Photothermal heterodyne imaging of individual nonfluorescent nanoclusters and nanocrystals

We introduce a new, highly sensitive, and simple heterodyne optical method for imaging individual nonfluorescent nanoclusters and nanocrystals. A 2 order of magnitude improvement of the signal is achieved compared to previous methods. This allows for the unprecedented detection of individual small absorptive objects such as metallic clusters (of 67 atoms) or nonluminescent semiconductor nanocrystals. The measured signals are in agreement with a calculation based on the scattering field theory from a photothermal-induced modulated index of refraction profile around the nanoparticle

Tethered Particle Motion Reveals that LacI·DNA Loops Coexist with a Competitor-Resistant but Apparently Unlooped Conformation

AbstractThe lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound

Photothermal Heterodyne Imaging of Individual Metallic Nanoparticles: Theory versus Experiments

We present the theoretical and detailed experimental characterizations of Photothermal Heterodyne Imaging. An analytical expression of the photothermal heterodyne signal is derived using the theory of light scattering from a fluctuating medium. The amplitudes of the signals detected in the backward and forward configurations are compared and their frequency dependences are studied. The application of the Photothermal Heterodyne detection technique to the absorption spectroscopy of individual gold nanoparticles is discussed and the detection of small individual silver nanoparticles is demonstrated

Sub-nanometer measurement of transient structural changes in dye-doped polystyrene microspheres

We present an interferometric spectral-domain optical coherence tomography microscopy setup to detect structural changes using interference of light reflected from different interfaces of the sample. We induce a reproducible nanometer-scale size change in dye-doped 10-µm polystyrene microspheres by the release of Stokes shift energy of dye molecules inside the microspheres, excited by a modulated 532-nm laser. The resulting optical path length difference was measured with a sensitivity of 0.4 pm/√Hz limited by photodetection noise, and reveals elastic as well as inelastic responses, which opens up possibilities for measuring the response of cell-sized biological objects

A Master equation approach to modeling an artificial protein motor

Linear bio-molecular motors move unidirectionally along a track by coordinating several different processes, such as fuel (ATP) capture, hydrolysis, conformational changes, binding and unbinding from a track, and center-of-mass diffusion. A better understanding of the interdependencies between these processes, which take place over a wide range of different time scales, would help elucidate the general operational principles of molecular motors. Artificial molecular motors present a unique opportunity for such a study because motor structure and function are a priori known. Here we describe use of a Master equation approach, integrated with input from Langevin and molecular dynamics modeling, to stochastically model a molecular motor across many time scales. We apply this approach to a specific concept for an artificial protein motor, the Tumbleweed.Comment: Submitted to Chemical Physics; 9 pages, 7 figure

Single NanoParticle Photothermal Tracking (SNaPT) of 5 nm gold beads in live cells

Tracking individual nano-objets in live cells during arbitrary long times is an ubiquitous need in modern biology. We present here a method for tracking individual 5 nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against Single fluorescent Molecule Tracking in live COS7 cells on subsecond time scales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. SNaPT has the unique potential to record arbitrary long trajectory of membrane proteins using non-fluorescent nanometer sized labels