Philipp Kukura

Conical intersection dynamics of the primary photoisomerization event in vision

Ever since the conversion of the 11-cis retinal chromophore to its all-trans form in rhodopsin was identified as the primary photochemical event in vision, experimentalists and theoreticians have tried to unravel the molecular details of this process. The high quantum yield of 0.65 (ref. 2), the production of the primary ground-state rhodopsin photoproduct within a mere 200 fs (refs 3–7), and the storage of considerable energy in the first stable bathorhodopsin intermediate all suggest an unusually fast and efficient photoactivated one-way reaction. Rhodopsins unique reactivity is generally attributed to a conical intersection between the potential energy surfaces of the ground and excited electronic states enabling the efficient and ultrafast conversion of photon energy into chemical energy. But obtaining direct experimental evidence for the involvement of a conical intersection is challenging: the energy gap between the electronic states of the reacting molecule changes significantly over an ultrashort timescale, which calls for observational methods that combine high temporal resolution with a broad spectral observation window. Here we show that ultrafast optical spectroscopy with sub-20-fs time resolution and spectral coverage from the visible to the near-infrared allows us to follow the dynamics leading to the conical intersection in rhodopsin isomerization. We track coherent wave-packet motion from the photoexcited Franck–Condon region to the photoproduct by monitoring the loss of reactant emission and the subsequent appearance of photoproduct absorption, and find excellent agreement between the experimental observations and molecular dynamics calculations that involve a true electronic state crossing. Taken together, these findings constitute the most compelling evidence to date for the existence and importance of conical intersections in visual photochemistry.

Structural Observation of the Primary Isomerization in Vision with Femtosecond-Stimulated Raman

The primary event that initiates vision is the light-induced 11-cis to all-trans isomerization of retinal in the visual pigment rhodopsin. Despite decades of study with the traditional tools of chemical reaction dynamics, both the timing and nature of the atomic motions that lead to photoproduct production remain unknown. We used femtosecond-stimulated Raman spectroscopy to obtain time-resolved vibrational spectra of the molecular structures formed along the reaction coordinate. The spectral evolution of the vibrational features from 200 femtoseconds to 1 picosecond after photon absorption reveals the temporal sequencing of the geometric changes in the retinal backbone that activate this receptor.

Femtosecond broadband stimulated Raman spectroscopy: Apparatus and methods

The laser, detection system, and methods that enable femtosecond broadband stimulated Raman spectroscopy (FSRS) are presented in detail. FSRS is a unique tool for obtaining high time resolution (<100 fs) vibrational spectra with an instrument response limited frequency resolution of <10 cm(-1). A titanium:Sapphire-based laser system produces the three different pulses needed for FSRS: (1) A femtosecond visible actinic pump that initiates the photochemistry, (2) a narrow bandwidth picosecond Raman pump that provides the energy reservoir for amplification of the probe, and (3) a femtosecond continuum probe that is amplified at Raman resonances shifted from the Raman pump. The dependence of the stimulated Raman signal on experimental parameters is explored, demonstrating the expected exponential increase in Raman intensity with concentration, pathlength, and Raman pump power. Raman spectra collected under different electronic resonance conditions using highly fluorescent samples highlight the fluorescence rejection capabilities of FSRS. Data are also presented illustrating our ability: (i) To obtain spectra when there is a large transient absorption change by using a shifted excitation difference technique and (ii) to obtain high time resolution vibrational spectra of transient electronic states.

A planar dielectric antenna for directional single-photon emission and near-unity collection efficiency

Single-photon sources have been discussed as the building blocks of quantum cryptography, optical quantum computation, spectroscopy, and metrology. However, when using sources based on single emitters, the success of these proposals depends on the ability to achieve near-unity collection efficiency into well-defined modes. Some of the current state-of-the-art efforts aimed at achieving these criteria have been demonstrated, but despite an impressive progress the results still fall short. In particular, a collection efficiency of 38% were reported using microresonators [1], while a nanowire device reached an efficiency of 72% at cryogenic temperatures [2]. Here we report on a broad-band room-temperature scheme, which uses a layered dielectric antenna for realizing ultra-bright single photon sources with near-unity collection efficiency.

High-speed nanoscopic tracking of the position and orientation of a single virus.

Optical studies have revealed that, after binding, virions move laterally on the plasma membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings, which are important for cell infection. Here we present a colocalization methodology that combines scattering interferometry and single-molecule fluorescence microscopy to visualize both position and orientation of single quantum dot–labeled Simian virus 40 (SV40) particles. By achieving nanometer spatial and 8 ms temporal resolution, we observed sliding and tumbling motions during rapid lateral diffusion on supported lipid bilayers, and repeated back and forth rocking between nanoscopic regions separated by 9 nm. Our findings suggest recurrent swap of receptors and viral pentamers as well as receptor aggregation in nanodomains. We discuss the prospects of our technique for studying virus-membrane interactions and for resolving nanoscopic dynamics of individual biological nano-objects.

Geometry-induced electrostatic trapping of nanometric objects in a fluid

The ability to trap an object—whether a single atom or a macroscopic entity—affects fields as diverse as quantum optics, soft condensed-matter physics, biophysics and clinical medicine. Many sophisticated methodologies have been developed to counter the randomizing effect of Brownian motion in solution, but stable trapping of nanometre-sized objects remains challenging. Optical tweezers are widely used traps, but require sufficiently polarizable objects and thus are unable to manipulate small macromolecules. Confinement of single molecules has been achieved using electrokinetic feedback guided by tracking of a fluorescent label, but photophysical constraints limit the trap stiffness and lifetime. Here we show that a fluidic slit with appropriately tailored topography has a spatially modulated electrostatic potential that can trap and levitate charged objects in solution for up to several hours. We illustrate this principle with gold particles, polymer beads and lipid vesicles with diameters of tens of nanometres, which are all trapped without external intervention and independently of their mass and dielectric function. The stiffness and stability of our electrostatic trap is easily tuned by adjusting the system geometry and the ionic strength of the solution, and it lends itself to integration with other manipulation mechanisms. We anticipate that these features will allow its use for contact-free confinement of single proteins and macromolecules, and the sorting and fractionation of nanometre-sized objects or their assembly into high-density arrays.

Theory of femtosecond stimulated Raman spectroscopy

Femtosecond broadband stimulated Raman spectroscopy (FSRS) is a new technique that produces high-resolution (time-resolved) vibrational spectra from either the ground or excited electronic states of molecules, free from background fluorescence. FSRS uses simultaneously a narrow bandwidth approximately 1-3 ps Raman pump pulse with a continuum approximately 30-50 fs Stokes probe pulse to produce sharp Raman gains, at positions corresponding to vibrational transitions in the sample, riding on top of the continuum Stokes probe spectrum. When FSRS is preceded by a femtosecond actinic pump pulse that initiates the photochemistry of interest, time-resolved Raman spectroscopy can be carried out. We present two theoretical approaches to FSRS: one is based on a coupling of Raman pump and probe light waves with the vibrations in the medium, and another is a quantum-mechanical description. The latter approach is used to discuss the conditions of applicability and limitations of the coupled-wave description. Extension of the quantum-mechanical description to the case where the Raman pump beam is on resonance with an excited electronic state, as well as when FSRS is used to probe a nonstationary vibrational wave packet prepared by an actinic pump pulse, is also discussed.

Femtosecond broadband stimulated Raman: a new approach for high-performance vibrational spectroscopy.

Femtosecond stimulated Raman spectroscopy (FSRS) is a new technique that produces high-quality vibrational spectra free from background fluorescence. FSRS combines a narrow-bandwidth picosecond Raman pump pulse with an ∼80 fs continuum probe pulse to produce stimulated Raman spectra from the pump-induced gain in the probe spectrum. The high intensity of the Raman pump combined with the broad bandwidth of the probe produces high signal-to-noise vibrational spectra with very short data acquisition times. FSRS spectra of standard solutions and solvents such as aqueous Na2SO4, aqueous KNO3, methanol, isopropanol, and cyclohexane are collected in seconds. Furthermore, stimulated Raman spectra can be obtained using just a single pump–probe pulse pair that illuminates the sample for only ∼1 ps. Fluorescence rejection is demonstrated by collecting FSRS spectra of dyes (rhodamine 6G, chlorophyll a, and DTTCI) with varying degrees of fluorescence background and resonance enhancement. The high signal-to-noise, short data acquisition time, fluorescence rejection, and high spectral and temporal resolution of femtosecond stimulated Raman spectroscopy make it a valuable new vibrational spectroscopic technique.

Direct observation of the ultrafast intersystem crossing in tris(2,2′-bipyridine)ruthenium(II) using femtosecond stimulated Raman spectroscopy

Time-resolved femtosecond stimulated Raman spectroscopy (FSRS) is used to explore the ultrafast intersystem crossing between the metal-to-ligand charge-transfer (MLCT) states of tris(2,2′-bipyridine)ruthenium(II) ( ). Excitation at 480 nm by a ∼35 fs actinic pump pulse initiates electron transfer from the metal to the bipyridine ligands and the subsequent changes in the vibrational structure of the ligands are probed by FSRS with high spectral (10 cm−1) and temporal (70 fs) resolution. The unique Raman spectral features of the 3MLCT state of appear with rise times from 100 to 130 fs. An upper limit for the initial 1MLCT state lifetime of <30 fs is determined by analysis of the spontaneous emission spectra and quantum yield. The ultrashort lifetime of the 1MLCT state is attributed to fast Franck–Condon vibrational and solvent relaxation of the excited singlet state into near degeneracy with the triplet state, leading to fast and efficient intersystem crossing.

Imaging a Single Quantum Dot When It Is Dark

We have succeeded in recording extinction images of individual cadmium selenide quantum dots at ambient condition. This is achieved by optimizing the interference between the light that is coherently scattered from the quantum dot and the reflection of the incident laser beam. The ability to interrogate the dot in the absence of fluorescence has revealed that its extinction cross section diminishes in the photobleached state, but interestingly, it remains unchanged during fluorescence blinking off times. Our methodology makes optical imaging and spectroscopy accessible to the study of ultrasmall nanoscopic objects such as nonfluorescent macromolecules and single emitters with very low quantum efficiencies.