Taisuke Ota

Methods for the characterization of deformable membrane mirrors.

We demonstrate two methods for the characterization of deformable membrane mirrors and the training of adaptive optics systems that employ these mirrors. Neither method employs a wave-front sensor. In one case, aberrations produced by a wave-front generator are corrected by the deformable mirror by use of a rapidly converging iterative algorithm based on orthogonal deformation modes of the mirror. In the other case, a simple interferometer is used with fringe analysis and phase-unwrapping algorithms. We discuss how the choice of singular values can be used to control the pseudoinversion of the control matrix.

Dynamic axial-position control of a laser-trapped particle by wave-front modification

The axial position of a laser-trapped particle has been controlled by modification of the wave front by means of a membrane deformable mirror. The mirror gives wave-front modulation in terms of Zernike polynomials. By modulation of the Zernike defocus term we can modulate the particle position under conditions of laser trapping. A polystyrene particle of 1-microm diameter was moved along the optical axis direction for a distance of 2370 nm in minimum steps of 55.4 nm. We also demonstrated particle oscillation along the optical axis by changing the focal position in a sinusoidal manner. From the frequency dependency of the amplitude of particle oscillation we determined the spring constant as 91.7 nN/m.

Rupture force measurement of biotin-streptavidin bonds using optical trapping

Optical trapping has been applied to investigate the bond rupture force between biotin attached to a microparticle and streptavidin attached to a substrate. The force for rupturing the bonds was loaded vertically by pulling the particle from the substrate and detected by measuring the displacement of the particle from the equilibrium position in the optical trap. The displacement was measured by detecting light the light scattered from the particle in an evanescent field, and the field was generated by total internal reflection of a beam output from a high-NA objective lens. From the histogram yielded from the rupture force measurements, the most frequent rupture force was determined to be between 3.6 and 5.4 pN with a loading rate of 7.7pN∕s.

Enhancement of laser trapping force by spherical aberration correction using a deformable mirror

We have developed a method to enhance axial trapping force in optical tweezers by aberration correction of a laser beam with a membrane deformable mirror. The axial trapping force is strongly dependent on the quality of the laser beam spot, which is deteriorated by aberration due to the refractive index mismatch between a cover glass and water. The aberration correction, therefore, is crucial for stable trapping of a particle and for weak-force measurement with laser trapping. We have evaluated spring constants of the trapping force in the axial direction with and without aberration correction. The enhancement factor of the spring constants by the aberration correction has been achieved as 1.35 for the case of 5-mm sample thickness and as 1.83 for the case of 10-mm sample thickness. The numerical simulation is coincident with the experimental results. [DOI: 10.1143/JJAP.42.L701]

In situ fluorescence imaging of organs through compact scanning head for confocal laser microscopy.

We develop a compact scanning head for use in laser confocal fluorescence microscopy for in situ fluorescence imaging of organs. The head, cylindrical in shape, has 3.5 mm diameter and 30 mm length, and is thus small enough to operate in a living rat heart. The lateral and axial resolutions, defined as full widths at half maximum (FWHM) of a point spread function (PSF), measures 1.0 and 5.0 microm, respectively, for 488-nm excitation and 1.0 and 5.4 microm, respectively, for 543-nm excitation. The chromatic aberration between 488- and 543-nm laser beams is well suppressed. We perform Ca2+ imaging in cardiomyocytes through the right ventricular chamber of a perfused rat heart in line-scan mode with 2.9-ms time resolution. We also carried out two-color imaging of a fixed mouse heart and liver with subcellular resolution. The compact head of the microscope equipped with a line-scan imaging mode and two-color imaging mode is useful for in situ imaging in living organs with subcellular resolution and can advantageously be applied to in vivo research.

Imaging of anticancer agent distribution by a slit-scanning Raman microscope

In recent years, various types of molecular imaging technologies have been developed, but many of them require probes and may have some influence on the distribution of the target molecules. In contrast, Raman microscopic analysis is effective for molecular identification of materials, and molecular imaging methods employing Raman scattering light can be applied to living organisms without use of any exogenous probes. Unfortunately, Raman microscopic imaging is rarely used in the biomedical field due to the weakness of Raman signals. When the conventional Raman microscopes are used, the acquisition of an image of a cell usually takes several hours. Recently, a slit-scanning confocal Raman microscope has been developed. It can acquire images of living cells and tissues with faster scanning speed. In this study, we used the slit-scanning confocal Raman microscope (RAMAN-11) to image the distribution of a drug in living cells. We could acquire images of the distribution of an anticancer reagent in living cells within several minutes. Since the wavelength of Raman scattering light is determined by the frequency of molecular vibration, the in situ mapping of the intracellular drugs without use of a probe is possible, suggesting that laser Raman imaging is a useful method for a variety of pharmacokinetic studies.

Surface-force measurement with a laser-trapped microprobe in solution

Surface force between a micron-sized polystyrene and dielectric substrate in solution has been analyzed by measuring the displacement of the laser-trapped probe. The amount of displacement is measured by detecting the intensity of light scattering at the probe illuminated by evanescent field. The sensitivity of 27 fN and the range of 5.2 pN have been achieved in the experiment with various concentrations of electrolyte trisaminomethane. The distribution of the surface force of 3-aminopropyltriethoxysilane coated on the glass substrate was also detected.

Measurement of Young's modulus of primary cilia by using optical tweezers

Most renal tubular epithelial cells possess primary cilia that protrude into the tubular lumen, where they are exposed to urine flow. The cilia of the epithelia curve with the urine flow within the renal tubules. The primary cilium of renal epithelium has been assumed to be a mechano-sensor of the urine flow. However, the mechanical characteristics of the cilium of an each cell are not well assessed. In this study, we measured Youngs modulus of each primary cilium by using optical tweezers. First, we developed a system to manipulate each primary cilium of living tubular epithelial cells in situ by using optical tweezers. We adopted flexible-substratum technique to visualize individual micron-sized primary cilia of the Madin-Darby canine kidney (MDCK) cell lines. Through the use of the flexible-substratum technique, the primary cilia of MDCK cells could be clearly imaged from the side. We showed that we could apply minute force to polystyrene microspheres stuck on the tip of the cilia by using optical tweezers. The Youngs modulus of each primary cilium was successfully examined by measuring the displacement of the microsphere. We concluded that this methodology enables us to manipulate each primary cilium minutely and to estimate the elasticity of an individual primary cilium.

Manipulation of primary cilia using optical tweezers

Nonmotile primary cilia are expressed in most renal epithelial cells. The primary cilia of renal epithelia bend with the urinary flow within the renal tubules. It has been postulated that the cilia are a mechano-sensor of urinary flow. However, the mechanical characteristics of an individual primary cilium are not fully understood. In the present study, we successfully manipulated micron-sized primary cilia of the Madin-Darby canine kidney (MDCK) cell lines derived from the collecting duct, by using optical tweezers. The primary cilia of MDCK cells, cultured on flexible plastic films, could be clearly visualized from the side by video microscopy. Optical tweezers utilize the radiation pressure from the concentrated laser beam to trap and manipulate a small microparticle, and forces on the objects in the trap can be evaluated. We showed that we could apply forces in the piconewton range to a polystyrene bead fixed on the tip of the primary cilium by using optical tweezers; by that means, the primary cilium could be bent and stretched. The elasticity of each primary cilium was analyzed by measuring the displacement of the bead

Chapter 22 Pico-newton force-measurement of biomolecular interaction using laser trapping

Publisher Summary This chapter discusses the pico-newton force-measurement of biomolecular interaction using laser trapping. A micro or nano-particle is trapped and is manipulated as a force-probe on the substrate. When the trapped particle approaches the substrate, the particle senses the force, known as “surface force,” from the substrate. The force is measured when the interaction bonds are ruptured while the particle retracts. Surface force works on a particle when a particle is located near a substrate within several hundreds nanometer (nm) from the surface. The trapped particle is displaced because of the surface forces within the focus spot. The required resolution of the displacement measurement for sensing the force is estimated as a few nm. For achieving the precise measurement of the displacement, evanescent light illumination is adopted to monitor the particle position from the substrate. The system of surface force measurement is applied to measure the force between biotin and streptavidin as a prototypical sample of ligand–receptor specific interaction.