Vibrationally coherent crossing and coupling of electronic states during internal conversion in beta-carotene

Coupling of nuclear and electronic degrees of freedom mediates energy flow in molecules after optical excitation. The associated coherent dynamics in polyatomic systems, however, remain experimentally unexplored. Here, we combined transient absorption spectroscopy with electronic population control to reveal nuclear wavepacket dynamics during the S2-S1 internal conversion in beta-carotene. We show that passage through a conical intersection is vibrationally coherent and thereby provides direct feedback on the role of different vibrational coordinates in the breakdown of the Born-Oppenheimer approximation

Sub-10 fs pulses tunable from 480 to 980 nm from a NOPA pumped by a Yb:KGW source

We describe two noncollinear optical parametric amplifier (NOPA) systems pumped by either the second (515 nm) or the third (343 nm) harmonic of an Yb:KGW amplifier, respectively. Pulse durations as short as 6.8 fs are readily obtained by compression with commercially available chirped mirrors. The availability of both second and third harmonic for NOPA pumping allows for gap-free tuning from 520 to 980 nm. The use of an intermediate NOPA to generate seed light at 780 nm extends the tuning range of the third-harmonic pumped NOPA towards 450 nm

Direct detection of single molecules by optical absorption

The advent of single molecule optics has had a profound impact in fields ranging from biophysics to material science, photophysics, and quantum optics. However, all existing room-temperature single molecule methods have been based on fluorescence detection of highly efficient emitters. Here we demonstrate that standard, modulation-free measurements known from conventional absorption spectrometers can indeed detect single molecules. We report on quantitative measurements of the absorption cross section of single molecules under ambient condition even in their dark state, for example during photoblinking or strong quenching. Our work extends single-molecule microscopy and spectroscopy to a huge class of materials that absorb light but do not fluoresce efficiently.Comment: 15 pages, 4 figure

Sensing force and charge at the nanoscale with a single-molecule tether

Measuring the electrophoretic mobility of molecules is a powerful experimental approach for investigating biomolecular processes. A frequent challenge in the context of single-particle measurements is throughput, limiting the obtainable statistics. Here, we present a molecular force sensor and charge detector based on parallelised imaging and tracking of tethered double-stranded DNA functionalised with charged nanoparticles interacting with an externally applied electric field. Tracking the position of the tethered particle with simultaneous nanometre precision and microsecond temporal resolution allows us to detect and quantify electrophoretic forces down to the sub-piconewton scale. Furthermore, we demonstrate that this approach is capable of detecting changes to the particle charge state, as induced by the addition of charged biomolecules or changes to pH. Our approach provides an alternative route to studying structural and charge dynamics at the single-molecule level.Comment: 23 pages, 7 figure

Direct observation of the coherent nuclear response after the absorption of a photon

How molecules convert light energy to perform a specific transformation is a fundamental question in photophysics. Ultrafast spectroscopy reveals the kinetics associated with electronic energy flow, but little is known about how absorbed photon energy drives nuclear or electronic motion. Here, we used ultrabroadband transient absorption spectroscopy to monitor coherent vibrational energy flow after photoexcitation of the retinal chromophore. In the proton pump bacteriorhodopsin we observed coherent activation of hydrogen wagging and backbone torsional modes that were replaced by unreactive coordinates in the solution environment, concomitant with a deactivation of the reactive relaxation pathway

Single-protein optical holography

Light scattering by nanoscale objects is a fundamental physical property defined by their scattering cross-section and thus polarisability. Over the past decade, a number of studies have demonstrated single-molecule sensitivity by imaging the interference between coherent scattering from the object of interest and a reference field. This approach has enabled mass measurement of single biomolecules in solution owing to the linear scaling of the image contrast with the molecular polarisability. Nevertheless, all implementations to date are based on a common-path interferometer and cannot separate and independently tune the reference and scattered light field, thus prohibiting access to the rich toolbox available to holographic imaging. Here, we demonstrate comparable sensitivity using a non-common path geometry based on a dark-field scattering microscope, similar to a Mach-Zehnder interferometer. We separate the scattering and reference light into four parallel, inherently phase stable detection channels, delivering a five orders of magnitude boost in sensitivity in terms of scattering cross-section over state-of-the-art holographic methods. We demonstrate the detection, resolution and mass measurement of single proteins with mass below 100 kDa. Separate amplitude and phase measurement also yields direct information on sample identity and experimental determination of the polarisability of single biomolecules

Wide-Field Detected Fourier Transform CARS Microscopy.

We present a wide-field imaging implementation of Fourier transform coherent anti-Stokes Raman scattering (wide-field detected FT-CARS) microscopy capable of acquiring high-contrast label-free but chemically specific images over the full vibrational 'fingerprint' region, suitable for a large field of view. Rapid resonant mechanical scanning of the illumination beam coupled with highly sensitive, camera-based detection of the CARS signal allows for fast and direct hyperspectral wide-field image acquisition, while minimizing sample damage. Intrinsic to FT-CARS microscopy, the ability to control the range of time-delays between pump and probe pulses allows for fine tuning of spectral resolution, bandwidth and imaging speed while maintaining full duty cycle. We outline the basic principles of wide-field detected FT-CARS microscopy and demonstrate how it can be used as a sensitive optical probe for chemically specific Raman imaging

Myosin II filament dynamics in actin networks revealed with interferometric scattering microscopy

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood due to difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering (iSCAT) microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect, and image individual membrane attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network

Lifting the concentration limit of mass photometry by PEG nanopattering

Mass photometry (MP) is a rapidly growing optical technique for label-free mass measurement of single biomolecules in solution. The underlying measurement principle provides numerous advantages over ensemble-based methods but has been limited to low analyte concentrations due to the need to uniquely and accurately quantify the binding of individual molecules to the measurement surface, which results in diffraction-limited spots. Here, we combine nanoparticle lithography with surface PEGylation to dramatically lower surface binding, resulting in a two-order magnitude improvement in the upper concentration limit associated with mass photometry. We demonstrate facile tunability of the degree of passivation, enabling measurements at increased analyte concentrations. These advances enable us to rapidly quantify protein- protein interactions in the high nM to low μM range, substantially expanding the application space of mass photometry