Marco Capitanio

Calibration of optical tweezers with differential interference contrast signals

A comparison of different calibration methods for optical tweezers with the differential interference contrast (DIC) technique was performed to establish the uses and the advantages of each method. A detailed experimental and theoretical analysis of each method was performed with emphasis on the anisotropy involved in the DIC technique and the noise components in the detection. Finally, a time of flight method that permits the reconstruction of the optical potential well was demonstrated.

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 μs, allows detection of interactions as brief as ∼100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

Three-dimensional magneto-optic trap for micro-object manipulation

A magneto-optic trap for micro-objects is described. Magnetic beads were trapped by optical tweezers while being rotated by a new integrated magnetic manipulator. Rotation was achieved with eight electromagnets with tip-pole geometry. The time orbital potential technique was used to achieve rotation of magnetic beads. Trapping in three dimensions and rotation of magnetic beads on three axes are demonstrated with forces up to 230 pN and force momenta of up to 10(-16)N m . A position-detection apparatus based on an interferometric scheme provides nanometer sensitivities in a few milliseconds.

Force and torque measurements using magnetic micro beads for single molecule biophysics

Characterization of optical trapping with laser tweezers of super-paramagnetic beads has been performed for the first time. In particular, we have compared the force action in optical trapping on super-paramagnetic beads with respect to the polystyrene ones. Using a novel magneto-optical manipulator we have developed a direct rotation detection scheme for trapped beads characterized by a high sensitivity and fast time response. Finally a calibration procedure able to directly measure an applied external torque on the trapped bead is shown. This demonstrates the capability of this apparatus to perform direct torque measurement (torsional rigidity) on a single bio-molecule attached to a super-paramagnetic bead.

FIONA in the trap: the advantages of combining optical tweezers and fluorescence

Technologies for the manipulation of single molecules have reached the resolution for the measurement of nanometre and sub-nanometre displacements and piconewton forces. In parallel with manipulation techniques, an array of single-molecule fluorescence detection methods have been developed to measure with great precision the position and/or the orientation of single biomolecules, as well as their conformational fluctuations. A new generation of instruments devoted to single-molecule biophysics is now emerging from the combination of two or more single-molecule techniques into one set-up. Particularly fruitful is the combination of manipulation techniques with single-molecule fluorescence techniques, allowing the detection of biomolecule position, conformation or biochemical state simultaneously with the measurement (or the external control) of mechanical output. Here we present the combination of optical tweezers and fluorescence imaging with nanometre accuracy (FIONA). The apparatus was tested on an actin filament labelled with a quantum dot and suspended in solution in a dumbbell configuration using the laser tweezers. This apparatus allows control of the mechanical conditions of a track (actin, microtubules, nucleic acids) while monitoring, by fluorescence, locomotion (and, possibly, biochemical state) of a motor on the track, thus being applicable to a large variety of biological systems.

Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level

The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates, explained in current models by a combination of 3D and 1D diffusion. Here, we present a review of the single-molecule approaches at the forefront of the study of protein-DNA interaction dynamics and target search in vitro and in vivo. Flow stretch, optical and magnetic manipulation, single fluorophore detection and localization as well as combinations of different methods are described and the results obtained with these techniques are discussed in the framework of the current facilitated diffusion model.

New techniques in linear and non-linear laser optics in muscle research

This review proposes a brief summary of two applications of lasers to muscle research. The first application (laser tweezers), is now a well-established technique in the field, adopted by several laboratories in the world and producing a constant stream of original data, fundamental for our improved understanding of muscle contraction at the level of detail that only single molecule measurements can provide. As an example of the power of this technique, here we focus on some recent results, revealing the performance of the working stroke in at least two distinct steps also in skeletal muscle myosin. A second laser-based technique described here is second-harmonic generation; the application of this technique to muscle research is very recent. We describe the main results obtained thus far in this area and the potentially remarkable impact that this technology may have in muscle research.

An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

•  Force and shortening in muscle are due to the ATP‐powered motor protein myosin II, polymerized in two bipolar arrays of motors that pull the two overlapping actin filaments toward the centre of the sarcomere. •  The parameters of the myosin motor in situ have been best characterized for the skeletal muscle of the frog, from which single intact cells can be isolated allowing fast sarcomere level mechanics to be applied. •  Up to now no reliable methods have been developed for the study of frog myosin with single molecule techniques. •  In this work a new protocol for extraction and conservation of frog muscle myosin allows us to estimate the sliding velocity of actin on myosin (VF) and its modulation by pH, myosin density, temperature and substrate concentration. •  By integrating in vitro and in situ parameters of frog muscle myosin we can relate kinetic and mechanical steps of the acto‐myosin ATPase.

3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition.

Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories.

High-precision measurements of light-induced torque on absorbing microspheres

Laser beams have been demonstrated to be capable of exerting torque as well as forces on microparticles. Using a custom magneto-optic manipulator, we directly measured the torque exerted by laser light on absorbing microspheres as a result of the transfer of spin angular momentum. A general method for measuring torque has been developed, and the experimental apparatus has shown a sensitivity of approximately 1 pN/nm.