Matthew J. Comstock

Ultrahigh-resolution optical trap with single-fluorophore sensitivity

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore–labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).

Multiphoton intrapulse interference 6; binary phase shaping

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.

Direct observation of structure-function relationship in a nucleic acid processing enzyme

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 Both structure and function can be studied at the same time while an enzyme unzips DNA. The relationship between protein three-dimensional structure and function is essential for mechanism determination. Unfortunately, most techniques do not provide a direct measurement of this relationship. Structural data are typically limited to static pictures, and function must be inferred. Conversely, functional assays usually provide little information on structural conformation. We developed a single-molecule technique combining optical tweezers and fluorescence microscopy that allows for both measurements simultaneously. Here we present measurements of UvrD, a DNA repair helicase, that directly and unambiguously reveal the connection between its structure and function. Our data reveal that UvrD exhibits two distinct types of unwinding activity regulated by its stoichiometry. Furthermore, two UvrD conformational states, termed “closed” and “open,” correlate with movement toward or away from the DNA fork.

Measuring reversible photomechanical switching rates for a molecule at a surface

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.

Multiphoton Intrapulse Interference 8. Coherent control through scattering tissue.

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.

Self-patterned molecular photoswitching in nanoscale surface assemblies

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 molecules nanoscale environment in determining its switching properties.

Femtosecond ground state dynamics of gas phase N2O4 and NO2

Abstract 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.

Femtosecond photon echo measurements of electronic coherence relaxation between the X(1Σg+) and B(3Π0u+) states of I2 in the presence of He, Ar, N2, O2, C3H8

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.

Ultrafast transient-grating study of molecules after high intensity excitation,

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.

Two-dimensional (time-frequency) femtosecond four-wave mixing at 1014 W/cm2: molecular and electronic response

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.