Strategies for Overcoming the Single-Molecule Concentration Barrier

Abstract

Fluorescence-based single-molecule approaches have helped revolutionize our understanding of chemical and biological mechanisms. Unfortunately, these methods are only suitable at low concentrations of fluorescent molecules so that single fluorescent species of interest can be successfully resolved beyond background signal. The application of these techniques has therefore been limited to high-affinity interactions despite most biological and chemical processes occurring at much higher reactant concentrations. Fortunately, recent methodological advances have demonstrated that this concentration barrier can indeed be broken, with techniques reaching concentrations as high as 1 mM. The goal of this Review is to discuss the challenges in performing single-molecule fluorescence techniques at high-concentration, offer applications in both biology and chemistry, and highlight the major milestones that shatter the concentration barrier. We also hope to inspire the widespread use of these techniques so we can begin exploring the new physical phenomena lying beyond this barrier.

Figure 1

Concentration barrier of single-molecule fluorescence measurements. (Top left) Cartoon showing a surface tethered receptor binding a fluorescently labeled ligand. At low ligand concentrations, individual binding events can be resolved by TIRF. (Top right) At concentrations exceeding the concentration barrier, TIRF excites freely diffusing fluorescent-ligands in solution, thereby obscuring the resolution of specific binding events. (Bottom) Histogram of over 30 000 Michaelis–Menten constants (K_M) obtained from the BRENDA database. The vertical black line indicates the 10 nM concentration barrier of TIRF. The accessible concentration range of many of the techniques discussed herein are overlaid.

Figure 2

Defining excitation, detection, observation, and physical volumes in single-molecule fluorescence experiments (a) Excitation volume is the volume of the excitation light (yellow). (b) Detection volume (yellow) refers to the volume where light is collected and may occupy a smaller space than the excitation volume. The observation volume is given by the overlap of the excitation and detection volumes. (c) Physical volume related to the physical confinement of the molecule of interest.

Figure 3

Fluorogenic reactions. (a) Cartoon depiction of enzyme immobilization and conversion of resorufin-β-d-galactopyranoside (RGP) to fluorescent resorufin (R). (b) Turnover time trace and histogram of single enzyme molecules at 100 μM RGP. Threshold to determining wait times between adjacent bursts depicted by dashed horizontal line. Adapted with permission from ref (37). Copyright (2006) Springer Nature. (c) Experimental setup and fluorescence time trace also using the conversion of resazurin to resorufin to study the catalysis of gold nanoparticles. Adapted with permission from ref (12). Copyright (2008) Springer Nature. (d) An abiotic example of a fluorogenic reaction using a boron dipyrromethene dye. The wavelength shift is catalyzed by a mesoporous titanosilicate catalyst which epoxidizes the connection between the BODIPY core and a conjugated substituent. Reprinted with permission from ref (44). Copyright (2010) John Wiley and Sons.

Figure 4

FRET. (a,b) Fluorescence time trajectory of individual quantum dots (green trace) with 10 μM Cy3-ATP (a) or 60 μM Cy3-ATP (magenta trace corresponds to Cy3-ATP signal). Individual turnover can still be clearly observed at 10 μM, but not at 60 μM, where there is continuous signal in the acceptor channel. Reprinted with permission from ref (48). Copyright (2010) John Wiley and Sons. (c) White-light image of a piece of Cu/C catalyst. (d) Fluorescence time trace for area highlighted in the red box. (e1) Expanded accumulated TIRF image. (e2) TIRF image overlaid with (a). (e3) Reconstructed super-resolution image of the same area overlaid with (a). (f1–f3) Three-dimensional representation of e1-e3, respectively. Reprinted from ref (49). Copyright (2015) American Chemical Society.

Figure 5

PhADE. (a) Cartoon depicting the process of PhADE. (b) Images of 6xHis-mKikGR bound to anti-6xHis antibody, which was immobilized onto the surface using a biotin-streptavidin interaction, in the presence of 2 μM 6xHis-mKikGR in solution. mKikG was first imaged using 488 nm light (‘pre’) then mKikR was imaged using the pulse sequence shown before (‘0s’) and after 405 nm photoactivation (‘1s’ through ‘5s’). White arrows showcase an intensity change characteristic of a single mKikR molecule. Scale bar, 1 μm. (c) Conventional concentrations used for single-molecule imaging is not able to detect Fen1^KikGR at replication forks, while PhADE is able to detect it at concentrations above the concentration barrier. Doubly tethered λ DNA was replicated in the presence of 250nm (left side) and 10 nM WT Fen1^KikGR (right side). (i) Images of Fen1^KikG during replication (488 nm excitation, 37 W cm^–2, 100 ms). (ii) Fen1^KikR in the same region from (i) was imaged 10s later (562 nm excitation, 47 W cm^–2, 100 ms). (iii) Fen1^KikR was imaged again 700 ms following photoactivation (405 nm, 30 W cm^–2, 300 ms, ∼90% of molecules activated). (iv) Extract was removed and replicated DNA stained with α-Dig immediately following (iii). (v) Total DNA stained with SYTOX Orange, an intercalating dye. Scale bar, 1 μm. Adapted with permission from ref (54). Copyright (2012) Springer Nature.

Figure 6

COMPEITS. (a) Schematic representation of the principle of COMPEITS. (b) Two-dimensional histogram of the number of detected amplex red oxidation products (n_p, the auxiliary fluorogenic reaction) over 22.5 min without hydroquinone (the starting material of the nonfluorescent target reaction). (c) Two-dimensional histogram of n_p over 22.5 min with 50 μM (i), 100 μM (ii), 250 μM (iii), and 500 μM (iv) hydroquinone. The lines are structural features determined from the SEM image (f). (d) Δn_p between b and ci-civ, respectively. White pixels represent negative values. (e) COMPEITS image, Δ(n_p^–1) between b and ci–civ, respectively. (f) SEM image of the BiVO₄ particle being studied in (b)–(e). (g) Difference between basal {010} and lateral {110} facets on the particle. (h) Defining a type-I (50 nm width on either end of black edge line) and type-II edge region. (i–l) Plot of the inverse amplex red oxidation auxiliary reaction rate (v_AR^–1) versus hydroquinone concentration for the basal {010} (i) and lateral {110} facets (j) and the type-I (k) and type-II (l) edges. The data points for the particle shown in (b)–(e) are shown as solid circles and the open squares represent the averages for 42 particles. Scale bars, 500 nm. Error bars are standard error of the mean. Adapted with permission from ref (58). Copyright (2019) Springer Nature.

Figure 7

Nanoantennas. (a) SEM image of a gold bowtie nanoantenna. Scale bar, 100 nm. (b) Local intensity enhancement as calculated by the finite-difference time-domain method. Scale bar, 100 nm. (c) Confocal scan without bowtie nanoantennas at low concentration (less than 1 molecule/diffraction limited spot). Scale bar, 4 μm. (d) Confocal scale with bowtie nanoantennas at high concentrations (∼1000 molecules/diffraction limited spot). Scale bar, 4 μm. (e) Fluorescence time trace of a single molecule, unenhanced. (f) Fluorescence time trace of a molecule coated bowtie nanoantenna. (g) Scatter plot of 129 single-molecule fluorescence brightness enhancements (f_F) as a function of bowtie gap size. Adapted with permission from ref (60). Copyright (2009) Springer Nature.

Figure 8

Nanoantennas and DNA origami. (a) Cartoon schematic of a nanoantenna/DNA origami structure with a top town view inset. (b) Numerical simulation of electric field intensity at the equatorial plane of the dimer structure (interparticle distance 12nm, λ = 640 nm, incident electric field is polarized parallel to dimer orientation). (c) Numerical simulation of the effective quantum yield of fluorophores at the nanoantenna hotspot (aligned with the dimer orientation, λ = 669 nm) as a function of intrinsic quantum yield. (d) Fluorescence time traces for a dimer nanoantenna (black) and for a DNA origami structure without the dimer nanoantenna (red). Red trace was collected using ten times more excitation intensity. The excitation polarization is being rotated at 20 Hz. Adapted from ref (63). Copyright (2015) American Chemical Society.

Figure 9

Nanoconfinement. (a) 9-bp DNA duplex studied (Cy3 FRET donor green circle, Cy5 FRET acceptor pink circle). (b) Cartoon of DNA encapsulation within a nanovesicle. (c–i) Single-molecule time traces and histograms for more than 100 vesicles at increasing salt levels (c, d, g at 5 mM; e, h at 20 mM, and f, i at 50 mM; donor trace is in green, and acceptor trace is in pink). (j–l) Rates as a function of salt concentration, calculated using 541 vesicles (K_D ≡ k_off/k_on). Error bars represent standard error of the mean from experiments in triplicate at 23 °C. Reprinted with permission from ref (73). Copyright (2012) Springer Nature.

Figure 10

CLiC. (a) Cartoon schematic of the concept of CLiC. (b) Signal-to-background ratio as a function of displacement from point of contact. (c,d) CliC image of surface tethered DNA oligonucleotides in the presence of 0.2 μM (c) and 2 μM (d) fluorophore. Scale bar 10 μm. (e,f) Photobleaching time traces from (c) and (d), respectfully. Reprinted from ref (77). Copyright (2010) American Chemical Society.

Figure 11

Zero-mode waveguides. (a) Bright field image of a ZMW array. There are approximately 1600 ZMWs in the field of view imaged with a 512 × 512 EMCCD and a 100× objective. (b) Representative scanning electron microscopy (SEM) image of a ZMW array. (c) Cartoon showing a high concentration of a fluorescently labeled ligand binding to a surface tethered receptor inside a ZMW. The observation volume decays rapidly from the surface (∼25 nm). Only molecules diffusing within the small observation volume are observed.

Figure 12

Use of ZMWs to break the concentration barrier. Top: (a) Cartoon of a ZMW with DNA polymerase immobilized onto the bottom of the well and bound to a single molecule of DNA in a bulk solution of fluorescently labeled phospholinked nucleotide substrates. (b) Diagram of the phospholinked dNTP incorporation cycle and corresponding simulated fluorescence time trace that would be observed with the above cycle. (c) Single-molecule, real-time, four-color DNA sequencing. Fluorescence time traces for all four dye-weighted channels, colored corresponding to algorithmic least-squares fitting for all 150 bases of the linear template DNA strand. (d) Expanded time trace for 28 base incorporations and three errors. Expected template above with dashed lines corresponding to matches and lowercase corresponding to errors. Adapted with permission from ref (89). Copyright (2009) The American Association for the Advancement of Science. Middle: (e) Scanning electron microscopy (SEM) of ZMWs. (f) Cartoon of a ZMW with eGFP-tagged HCN deposited into the well for studying the binding of fcAMP (eGFP, Protein Data Bank (PDB) 2Y0G; HCN1, PDB 6UQG). (g–j) Fluorescence time trajectories of fcAMP binding to HCN1SM and HCN2SM at 250 nM (g, i) and 750 nM (h, j) with idealized fit overlaid (black), showing identification of 5 different binding states. Adapted with permission from ref (88). Copyright (2021) Springer Nature. Bottom: (k) Cartoon schematic of a ZMW with a ribosomal complex containing Cy3-labeled fMet-tNA^fMet deposited into the well. Ternary complexes Cy5-labeled Phe-tRNAPhe-EF-Tu(GTP) and Cy2-labeled Lys-tRNALys-EF-Tu(GTP), as well as EF-G(GTP) are introduced to the well. (l) Expected fluorescence time trace for low and high concentration of ternary complex. (m) Experimental fluorescence time traces for the translation of two heteropolymeric mRNAs (M(FK)₆ and M(FKK)₄) encoding 13 amino acids. (200 nM Phe-(Cy5)tRNA^Phe, 200 nM Lys-(Cy2)tRNA^Lys ternary complex and 500 nM EF-G). The fluorescent pulses observed mirror the mRNA sequence. Adapted with permission from ref (90). Copyright (2010) Springer Nature.

Figure 13

ZMW-FRET. Single-molecule ligand binding at mM concentrations with ZMW-FRET. Fluorescence time series for fcGMP binding events at a single CNBD with freely diffusing fcGMP at a concentration of 1 mM. Simultaneous emission from the donor (blue, fcGMP) and acceptor (red) upon interleaved donor (excitation at 532 nm) and acceptor (excitation at 640 nm) excitation. The acceptor emission is overlaid with the idealized time series (black). Adapted with permission from ref (114). Copyright (2017) John Wiley and Sons.