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. Rhodopsin's 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.

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.

The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.

Femtosecond stimulated Raman spectroscopy (FSRS) is a new ultrafast spectroscopic technique that provides vibrational structural information with high temporal (50-fs) and spectral (10-cm(1)) resolution. As a result of these unique capabilities, FSRS studies of chemical and biochemical reaction dynamics are expected to grow rapidly, giving previously unattainable insight into the structural dynamics of reactively evolving systems with atomic spatial and femtosecond temporal resolution. This review discusses the experimental and theoretical concepts behind FSRS, with an emphasis on the origins of its unique temporal and spectral capabilities. We illustrate these capabilities with vibrational studies of ultrafast electronic dynamics, as well as the direct structural observation of nonstationary vibrational wave-packet motion in small molecules and in complex biochemical reaction dynamics.

Singlet exciton fission is the process in organic semiconductors through which a spin-singlet exciton converts into a pair of spin-triplet excitons residing on different chromophores, entangled in an overall spin-zero state. For some systems, singlet fission has been shown to occur on the 100 fs timescale and with a 200% yield, but the mechanism of this process remains uncertain. Here we study a model singlet fission system, TIPS-pentacene, using ultrafast vibronic spectroscopy. We observe that vibrational coherence in the initially photogenerated singlet state is transferred to the triplet state and show that this behaviour is effectively identical to that observed in ultrafast internal conversion for polyenes in solution. This similarity in vibronic dynamics suggest that both multi-molecular singlet fission and single-molecular internal conversion are mediated by the same underlying relaxation processes, based on strong coupling between nuclear and electronic degrees of freedom. In its most efficient form this leads to a conical intersection between the coupled electronic states.

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.

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.

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.