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We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy. Biological macromolecules exhibit complex conformation and stoichiometry changes in coordination with their motion and activity. To further our understanding of the complex machinery of life, we need methods that can simultaneously probe more than one degree of freedom of single molecules and complexes. Fluorescence optical tweezers, or "fleezers," combine the capabilities of optical tweezers and single-molecule fluorescence microscopy into a single instrument. Here we present the latest generation of a high-resolution fleezers instrument integrated with multicolor fluorescence spectroscopy. The tweezers portion of the instrument can manipulate biological macromolecules with pN scale forces while measuring subnanometer distances. Simultaneous with tweezers measurements, the multicolor fluorescence capability allows the direct observation of multiple molecules or multiple degrees of freedom which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, all sourced from a single-mode optical fiber allowing a reliable alignment scheme, that allows, for example, three independent fluorescent probes or fluorescence resonance energy transfer (FRET) measurements and also increases flexibility in the choice of fluorescent probes. To avoid photobleaching and improve tweezers stability, the instrument implements a timesharing (using a single trap laser to produce a pair of traps via rapid switching between two locations) and interlacing (turning the trapping beam off when the fluorescence excitation beams are on and vice versa) scheme using acousto-optic modulators (AOM) to rapidly and precisely modulate lasers. Our latest "random phase" trap AOM control method obliterates previous residual trap positioning and bead position measurement errors. Here we present the general design principles and detailed construction and testing protocols for the instrument.

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a newly developed single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single-molecule assembly. The multicolor allows the simultaneous observation of multiple molecules or multiple degrees of freedom, which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, with a reliable alignment scheme, which will allow three independent fluorescent probe or FRET measurements and also increases flexibility in the choice of fluorescent molecules. We demonstrate the ability to simultaneously measure angstrom-scale changes in tether extension and fluorescence signals. Simultaneous tweezers and fluorescence measurement are particularly challenging because of fluorophore photobleaching, even more so if multiple fluorophores are to be measured. Therefore, (1) fluorescence excitation and detection is interlaced with time-shared dual optical traps. (2) We investigated the photostability of common fluorophores. The mean number of photons emitted before bleaching was unaffected by the trap laser and decreased only slightly with increasing excitation laser intensity. Surprisingly, we found that Cy5 outperforms other commonly used fluorophores by more than fivefold. (3) We devised computer-controlled automation, which conserves fluorophore lifetime by quickly detecting fluorophore-labeled molecule binding, turning off lasers, and moving to add the next fluorophore-labeled component. The single-molecule assembly line enables the precise assembly of multimolecule complexes while preserving fluorophores.

Fundamental processes of life are carried out within cells by nanometer-scale molecular machines. Understanding how these tiny machines work reveals the basic physical underpinnings of life as well as provides opportunities for technological and medical advances. Single molecule biophysics, including optical tweezers, provides powerful experimental methods allowing us to directly observe the actions of individual molecules in real time. I will present new results from methods that combine two of the most powerful techniques: angstrom-resolution optical tweezers and single molecule fluorescence microscopy. I will describe some of the technical innovations involved in the research including tweezers stability and accuracy advances due to new acousto optic trap positioning device methods. I will then present very recent results where we have been able to perform high-resolution measurements of human telomerase protein machines extending DNA amid folding and unfolding DNA structures.

Over the past two decades, one of the standard models of protein folding has been the "two-state" model, in which a protein only resides in the folded or fully unfolded states with a single pathway between them. Recent advances in spatial and temporal resolution of biophysical measurements have revealed "beyond-two-state" complexity in protein folding, even for small, single-domain proteins. In this work, we used high-resolution optical tweezers to investigate the folding/unfolding kinetics of the B1 domain of immunoglobulin-binding protein G (GB1), a well-studied model system. Experiments were performed for GB1 both in and out of equilibrium using force spectroscopy. When the force was gradually ramped, simple single-peak folding force distributions were observed, while multiple rupture peaks were seen in the unfolding force distributions, consistent with multiple force-dependent parallel unfolding pathways. Force-dependent folding and unfolding rate constants were directly determined by both force-jump and fixed-trap measurements. Monte Carlo modeling using these rate constants was in good agreement with the force ramp data. The unfolding rate constants exhibited two different behaviors at low vs high force. At high force, the unfolding rate constant increased with increasing force, as previously reported by high force, high pulling speed force ramp measurements. However, at low force, the situation reversed and the unfolding rate constant decreased with increasing force. Taken together, these data indicate that this small protein has multiple distinct pathways to the native state on the free energy landscape.

Despite its fundamental importance in cellular processes and abundant use in biotechnology, we lack a detailed understanding of the kinetics of nucleic acid hybridization. In particular, the identity of the transition state, which determines the kinetics of the two-state reaction, remains poorly characterized. Here, we used optical tweezers with single-molecule fluorescence to observe directly the binding and unbinding of short oligonucleotides (7-12 nt) to a complementary strand held under constant force. Binding and unbinding rate constants measured across a wide range of forces (1.5-20 pN) deviate from the exponential force dependence expected from Bell's equation. Using a generalized force dependence model, we determined the elastic behavior of the transition state, which we find to be similar to that of the pure single-stranded state. Our results indicate that the transition state for hybridization is visited before the strands form any significant amount of native base pairs. Such a transition state supports a model in which the rate-limiting step of the hybridization reaction is the alignment of the two strands prior to base pairing.

A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Forster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. The proposed method is particularly promising as it enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force.

Acousto-optic (AO) devices have been used extensively in optical tweezers because of their flexibility and speed; however, these devices have trap positioning inaccuracies that limit their usefulness, especially for high-resolution applications. We show that these inaccuracies are due to interference patterns within the AO device sound fields. We have devised a method that removes these inaccuracies by reducing the coherence of the sound fields by directly controlling and randomizing the phase of the radio frequency voltage input signal. We demonstrate that the trapping inaccuracies are eliminated, for both constant trap position and force-ramp measurements, and that no additional noise is added. We show that this random phase method is applicable to both acousto-optic modulator and deflector type devices and can be easily integrated via software upgrade into existing instruments.

Despite their importance in biology and use in nanotechnology, the elastic behavior of nucleic acids on "ultrashort" (15 nt) length scales remains poorly understood. Here, we use optical tweezers combined with fluorescence imaging to observe directly the hybridization of oligonucleotides (7-12 nt) to a complementary strand under tension and to measure the difference in end-to-end extension between the single-stranded and duplex states. Data are consistent with long-polymer models at low forces (8 pN) but smaller than predicted at higher forces (8 pN), the result of the sequence-dependent duplex edge effects.

Engineering superenzyme function Understanding how protein domains and subunits operate is critical for engineering novel functions into proteins. Arslan et al. introduced intramolecular crosslinks between two domains of the Escherichia coli helicase Rep, which unwinds DNA. By inserting linkers of different lengths, the domains can be held either “open” or “closed.” The closed conformation activates the helicase, but it can also generate super-helicases capable of unzipping long stretches of DNA at high speed and with considerable force. Comstock et al. used optical tweezers and fluorescence microscopy to simultaneously measure the structure and function of the bacterial helicase UvrD. They monitored its DNA winding and unwinding activity and its shape during these activities. The motor domain also has a “closed” conformation during DNA unwinding and switches to a reversed “open” conformation during the zipping-up interaction. Science , this issue p. 344 and p. 352

We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument’s capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme.

Recent advances in optical tweezers have greatly expanded their measurement capabilities. A new generation of hybrid instrument that combines nanomechanical manipulation with fluorescence detection-fluorescence optical tweezers, or "fleezers"-is providing a powerful approach to study complex macromolecular dynamics. Here, we describe a combined high-resolution optical trap/confocal fluorescence microscope that can simultaneously detect sub-nanometer displacements, sub-piconewton forces, and single-molecule fluorescence signals. The primary technical challenge to these hybrid instruments is how to combine both measurement modalities without sacrificing the sensitivity of either one. We present general design principles to overcome this challenge and provide detailed, step-by-step instructions to implement them in the construction and alignment of the instrument. Lastly, we present a set of protocols to perform a simple, proof-of-principle experiment that highlights the instrument capabilities.

Helicases are a diverse set of enzymes that play an essential role in genome maintenance by converting chemical energy into the mechanical work of unzipping nucleic acid duplexes. E. coli UvrD is a prototypical superfamily 1 helicase involved in methyl-directed mismatch repair and nucleotide excision repair. Recent measurements have shown that it is able to both unwind and rezip the DNA duplex. Structural, ensemble kinetic, and single-molecule studies have provided insights into the unwinding mechanism of this helicase, but there has not yet been a direct observation of the individual motor steps involved in the process. Here, we use high-resolution optical tweezers to observe directly the stepping behavior of UvrD at a single-molecule level as it unwinds and rezips a DNA hairpin. Interestingly, we measure a step size of 3-4 base pairs for both unwinding and rezipping activities. Furthermore, our analysis of the stepping kinetics indicates that a single rate-limiting step governs the process, which is consistent with ensemble studies measuring an unwinding step size of 4-5 base pairs per rate-limiting step. However, previous studies have determined that 1 ATP molecule is hydrolyzed per base pair translocated, and would predict 3-4 rate-limiting steps per unwinding step. Our results therefore suggest a mechanism whereby UvrD rapidly unwinds 3-4 base pairs, and then must slowly “reset” itself in order to continue its unwinding cycle.

Photon echo and reverse transient grating measurements of the loss of electronic coherence for molecular iodine are presented. Systematic measurements of the coherence decay rate were made as a function of buffer gas. From the dependence of decay rate on numerical density, we calculated experimental cross sections of decoherence. These values range from 135 A2 for helium to 1170 A2 for I2. We find Lennard-Jones parameters for the long-range interactions responsible for decoherence which can be modeled by dispersion forces.

Phase modulation of ultrafast laser pulses has been demonstrated recently by taking advantage of the time-dependent refractive index that takes place during rotational wave packet revivals in a gaseous sample [Phys. Rev. Lett. 88 (2002) 013903]. Here we explore the effect of laser intensity, physical properties of molecules, and temperature on rotational wavepacket revivals by femtosecond transient grating spectroscopy. Theoretical analysis of experimental data is used to predict the optimum conditions for the phase modulation and compression of ultrafast laser pulses.

Non-resonant femtosecond time-resolved four-wave mixing (FWM) data on gas phase NO 2 and N 2 O 4 are presented. The initial rotational dephasing is observed during the first picosecond after excitation. Fast vibrational dynamics (average value 133±1 fs beats) are observed for dinitrogen tetroxide and are assigned to the ν 3 N–N stretching mode in the ground state ( 1 A g ) . Rotational revivals on the picosecond time scale are recorded for both samples. No evidence of a long-lived excited state participation was found using photon echo (PE) or virtual echo (VE) pulse sequences with 800 nm laser pulses, however, NO 2 photoproducts were observed following N 2 O 4 excitation.

The hybridization of short oligonucleotides plays a critical role in many biological systems, from DNA replication to gene silencing. Despite extensive studies, details on the mechanism of this process on the shortest length scale (∼10 bp) remain poorly understood. We use high-resolution optical tweezers with simultaneous fluorescence microscopy to investigate the hybridization of single oligonucleotides under tension1. We measure the change in end-to-end extension upon annealing and melting as well as the unbinding kinetics of short (7-12 bp), fluorescently labeled oligonucleotides hybridizing to a complementary DNA sequence tethered between trapped beads. Our results allow us to construct an energy landscape of oligonucleotide hybridization along well-defined reaction coordinates. Interestingly, our measurement of the distance to the transition state for DNA melting as a function of oligonucleotide length suggests that the extension of the transition state matches that of a state with 6 bp hybridized. Lastly, we find that the change in extension upon hybridization deviates from extensible-wormlike-chain behavior at forces >10 pN. We discuss possible models to explain these results.1. M. J. Comstock, Taekjip Ha, and Y. R. Chemla, Nat. Meth. 8, 4 (2011).

We present single-molecule high-resolution optical trapping measurements of RNA unwinding by the Ski2-like nuclear helicase Mtr4p both alone and within the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex. RNA helicases such as Mtr4p play key roles in RNA metabolism. Mtr4p is thought to assist RNA degradation by the exosome presumably by unwinding RNA structures. In addition, the TRAMP complex appends short 3′ poly(A) tails to RNA meant for degradation by the exosome. Mtr4p within the TRAMP complex plays a crucial role in regulating the polyadenylation activity, limiting the poly(A) tail length. The unwinding activity of Mtr4p is required and indeed within the TRAMP complex, Mtr4p unwinds duplex RNA 10x faster than when alone. However, the dynamic details of the RNA unwinding activity and interaction of Mtr4p and TRAMP and indeed the Ski2-like family of RNA helicases are unknown. We have performed high-resolution RNA hairpin unwinding experiments for both Mtr4p and TRAMP. We find that Mtr4p unwinds RNA duplexes in a single or multiple bursts with a mean step size of 9 base pairs. The unwinding is irreversible suggesting that Mtr4p remains stably bound after unwinding the duplex, preventing re-annealing. TRAMP unwinding experiments reveal a faster molecular recognition by the Mtr4p helicase when in complex as well as enhanced RNA duplex unwinding dynamics. Combined high-resolution trapping and fluorescence experiments underway may reveal how the competition within TRAMP between the 3′-to-5′ Mtr4p helicase and the 5′-to-3′ Trf4p polymerase is resolved dynamically.

Single-molecule-resolved scanning tunneling microscopy of tetra-tert-butyl azobenzene (TTB-AB) molecules adsorbed onto Au(111) reveals chirality selection rules in their photoswitching behavior. This observation is enabled by the fact that trans-TTB-AB molecules self-assemble into homochiral domains. Cis-TTB-AB molecules produced via photoisomerization are found in two distinct conformations with final state chirality determined by the initial trans isomer chirality. Based on these observations and ab initio calculations, we propose a new inversion-based dynamical photoswitching mechanism for azobenzene molecules at a surface.

We demonstrate experimentally that selective two-photon probe excitation using phase shaped pulses can be achieved even when the laser propagates through scattering tissue. The pre-optimized phase tailored femtosecond pulses were able to identify acidic and basic solutions of a pH sensitive chromophore hidden behind a slab of scattering tissue. This observation has important implications for future applications of coherent control for biomedical imaging and photodynamic therapy.

We demonstrate a new approach to laser control using binary phase shaping. We apply this method to the problem of spectrally narrowing multiphoton excitation using shaped laser pulses as required for selectivity in two-photon microscopy. The symmetry of the problem is analyzed from first principles and a rational solution is proposed. Successful experimental implementation and simulations are presented using 10 fs ultrashort pulses. The proposed solution is a factor of 6 better than the sinusoidal phase used previously by our group. An evolutionary learning algorithm was used to efficiently improve the solution by a further factor of 2.5 because of the greatly reduced search space afforded by binary phase shaping.

Photomechanical switching (photoisomerization) of molecules at a surface is found to strongly depend on molecule-molecule interactions and molecule-surface orientation. Scanning tunneling microscopy was used to image photoswitching behavior in the single-molecule limit of tetra-tert-butyl-azobenzene molecules adsorbed onto Au(111) at 30 K. Photoswitching behavior varied strongly with surface molecular island structure, and self-patterned stripes of switching and nonswitching regions were observed having approximately 10 nm pitch. These findings can be summarized into photoswitching selection rules that highlight the important role played by a molecule's nanoscale environment in determining its switching properties.

We have observed reversible light-induced mechanical switching for a single organic molecule bound to a metal surface. Scanning tunneling microscopy (STM) was used to image the features of an individual azobenzene molecule on Au(111) before and after reversibly cycling its mechanical structure between trans and cis states using light. Azobenzene molecules were engineered to increase their surface photomechanical activity by attaching varying numbers of tert-butyl (TB) ligands ("legs") to the azobenzene phenyl rings. STM images show that increasing the number of TB legs "lifts" the azobenzene molecules from the substrate, thereby increasing molecular photomechanical activity by decreasing molecule-surface coupling., related theoretical paper: cond-mat/0612202

We have manipulated single azobenzene molecules using several different techniques with a variable-temperature scanning tunneling microscope (STM). Isolated azobenzene molecules were adsorbed and manipulated on the Au(111) surface at low temperature $(T=34\phantom{\rule{0.3em}{0ex}}\mathrm{K})$. STM tip voltage pulses reversibly switched the azobenzene molecule between two stable orientational configurations. A sliding technique was used to move single azobenzene molecules between stable sites on the surface, enabling construction of molecular chains. Stable sites (i.e., molecular anchor points) were created on the Au(111) surface through STM tip voltage pulses.

We have used scanning tunneling spectroscopy to probe the local electronic properties of Si(100) near room temperature. In the low-bias filled-state regime we observe a band that is spatially localized to the region between Si dimer rows and that is closer to the Fermi energy than features previously associated with the dangling-bond $\ensuremath{\pi}$ band. In the high-bias empty-state regime we observe strong tip-height dependence for the energy of a band typically associated with antibonding back-bond $({B}^{*})$ and dimer-bond $({\ensuremath{\sigma}}^{*})$ states. This latter effect can be explained through tip-induced band bending.

We demonstrate experimentally selective two-photon functional imaging through a scattering medium (a thin slice of chicken) based on concepts of coherent control. The selectivity, achieved using binary phase shaping, is maintained by the ballistic photons.

We test whether coherent control methods based on ultrashort-pulse phase shaping can be applied when the laser light propagates through biological tissue. Our results demonstrate experimentally that the spectral-phase properties of shaped laser pulses optimized to achieve selective two-photon excitation survive as the laser pulses propagate through tissue. This observation is used to obtain functional images based on selective two-photon excitation of a pH-sensitive chromophore in a sample that is placed behind a slice of biological tissue. Our observation of coherent control through scattering tissue suggests possibilities in multiphoton-based imaging and photodynamic therapy.

N2O4 is studied by heterodyne detected FWM. Vibrational dynamics in the ground state are observed and assigned to N-N stretch and torsion. Rotational revivals on picosecond time were used to get information about the geometry.

The response of atoms and molecules to intense 1013 — 1015 W/cm2 lasers was investigated using two-dimensional (time-frequency) four-wave mixing. The data reveal a transition from a molecular response to a purely electronic (plasma) response.

We report a method based on transient-grating spectroscopy to assess the degree of alignment and electronic state mixing caused by intense femtosecond laser pulses. Molecular deformations are determined by analysis of the filed-free rotational recurrences.

We present single-molecule in vitro measurements of the DNA unwinding activity of the Superfamily 1 UvrD helicase that reveal directly how helicase stoichiometry and conformation correspond to motor activity. Although a single UvrD molecule can translocate along ssDNA, it has been postulated that only a dimer is capable of unwinding duplex DNA. UvrD is known to exhibit two different conformations: the ‘closed' and ‘open' orientation of the 2B regulatory subdomain. The function of these conformations has remained elusive. using a new instrument combining high-resolution optical tweezers with simultaneous single-molecule fluorescence microscopy (Comstock, Ha, and Chemla, Nature Methods 2011), we obtain an unprecedented detailed view of helicase motor dynamics by recording single base pair-scale DNA unwinding activity (optical tweezers) simultaneously with helicase stoichiometry and conformation (fluorescence). We observe two classes of DNA unwinding by UvrD: long distance continuous unwinding (∼100 bp) and repetitive, non-processive (∼12 bp) unwinding. We determine directly the stoichiometry of UvrD molecules participating in unwinding by quantifying the fluorescence intensity using singly-labeled UvrD molecules. We observe that pairs of UvrD molecules are required for long distance unwinding but individual molecules exhibit limited non-processive unwinding activity. Furthermore, we observe directly the conformation of the 2B subdomain of individual UvrD molecules during unwinding by measuring the fluorescence of FRET-labeled proteins. We observe that UvrD is in the ‘closed' conformation during DNA unwinding but surprisingly switches to the ‘open' conformation when UvrD translocates on the opposing ssDNA strand away from the junction with the DNA junction re-annealing behind it. These measurements may explain UvrD helicase's remarkable ability to switch translocation strands. We hypothesize that the UvrD 2B subdomain acts as a switch that controls DNA unwinding vs. re-annealing.

We have investigated the temperature-dependent behavior of thiolated azobenzene molecules on Au(111) using scanning tunneling microscopy. The addition of a thiol functional group to azobenzene molecules leads to increased surface anchoring of single azobenzene molecules to gold. Thiolated azobenzene shows diverse surface morphology and does not form well-ordered structures at low coverage. At elevated temperatures, anchored molecules are observed to spin in place via hindered rotation. By measuring the number of rotating molecules as a function of temperature and using a simple model, we are able to estimate the energy barrier and attempt frequency for thermally induced hindered rotation to be 102+/-3 meV and 110+/-2 GHz, respectively.

We have used single-molecule-resolved scanning tunneling microscopy to measure the photomechanical switching rates of azobenzene-derived molecules at a gold surface during exposure to UV and visible light. This enables the direct determination of both the forward and reverse photoswitching cross sections for surface-mounted molecules at different wavelengths. In a dramatic departure from molecular behavior in solution-based environments, visible light does not efficiently reverse the reaction for azobenzene-derived molecules at a gold surface.

Three shaped non-degenerate pulses can initialize a manifold of rovibronic states, store information, and perform a computation. Results from experiments carried out on molecular iodine will be presented.