Engineering of IF1 -susceptive bacterial F1 -ATPase.

IF1 , an inhibitor protein of mitochondrial ATP synthase, suppresses ATP hydrolytic activity of F1 . One of the unique features of IF1 is the selective inhibition in mitochondrial F1 (MF1 ); it inhibits catalysis of MF1 but does not affect F1 with bacterial origin despite high sequence homology between MF1 and bacterial F1 . Here, we aimed to engineer thermophilic Bacillus F1 (TF1 ) to confer the susceptibility to IF1 for elucidating the molecular mechanism of selective inhibition of IF1 . We first examined the IF1 -susceptibility of hybrid F1 s, composed of each subunit originating from bovine MF1 (bMF1 ) or TF1 . It was clearly shown that only the hybrid with the ß subunit of mitochondrial origin has the IF1 -susceptibility. Based on structural analysis and sequence alignment of bMF1 and TF1 , the five non-conserved residues on the C-terminus of the ß subunit were identified as the candidate responsible for the IF1 -susceptibility. These residues in TF1 were substituted with the bMF1 residues. The resultant mutant TF1 showed evident IF1 -susceptibility. Reversely, we examined the bMF1 mutant with TF1 residues at the corresponding sites, which showed significant suppression of IF1 -susceptibility, confirming the critical role of these residues. We also tested additional three substitutions with bMF1 residues in α and γ subunits that further enhanced the IF1 -susceptibility, suggesting the additive role of these residues. We discuss the molecular mechanism by which IF1 specifically recognizes F1 with mitochondrial origin, based on the present result and the structure of F1 -IF1 complex. These findings would help the development of the inhibitors targeting bacterial F1 .

The series of conformational states adopted by rotorless F1-ATPase during its hydrolysis cycle.

F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalytic mechanism and isolated F1-ATPase subcomplexes can also hydrolyze ATP to generate rotation of their central γ rotor subunit. As ATP is hydrolyzed, the F1-ATPase cycles through a series of conformational states that mediates unidirectional rotation of the rotor. However, even in the absence of a rotor, the α and ß subunits are still able to pass through a series of conformations, akin to those that generate rotation. Here, we use cryoelectron microscopy to establish the structures of these rotorless states. These structures indicate that cooperativity in this system is likely mediated by contacts between the ß subunit lever domains, irrespective of the presence of the γ rotor subunit. These findings provide insight into how long-range information may be transferred in large biological systems.

Digital Cascade Assays for ADP- or ATP-Producing Enzymes Using a Femtoliter Reactor Array Device.

Digital enzyme assays are emerging biosensing methods for highly sensitive quantitative analysis of biomolecules with single-molecule detection sensitivity. However, current digital enzyme assays require a fluorogenic substrate for detection, which limits the applicability of this method to certain enzymes. ATPases and kinases are representative enzymes for which fluorogenic substrates are not available; however, these enzymes form large domains and play a central role in biology. In this study, we implemented a fluorogenic cascade reaction in a femtoliter reactor array device to develop a digital bioassay platform for ATPases and kinases. The digital cascade assay enabled quantitative measurement of the single-molecule activity of F1-ATPase, the catalytic portion of ATP synthase. We also demonstrated a digital assay for human choline kinase α. Furthermore, we developed a digital cascade assay for ATP-synthesizing enzymes and demonstrated a digital assay for pyruvate kinase. These results show the high versatility of this assay platform. Thus, the digital cascade assay has great potential for the highly sensitive detection and accurate characterization of various ADP- and ATP-producing enzymes, such as kinases, which may serve as disease biomarkers.

Molecular mechanism on forcible ejection of ATPase inhibitory factor 1 from mitochondrial ATP synthase.

IF1 is a natural inhibitor protein for mitochondrial FoF1 ATP synthase that blocks catalysis and rotation of the F1 by deeply inserting its N-terminal helices into F1. A unique feature of IF1 is condition-dependent inhibition; although IF1 inhibits ATP hydrolysis by F1, IF1 inhibition is relieved under ATP synthesis conditions. To elucidate this condition-dependent inhibition mechanism, we have performed single-molecule manipulation experiments on IF1-inhibited bovine mitochondrial F1 (bMF1). The results show that IF1-inhibited F1 is efficiently activated only when F1 is rotated in the clockwise (ATP synthesis) direction, but not in the counterclockwise direction. The observed rotational-direction-dependent activation explains the condition-dependent mechanism of IF1 inhibition. Investigation of mutant IF1 with N-terminal truncations shows that the interaction with the γ subunit at the N-terminal regions is crucial for rotational-direction-dependent ejection, and the middle long helix is responsible for the inhibition of F1.

Structural Elements Involved in ATP Hydrolysis Inhibition and ATP Synthesis of Tuberculosis and Nontuberculous Mycobacterial F-ATP Synthase Decipher New Targets for Inhibitors.

The F1FO-ATP synthase is required for the viability of tuberculosis (TB) and nontuberculous mycobacteria (NTM) and has been validated as a drug target. Here, we present the cryo-EM structures of the Mycobacterium smegmatis F1-ATPase and the F1FO-ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis. Mutational studies reveal that the extended C-terminal domain (αCTD) of subunit α is the main element for the self-inhibition mechanism of ATP hydrolysis for TB and NTM bacteria. Rotational studies indicate that the transition between the inhibition state by the αCTD and the active state is a rapid process. We demonstrate that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of α, γ, and δ are attractive targets, providing a platform for the discovery of species-specific inhibitors.

A microreactor sealing method using adhesive tape for digital bioassays.

Digital assays using microreactors fabricated on solid substrates are useful for carrying out sensitive assays of infectious diseases and other biological tests. However, sealing of the microchambers using fluid oil is difficult for non-experts, and thus hinders the widespread use of digital microreactor assays. Here, we propose the physical isolation of tiny reactors with adhesive tape (PITAT) using simple, commercially available pressure-sensitive adhesive (PSA) tape as a separator of the microreactors. We confirmed that PSA tape can effectively seal the microreactors and prevent molecules from diffusing out. By testing several types of adhesive tape, we found that rubber-based adhesives are the most suitable for this purpose. In addition, we demonstrated that single-molecule enzyme assays can be successfully performed inside microreactors sealed with PSA tape. The results obtained using PITAT are quantitatively comparable to conventional oil sealing, although it is quick and cost-effective. Finally, we demonstrated that single-particle virus counting of the influenza virus can be achieved using PITAT. Collectively, our results suggest that PITAT may be suitable for use in the design of sensitive tests for infectious diseases at the point of care, where no sophisticated equipment or machines are available.

The six steps of the complete F1-ATPase rotary catalytic cycle.

F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. Isolated F1-ATPase catalytic cores can hydrolyze ATP, passing through six intermediate conformational states to generate rotation of their central γ-subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in the F1-ATPase, the structure of an important conformational state (the binding-dwell) has remained elusive. Here, we exploit temperature and time-resolved cryo-electron microscopy to determine the structure of the binding- and catalytic-dwell states of Bacillus PS3 F1-ATPase. Each state shows three catalytic ß-subunits in different conformations, establishing the complete set of six states taken up during the catalytic cycle and providing molecular details for both the ATP binding and hydrolysis strokes. We also identify a potential phosphate-release tunnel that indicates how ADP and phosphate binding are coordinated during synthesis. Overall these findings provide a structural basis for the entire F1-ATPase catalytic cycle.

Kinetic analysis of the inhibition mechanism of bovine mitochondrial F1-ATPase inhibitory protein using biochemical assay.

ATPase inhibitory factor 1 (IF1) is a mitochondrial regulatory protein that blocks ATP hydrolysis of F1-ATPase, by inserting its N-terminus into the rotor-stator interface of F1-ATPase. Although previous studies have proposed a two-step model for IF1-mediated inhibition, the underlying molecular mechanism remains unclear. Here, we analysed the kinetics of IF1-mediated inhibition under a wide range of [ATP]s and [IF1]s, using bovine mitochondrial IF1 and F1-ATPase. Typical hyperbolic curves of inhibition rates with [IF1]s were observed at all [ATP]s tested, suggesting a two-step mechanism: the initial association of IF1 to F1-ATPase and the locking process, where IF1 blocks rotation by inserting its N-terminus. The initial association was dependent on ATP. Considering two principal rotation dwells, binding dwell and catalytic dwell, in F1-ATPase, this result means that IF1 associates with F1-ATPase in the catalytic-waiting state. In contrast, the isomerization process to the locking state was almost independent of ATP, suggesting that it is also independent of the F1-ATPase state. Further, we investigated the role of Glu30 or Tyr33 of IF1 in the two-step mechanism. Kinetic analysis showed that Glu30 is involved in the isomerization, whereas Tyr33 contributes to the initial association. Based on these findings, we propose an IF1-mediated inhibition scheme.

Multiparameter single-particle motion analysis for homogeneous digital immunoassay.

Digital homogeneous non-enzymatic immunosorbent assay (digital Ho-Non ELISA) is a new class of digital immunoassay that enables highly sensitive quantification of biomolecules using a simple protocol. In digital Ho-Non ELISA, nanoparticles are tethered onto the surface of femtoliter reactors via captured target molecules. The tethered particles capturing target molecules are identified as those showing a confined Brownian motion with root-mean-square displacement (RMSD) values in a defined range. The present work aims to improve the specificity to discriminate tethered particles via single-target molecules from non-specifically immobilized particles by analyzing two nanoparticle parameters. First, in order to suppress the broadening of RMSD due to the heterogeneity of bead size, we corrected the RMSD with the fluorescence intensity of the beads. Second, focusing on the shape of Brownian motion in the x-y trajectory, we classified motion patterns by aspect ratio of the trajectory. By using multiparameter single-particle motion analysis with corrected RMSD and aspect ratio, a 3.9-fold enhanced sensitivity in PSA assay was achieved compared to the conventional RMSD analysis approach. This new strategy would increase the potential of digital immunoassays.

The 3 × 120° rotary mechanism of Paracoccus denitrificans F1-ATPase is different from that of the bacterial and mitochondrial F1-ATPases.

The rotation of Paracoccus denitrificans F1-ATPase (PdF1) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or below Km, PdF1 showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF1 executes binding, hydrolysis, and probably product release at the same dwell. The comparison of ATP binding and catalytic pauses in single PdF1 molecules suggested that PdF1 executes both elementary events at the same rotary position. This point was confirmed in an inhibition experiment with a nonhydrolyzable ATP analog (AMP-PNP). Rotation assays in the presence of adenosine diphosphate (ADP) or inorganic phosphate at physiological concentrations did not reveal any obvious substeps. Although the possibility of the existence of substeps remains, all of the datasets show that PdF1 is principally a three-stepping motor similar to bacterial vacuolar (V1)-ATPase from Thermus thermophilus This contrasts with all other known F1-ATPases that show six or nine dwells per turn, conducting ATP binding and hydrolysis at different dwells. Pauses by persistent Mg-ADP inhibition or the inhibitory ζ-subunit were also found at the same angular position of the rotation dwell, supporting the simplified chemomechanical scheme of PdF1 Comprehensive analysis of rotary catalysis of F1 from different species, including PdF1, suggests a clear trend in the correlation between the numbers of rotary steps of F1 and Fo domains of F-ATP synthase. F1 motors with more distinctive steps are coupled with proton-conducting Fo rings with fewer proteolipid subunits, giving insight into the design principle the F1Fo of ATP synthase.

Multiplexed homogeneous digital immunoassay based on single-particle motion analysis.

Homogeneous digital immunoassay is a powerful analytical method for highly sensitive protein biomarker detection with a simple protocol. However, it has not been multiplexed yet. In this study, we developed a multiplexed homogeneous digital immunoassay based on single-particle motion analysis (digital homogeneous non-enzyme-linked immunosorbent assay, digital Ho-Non ELISA). In this assay, multiple target antigen molecules react with the optical subpopulation of magnetic nanobeads labeled with fluorescent dyes and capture antigen-specific antibodies. Then, these beads are magnetically pulled into femtoliter-sized reactors. The surface of these reactors is modified with multiple detection antibodies specific to each antigen by molecular tethers. Each antigen on the particles reacts with the detection antibodies anchored to the surface of the reactors. Magnetic force enhances the efficiency of bead encapsulation in the reactors, and subsequent physical compartmentalization of beads enhances the binding efficiency of the antigen-antibody reaction. The tethered beads show characteristic Brownian motion distinct from free diffusion or non-specific binding of the antigen-free beads. The color of the beads is attributed to target-identification, and the number of tethered beads is attributed to the concentration of the specific target. We measured two biomarkers (PSA and IL6) as model targets by multiplexed digital Ho-Non ELISA. Our method showed higher sensitivity compared to previous digital Ho-Non ELISA and could detect multiple targets simultaneously with the same performance as in single-plex detection. This new strategy has the potential to open a new avenue for next-generation multiplexed immunoassays in in vitro diagnostics.

Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments.

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant ßE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted "stall-and-release" experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.

Wash- and Amplification-Free Digital Immunoassay Based on Single-Particle Motion Analysis.

Digital enzyme-linked immunosorbent assay (ELISA) is a powerful analytical method for highly sensitive protein biomarker detection. The current protocol of digital ELISA requires multiple washing steps and signal amplification using an enzyme, which could be the potential drawback in in vitro diagnosis. In this study, we propose a digital immunoassay method, which we call "Digital HoNon-ELISA" (digital homogeneous non-enzymatic immunosorbent assay) for highly sensitive detection without washing and signal amplification. Target antigen molecules react with antibody-coated magnetic nanoparticles, which are then magnetically pulled into femtoliter-sized reactors. The antigens on the particles are captured by antibodies anchored on the bottom surface of the reactor via molecular tethers. Magnetic force enhances the efficiency of particle encapsulation in the reactors. Subsequent physical compartmentalization of the particles enhances the binding efficiency of antigen-carrying particles to the antibodies. The tethered particles show characteristic Brownian motion within a limited space by the molecular tethering, which is distinct from free diffusion or nonspecific binding of antigen-free particles. The number of tethered particles directly correlates with the concentration of the target antigen. Digital HoNon-ELISA was used with a prostate-specific antigen to achieve a detection of 0.093 pg/mL, which is over 9.0-fold the sensitivity of commercialized highly sensitive ELISA (0.84 pg/mL) and comparable to digital ELISA (0.055 pg/mL). This digital immunoassay strategy has sensitivity similar to digital ELISA with simplicity similar to homogeneous assay. Such similarity allows for potential application in rapid and simple digital diagnostic tests without the need for washing and enzymatic amplification.

Osmolyte-Enhanced Protein Synthesis Activity of a Reconstituted Translation System.

Molecular crowding is receiving great attention in cell-free synthetic biology because molecular crowding is a critical feature of natural cell discrimination from artificial cells. Further, it has significant and generic influences on biomolecular functions. Although there are reports on how the macromolecular crowder reagents affect cell-free systems such as transcription and translation, the second class of molecular crowder reagents with low molecular weight, osmolyte, was much less studied in cell-free systems. In the present study, we focused on trimethylamine- N-oxide (TMAO) and betaine, methylamine osmolytes, and investigated the effectiveness of these osmolytes on gene expression activity of reconstituted cell-free protein synthesis. The gene expression activity of the fluorescent proteins Venus and tdTomato and the enzymes ß-galactosidase and dihydrofolate reductase were tested. At 37 °C, 0.4 M TMAO showed the highest enhancement of translational activity by a factor of 1.6-3.8, regardless of protein type. In contrast, betaine showed only a moderate effect that was limited to fluorescent proteins. Excess amounts of osmolytes suppressed gene expression activity. An mRNA-start assay and SDS-PAGE quantitative analysis provided firm evidence that TMAO enhances the translation process, instead of transcription, folding, or the maturation of fluorescent proteins. Interestingly, at 26 °C, TMAO and betaine showed the highest enhancement of protein synthesis activity at lower concentrations than at 37 °C. These findings provide implications on how osmolytes assist translation in natural cells. Further, they provide guidelines for modulation of protein synthesis activity in artificial cells through osmolyte addition.

Ghost cytometry.

Ghost imaging is a technique used to produce an object's image without using a spatially resolving detector. Here we develop a technique we term "ghost cytometry," an image-free ultrafast fluorescence "imaging" cytometry based on a single-pixel detector. Spatial information obtained from the motion of cells relative to a static randomly patterned optical structure is compressively converted into signals that arrive sequentially at a single-pixel detector. Combinatorial use of the temporal waveform with the intensity distribution of the random pattern allows us to computationally reconstruct cell morphology. More importantly, we show that applying machine-learning methods directly on the compressed waveforms without image reconstruction enables efficient image-free morphology-based cytometry. Despite a compact and inexpensive instrumentation, image-free ghost cytometry achieves accurate and high-throughput cell classification and selective sorting on the basis of cell morphology without a specific biomarker, both of which have been challenging to accomplish using conventional flow cytometers.

Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase.

F1-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F1 exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal. However, the detailed mechanism has remained elusive. In this study, we performed stall-and-release experiments to elucidate how the ε subunit modulates ATP association/dissociation and hydrolysis/synthesis process kinetics and thermodynamics, key reaction steps for efficient ATP synthesis. The ε subunit significantly accelerated the rates of ATP dissociation and synthesis by two- to fivefold, whereas those of ATP binding and hydrolysis were not enhanced. Numerical analysis based on the determined kinetic parameters quantitatively reproduced previous findings of two- to fivefold coupling efficiency improvement by the ε subunit at the condition exhibiting the maximum ATP synthesis activity, a physiological role of F1-ATPase. Furthermore, fundamentally similar results were obtained upon ε subunit C-terminal domain truncation, suggesting that the N-terminal domain is responsible for the rate enhancement.

A Transient Rise in Free Mg2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation.

For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg2+, have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ increase is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell [33]. ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance.

Effects of non-equilibrium angle fluctuation on F1-ATPase kinetics induced by temperature increase.

F1-ATPase (F1) is an efficient rotary protein motor, whose reactivity is modulated by the rotary angle to utilize thermal fluctuation. In order to elucidate how its kinetics are affected by the change in the fluctuation, we have extended the reaction-diffusion formalism [R. Watanabe et al., Biophys. J., 2013, 105, 2385] applicable to a wider range of temperatures based on experimental data analysis of F1 derived from thermophilic Bacillus under high ATP concentration conditions. Our simulation shows that the rotary angle distribution manifests a stronger non-equilibrium feature as the temperature increases, because ATP hydrolysis and Pi release are more accelerated compared with the timescale of rotary angle relaxation. This effect causes the rate coefficient obtained from dwell time fitting to deviate from the Arrhenius relation in Pi release, which has been assumed in the previous activation thermodynamic quantities estimation using linear Arrhenius fitting. Larger negative correlation is also found between hydrolysis and Pi release waiting time in a catalytic dwell with the increase in temperature. This loss of independence between the two successive reactions at the catalytic dwell sheds doubt on the conventional dwell time fitting to obtain rate coefficients with a double exponential function at temperatures higher than 65 °C, which is close to the physiological temperature of the thermophilic Bacillus.

Direct Measurement of Single-Molecule Adenosine Triphosphatase Hydrolysis Dynamics.

F1-ATPase (F1) is a bidirectional molecular motor that hydrolyzes nearly all ATPs to fuel the cellular processes. Optical observation of labeled F1 rotation against the α3ß3 hexamer ring revealed the sequential mechanical rotation steps corresponding to ATP binding/ADP release and ATP hydrolysis/Pi release. These substeps originate from the F1 rotation but with heavy load on the γ shaft due to fluorescent labeling and the photophysical limitation of an optical microscope, which hampers better understanding of the intrinsic kinetic behavior of ATP hydrolysis. In this work, we present a method capable of electrically monitoring ATP hydrolysis of a single label-free F1 in real time by using a high-gain silicon nanowire-based field-effect transistor circuit. We reproducibly observe the regular current signal fluctuations with two distinct levels, which are induced by the binding dwell and the catalytic dwell, respectively, in both concentration- and temperature-dependent experiments. In comparison with labeled F1, the hydrolysis rate of nonlabeled F1 used in this study is 1 order of magnitude faster (1.69 × 108 M-1 s-1 at 20 °C), and the differences between two sequential catalytic rates are clearer, demonstrating the ability of nanowire nanocircuits to directly probe the intrinsic dynamic processes of the biological activities with single-molecule/single-event sensitivity. This approach is complementary to traditional optical methods, offering endless opportunities to unravel molecular mechanisms of a variety of dynamic biosystems under realistic physiological conditions.

Direct real-time detection of single proteins using silicon nanowire-based electrical circuits.

We present an efficient strategy through surface functionalization to build a single silicon nanowire field-effect transistor-based biosensor that is capable of directly detecting protein adsorption/desorption at the single-event level. The step-wise signals in real-time detection of His-tag F1-ATPases demonstrate a promising electrical biosensing approach with single-molecule sensitivity, thus opening up new opportunities for studying single-molecule biophysics in broad biological systems.