We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.

We use optical tweezers to perform stretching experiments on DNA molecules when interacting with the drugs daunomycin and ethidium bromide, which intercalate the DNA molecule. Our results show that the persistence length of the DNA-drug complexes increases strongly as the drug concentration increases up to some critical value. Above this critical value, the persistence length decreases abruptly and remains approximately constant for larger drug concentrations, at least in the concentration range used in our experiments. Measured intercalators critical concentrations for the persistence length transition coincide with the reported values for the helix-coil transition of DNA-drug complexes obtained from sedimentation experiments. The contour length of the molecules increases monotonically and saturates as the drug concentration increases. The neighbor exclusion model fits to our results for the total drug concentration as a function of the relative increase of the contour length.

Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional (2D) Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature (Laplacian of the local thickness) of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations (ruffles) on the surface of macrophages (cell of the innate immune system), and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.

Aiming at absolute force calibration of optical tweezers, following a critical review of proposed theoretical models, we present and test the results of MDSA (Mie-Debye-Spherical Aberration) theory, an extension of a previous (MD) model, taking account of spherical aberration at the glass/water interface. This first-principles theory is formulated entirely in terms of experimentally accessible parameters (none adjustable). Careful experimental tests of the MDSA theory, undertaken at two laboratories, with very different setups, are described. A detailed description is given of the procedures employed to measure laser beam waist, local beam power at the transparent microspheres trapped by the tweezers, microsphere radius and the trap transverse stiffness, as a function of radius and height in the (inverted microscope) sample chamber. We find generally very good agreement with MDSA theory predictions, for a wide size range, from the Rayleigh domain to large radii, including the values most often employed in practice, and at different chamber heights, both with objective overfilling and underfilling. The results asymptotically approach geometrical optics in the mean over size intervals, as they should, and this already happens for size parameters not much larger than unity. MDSA predictions for the trapping threshold, position of stiffness peak, stiffness variation with height, multiple equilibrium points and 'hopping' effects among them are verified. Remaining discrepancies are ascribed to focus degradation, possibly arising from objective aberrations in the infrared, not yet included in MDSA theory.