A simple optical tweezers design is proposed to manipulate particles in the axial direction and estimate particle position with nanometer sensitivity. Balb3T3 cell is probed using two different-sized particles, and the localized cell stiffness is evaluated using Hertz model. A series of experiments are performed to obtain the necessary parameters for the cell stiffness computation: particle displacement, trapping stiffness, force exertion, and cell deformation. The computed cell stiffness measurements are 17 and 40 Pa using 4 μm- and 2 μm-sized particles, respectively. Results suggest that the proposed optical tweezers scheme can measure the stiffness of a particular cell locale using Hertz model, offering insights about how cells respond to outside mechanical stimulus.
Mechanical property of a cell is investigated from reaction force generated on a particle fixed on a cell by moving with
optical tweezers. This system is called as cell palpation system. By using of this system we can measure mechanical
property at a desired location on a cell surface just by locating the probe particle with optical tweezers. We have
investigated focal adhesion formation through mechanical property measurement after initiation of touching by the probe
particle.
Optical tweezers is a technique that can trap and manipulate small objects using a highly focused laser beam. Because optical tweezers can also be used to measure small forces, it has been extensively used for the measurement of the mechanical forces of cells. Previous research works typically study particle manipulation and cell force measurement in the lateral direction, hence excluding valuable insights about the axial mechanical properties of cells. Other works that investigate axial cell force measurements utilize spatial light modulators and other devices that are expensive and complicate the setup. Thus, in our study, we designed a simple scheme that can axially manipulate particles by adjusting the position of one lens, called L1-lens, in our setup. Image processing techniques were utilized to determine the changes in the axial particle translation, providing nanometer sensitivity. We investigated the capability of our system using two different-sized particles and results show that for a given L1-lens default position and movement, a 2-micron particle and a 4.26-micron particle were moved axially for 7.68 µm and 4.83 µm, respectively. Axial trapping stiffness was also measured for the stated bead sizes in different magnification. Using the computed trapping sti_ness, we will investigate the axial reactive forces of cells.
Optical tweezers is a technique to trap and to manipulate micron sized objects under a microscope by radiation pressure
force exerted by a laser beam. Optical tweezers has been utilized for single-molecular measurements of force exerted by
molecular interactions and for cell palpation. To extend applications of optical tweezers we have developed a novel
optical tweezers system combined with a pulse laser. We utilize a pulse laser (Q-switched Nd: YAG laser, wavelength of
1064 nm) to assist manipulations by conventional optical tweezers with a continuous wave (CW) laser. The pulse laser
beam is introduced into the same optics for conventional optical tweezers. In principle, instantaneous radiation force is
proportional to instantaneous power of laser beam. As a result, pulse laser beam generates strong instantaneous force on
an object to be manipulated. If the radiation force becomes strong enough to get over an obstacle structure and/or to be
released from adhesion, the object will be free from these difficulties. We investigate the effect of pulse laser assistance
with changing pulse duration of the laser. We report optimum pulse duration of 100 ns to 200 ns deduced from motion
analysis of a particle in a beam spot. Our goal is to realize in-vivo manipulation and operation of a cell. For this purpose
we need to reduce light energy of pulse laser beam and to avoid laser induced breakdown caused by strong light field. So
we have developed a pulse laser with 160-ns pulse duration and have confirmed that availability on manipulation of
living cells.
Optical tweezers is a technique to trap and to manipulate micron sized objects under a microscope by radiation
pressure force exerted by a laser beam. Optical tweezers has been utilized for single-molecular measurements of force
exerted by molecular interactions and for cell palpation. To extend applications of optical tweezers we have developed a
novel optical tweezers system combined with a pulse laser. We utilize a pulsed laser (Q-switched Nd: YAG laser,
wavelength of 1064 nm) to assist manipulations by conventional optical tweezers achieved by a continuous wave (CW)
laser. The pulsed laser beam is introduced into the same optics for conventional optical tweezers. In principle,
instantaneous radiation force is proportional to instantaneous power of laser beam. As a result, pulsed laser beam
generates strong instantaneous force on an object to be manipulated. If the radiation force becomes strong enough to get
over an obstacle structure and/or to be released from adhesion, the object will be free from these difficulties. We have
named this technique as Pulse Laser beam Assisted optical Tweezers (PLAT). We have successfully demonstrated to
manipulate objects surface on a living cell for "in vivo manipulation."
During cell adhesion and migration, a cell forms focal adhesion, which connects cytoskeleton with extracellular matrix
(ECM) through integrin, and applies cytoskeletal force to the ECM through focal adhesion. In the initial phase of cell
adhesion (initial adhesion), protein related to cell adhesion recruits other components to reinforce adhesion force and
grows to focal complex. To study the mechanism of cell adhesion, we focused on relationship between variation of
mechanical property of cell adhesion and related protein for cell adhesion. Especially, we approached by understanding
mechanical property of initial adhesion. To measure this property, we developed a "cell palpation system", which utilizes
optical tweezers to apply mechanical stimulus to a cell and to investigate reactive force. As below, this system gives
information on the mechanical property (membrane support tension) and a time course of the property by using an
optically manipulated microbead through an analysis based on mechanical model of this microbead. To create cell
adhesion between the microbead and cell surface, the microbead was coated with collagen and we investigated the
mechanical property of initial adhesion. And we analyzed the processes in relation to maturation of initial adhesion at a
single molecular level.
Fluorescence correlation spectroscopy (FCS) is widely used for investigation of concentration, diffusion coefficients and dynamics of single molecules. To introduce spatial resolution in FCS measurement, we develop a novel FCS system, which uses an electron-multiplying charge-coupled device (EM-CCD) to get FCS data at each pixel. We tested 3 samples, which have different concentrations of fluorescent beads, and successfully investigated the difference of correlation coefficients of FCS signal.
In addition, we introduce a new illumination method for EM-CCD based FCS measurement, to limit depth of a observation volume. Although a evanescent field has a nature of limited penetration depth, the penetration depth which is 50 to 200nm in typical, is short in comparison with the resolution in the lateral direction.
As a result FCS measurement becomes too sensitive in the depth direction, but worse in lateral direction.
So we introduce a novel illumination method, in which a laser beam is incident with an angle slightly smaller than the critical angle to illuminate fluorescent molecule (critical-angle illumination). The depth of observation volume can be controlled with the angle of incidence. We expect this method to be applied to a measurement of local diffusion coefficient of molecules in living cells.
We have developed an optical cross sectional imaging method for scattering media with the aid of a pulse ultrasound wave. A converging pulse ultrasound wave and a He-Ne laser beam, which are set on the same axis and same direction, are irradiated simultaneously to a sample. As the pulse ultrasound wave travels into the sample, the instantaneous position of the wave changes the optical properties of a localized region of the sample and modulates the scattered intensity of the incident laser light. We detected the modulation of the scattered light that is transmitted through the sample to observe the absorptive features inside a thick sample. Depth resolving capability is achieved from the time-dependent measurement of the scattered light intensity. This system achieves 3D resolution determined by the focal spot of the focused ultrasound wave. The resolution of our system is measured at 1.5 mm in the axial direction and 0.3 mm in the transverse direction. We verify the system by observing an absorptive object, which is a silk thread stained with Victoria blue dye, that is embedded in silicone rubber. We also obtained a cross-sectional image of an absorbing object that is surrounded by a 10-mm thick highly scattering medium.
We have developed an optical measurement method to investigate the inside of scattering media by using focused ultrasound to specify the measuring point. This method enables us to observe the internal structure of human tissue non-invasively for biomedical applications. This method may give us various kinds of material information such as spectroscopic characteristics. To get the information from only particular region in a tissue sample with light, we use a focused ultrasound. At the focal region of the ultrasound, refractive index of the tissue changes with strong acoustic field. So a part of incident light reflects at the focal region of the ultrasound. The reflected light has information of the sample. According to the scanning of the sample, we can get an image of the sample. In our experiments, we have observed samples that consist of an agarose block which contains a piece of agarose cube stained with dye as absorbing material. The intensity of reflected light from the focal region of the ultrasound decreased when the measuring point was in the stained agarose section, because incident light was absorbed with the dye. Furthermore, we have observed a sample with a weak scattering object made of agarose in strong scattering agarose that contains PMMA particles.
KEYWORDS: Particles, Near field scanning optical microscopy, Gold, Glasses, Luminescence, Light scattering, Scattering, Near field optics, Microscopes, Molecules
We developed a near-field scanning optical microscope (NSOM) which uses an optically trapped metallic Rayleigh particle as a near-field probe. This microscope is suitable for observations of biological specimen under aqueous condition because the probe particle is held by non-contact force from radiation pressure. The spring constant of trapping force is so weak (several mN/m order typically) that induced damage on a sample rarely occurs by this force. We used a gold colloidal particle which had a diameter of 40 nm as a probe. The scattering efficiency of the particle (at 488-nm wavelength, nAu equals 0.916, KAu equals 1.840) is 348 times as great as that of a glass particle (nglass equals 1.5) in the same size under water. We investigated a surface of a cover glass with the gold colloidal particle and confirmed the ability to observe small irregularities of the surface in around 10 nm. We also observed aggregated gold colloidal particles (d equals 40 nm) on a cover glass and fluorescent beads on glass. The investigation of DNA stained with YOYO-1 iodide on glass under water conditions was also performed.
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