorescence enhancement based on self-assembled Ag@Au dimer plasmonic nanoantennas.

Dimer optical antennas (OAs) enable great fluorescence enhancement and excitation volume reduction and hence potentially can be a very useful tool for single-molecule detection. The realization of broadband fluorescence enhancement with a dimer OA remains an essential step for its usage in multi-color single-molecule fluorescence (SMF) detection. Although silver dimer OAs have been shown to be able to yield broadband fluorescence enhancement over the visible spectrum, they are amenable to oxidization, hard to functionalize, and could cause cytotoxicity. To overcome these limitations, in this work, we took advantage of nano-sized silver due to its optical properties and gold due to its chemical properties and developed an ameliorated Ag@Au dimer OA in terms of its overall performance. The Ag@Au nanoparticle in the dimer OA contains a 70 nm silver core and an ultra-thin (∼1-5 nm) gold shell which play a key role in its optical responses. Furthermore, we employed three typical dyes, i.e., FAM, TAMRA and Cy5, representing the blue, yellow and red ranges, respectively, and characterized their single-molecule fluorescence enhancements in the presence of Au or Ag@Au OAs. Our results indicate that, in contrast to its Au counterpart, the Ag@Au dimer OA prepared here can greatly improve its optical response in the blue range and eventually achieve broadband fluorescence enhancement throughout almost the whole visible spectral range. Meanwhile, it also maintains good chemical stability and accessibility to functionalization. Such Ag@Au dimer OAs are thus expected to have many important applications in the future, including single-molecule sequencing and multi-color biosensing.

Label- and enzyme-free plasmon-enhanced single molecule fluorescence detection of HIV DNA fragments based on a catalytic hairpin assembly.

We developed a label- and enzyme-free single molecule fluorescence counting strategy for HIV DNA fragments detection. The nucleic acid biosensor consists of a 5' terminal connected with a triangular gold nanoplate, 3' terminal rich in guanine hairpin probe (HP1) and a hairpin probe HP2 complementary to the partial sequence of HP1. Without the existence of the target DNA, the DNA fragment rich in the guanine region is locked in a hairpin structure and cannot form a G-quadruplex, hence NMM exhibits a low fluorescence signal. When the target DNA exists, the hairpin assembly will trigger a strand displacement amplification reaction that produces a great number of G-quadruplexes, and the fluorescence brightness of NMM will be enhanced. The plasmon resonance effect of the triangular gold nanoplates will further amplify the fluorescence signal. This method can analyze the target DNA with high sensitivity and selectivity, and the detection limit is 0.83 fM. The analysis of the HIV DNA fragments in diluted human serum samples was successfully achieved, and the recovery rate was 92%-104%. Because of its easy operation and low cost, it has broad development potential in biochemical analysis and clinical applications.

Distinct lipid membrane-mediated pathways of Tau assembly revealed by single-molecule analysis.

The conversion of intrinsically disordered Tau to highly ordered amyloid aggregates is associated with a wide range of neurodegenerative diseases termed tauopathies. The presence of lipid bilayer membranes is a critical factor that accelerates the abnormal aggregation of Tau protein. However, the lipid membrane-induced conformational changes of Tau and the mechanism for the accelerated fibrillation remain elusive. In this study, single-molecule Förster resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS) were applied to detect the conformational changes and intermolecular interactions of full-length Tau in the presence of different concentrations of 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) vesicles. The results show that the conformation of Tau becomes expanded with opening of the N-terminal and C-terminal domains of Tau upon binding to DMPS. At low DMPS concentrations, Tau forms oligomers with a partially extended conformation which facilitates the amyloid fibrillization process. At high DMPS concentrations, Tau monomer binds to lipid membranes in a fully expanded conformation at low density thus inhibiting intermolecular aggregation. Our study reveals the underlying mechanisms by which lipid membranes influence amyloid formation of Tau, providing a foundation for further understanding of the pathogenesis and physiology of the interplay between Tau protein and lipid membranes.

Probing DNA - Transcription Factor Interactions Using Single-Molecule Fluorescence Detection in Nanofluidic Devices.

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, this data on fluorescently labeled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements, and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA responseelement.

Engineering single-molecule fluorescence with asymmetric nano-antennas

As a powerful tool for studying molecular dynamics in bioscience, single-molecule fluorescence detection provides dynamical information buried in ensemble experiments. Fluorescence in the near-infrared (NIR) is particularly useful because it offers higher signal-to-noise ratio and increased penetration depth in tissue compared with visible fluorescence. The low quantum yield of most NIR fluorophores, however, makes the detection of single-molecule fluorescence difficult. Here, we use asymmetric plasmonic nano-antenna to enhance the fluorescence intensity of AIEE1000, a typical NIR dye, by a factor up to 405. The asymmetric nano-antenna achieve such an enhancement mainly by increasing the quantum yield (to ~80%) rather than the local field, which degrades the molecules’ photostability. Our coupled-mode-theory analysis reveals that the enhancements stem from resonance-matching between antenna and molecule and, more importantly, from optimizing the coupling between the near- and far-field modes with designer asymmetric structures. Our work provides a universal scheme for engineering single-molecule fluorescence in the near-infrared regime.

Optical tweezers in single-molecule biophysics.

Optical tweezers have become the method of choice in single-molecule manipulation studies. In this Primer, we first review the physical principles of optical tweezers and the characteristics that make them a powerful tool to investigate single molecules. We then introduce the modifications of the method to extend the measurement of forces and displacements to torques and angles, and to develop optical tweezers with single-molecule fluorescence detection capabilities. We discuss force and torque calibration of these instruments, their various modes of operation and most common experimental geometries. We describe the type of data obtained in each experimental design and their analyses. This description is followed by a survey of applications of these methods to the studies of protein-nucleic acid interactions, protein/RNA folding and molecular motors. We also discuss data reproducibility, the factors that lead to the data variability among different laboratories and the need to develop field standards. We cover the current limitations of the methods and possible ways to optimize instrument operation, data extraction and analysis, before suggesting likely areas of future growth.