Ming-Tzo Wei

A comparative study of living cell micromechanical properties by oscillatory optical tweezers

Micromechanical properties of biological cells are crucial for cells functions. Despite extensive study by a variety of approaches, an understanding of the subject remains elusive. We conducted a comparative study of the micromechanical properties of cultured alveolar epithelial cells with an oscillatory optical tweezer-based cytorheometer. In this study, the frequency-dependent viscoelasticity of these cells was measured by optical trapping and forced oscillation of either a submicron endogenous intracellular organelle (intra-cellular) or a 1.5microm silica bead attached to the cytoskeleton through trans-membrane integrin receptors (extra-cellular). Both the storage modulus and the magnitude of the complex shear modulus followed weak power-law dependence with frequency. These data are comparable to data obtained by other measurement techniques. The exponents of power-law dependence of the data from the intra- and extra- cellular measurements are similar; however, the differences in the magnitudes of the moduli from the two measurements are statistically significant.

Dynamic deformation of red blood cell in Dual-trap Optical Tweezers

Three-dimensional dynamic deformation of a red blood cell in a dual-trap optical tweezers is computed with the elastic membrane theory and is compared with the experimental results. When a soft particle is trapped by a laser beam, the particle is deformed depending on the radiation stress distribution whereas the stress distribution on the particle in turn depends on the deformation of its morphological shape. We compute the stress re-distribution on the deformed cell and its subsequent deformations recursively until a final equilibrium state solution is achieved. The experiment is done with the red blood cells in suspension swollen to spherical shape. The cell membrane elasticity coefficient is obtained by fitting the theoretical prediction with the experimental data. This approach allows us to evaluate up to 20% deformation of cells shape.

Three-dimensional optical force field on a Chinese hamster ovary cell in a fiber-optical dual-beam trap

We used a fiber-optical dual-beam trap (single-mode fiber, lambda = 532nm, trapping power ~ 22mW, the distance between the two fiber end-faces = 125mum) to capture a Chinese hamster ovary (CHO) cell with a diameter of approximately 15mum and tracked its three-dimensional Brownian motion via a pair of orthogonal quadrant photodiodes. By analyzing the Brownian motion of the trapped CHO cell, we determined the force constants of the optical force field on the CHO cell to be k(x)=6.75 pN/mum, k(y)=5.53 pN/mum, k(z)=1.96 pN/mum, and k(x)=2.91 pN/mum, k(y)=2.7 pN/mum, k(z)=0.79 pN/mum, respectively, before and after the CHO cell was treated with latrunculin, a toxic drug known to disrupt the cytoskeleton of the cell.

Three-dimensional tracking of Brownian motion of a particle trapped in optical tweezers with a pair of orthogonal tracking beams and the determination of the associated optical force constants

We report the first experimental results on quantitative mapping of three-dimensional optical force field on a silica micro-particle and on a Chinese hamster ovary cell trapped in optical tweezers by using a pair of orthogonal laser beams in conjunction with two quadrant photo-diodes to track the particles (or the cells) trajectory, analyze its Brownian motion, and calculate the optical force constants in a three-dimensional parabolic potential model. For optical tweezers with a 60x objective lens (NA = 0.85), a trapping beam wavelength lambda = 532nm, and a trapping optical power of 75mW, the optical force constants along the axial and the transverse directions (of the trapping beam) were measured to be approximately 1.1x10-8N/m and 1.3x10-7N/m, respectively, for a silica particle (diameter = 2.58microm), and 3.1x10-8 N/m and 2.3x10-7 N/m, respectively, for a Chinese hamster ovary cell (diameter ~ 10 microm to 15microm). The set of force constants (Kx, Ky, and Kz ) completely defines the optical force field E(x, y, z) = [Kx x2 + Ky y2 + Kz z2]/2 (in the parabolic potential approximation) on the trapped particle. Practical advantages and limitations of using a pair of orthogonal tracking beams are discussed.

Optical forced oscillation for the study of lectin-glycoprotein interaction at the cellular membrane of a Chinese hamster ovary cell

We report the application of a set of twin optical tweezers to trap and oscillate a ConA (lectin)- coated polystyrene particle and to measure its interaction with glycoprotein receptors at the cellular plasma membrane of a Chinese hamster ovary (CHO) cell. The particle was trapped between two quadratic potential wells defined by a set of twin optical tweezers and was forced to oscillate by chopping on and off one of the trapping beams. We tracked the oscillatory motion of the particle via a quadrant photodiode and measured with a lock-in amplifier the amplitude of the oscillation as a function of frequency at the fundamental component of the driving frequency over a frequency range from 10Hz to 600Hz. By analyzing the amplitude as a function of frequency for a free particle suspended in buffer solution without the presence of the CHO cell and compared with the corresponding data when the particle was interacting with the CHO cell, we deduced the transverse force constant associated with the optical trap and that associated with the interaction by treating both the optical trap and the interaction as linear springs. The force constants were determined to be approximately 2.15pN/mum for the trap and 2.53pN/mum for the lectin-glycoprotein interaction. When the CHO cell was treated with lantrunculin A, a drug that is known to destroy the cytoskeleton of the cell, the oscillation amplitude increased with time, indicating the softening of the cellular membrane, until a steady state with a smaller force constant was reached. The steady state value of the force constant depended on the drug concentration.

The interaction of lipopolysaccharide with membrane receptors on macrophages pre-treated with extract of Reishi polysaccharides measured by optical tweezers

Lipopolysaccharide (LPS), one of the cell wall components of Gram-negative bacteria, is recognized by and interacted with receptors on macrophages. In this paper, we report the trapping of LPS-coated polystyrene particles via optical tweezers and measured its interaction with murine macrophages (J774A.1 cells) for cells pre-treated with extract of Reishi polysaccharides (EORP) vs. those without EORP treatment. Our experimental results indicate that the cellular affinity for LPS increases when the macrophage is pretreated with EORP. We demonstrate for the first time by conventional biological methods and by tracking the dynamics of optically-trapped LPS-coated particles interacting with J774A.1 cells, that EORP not only enhances J774A.1 cells surface expression of TLR4 and CD14, two receptors on macrophages, as well as LPS binding and phagocytosis internalization, but also reduces the adhesion time constant and increases the force constant of the binding interaction. The application of optical tweezers allows us to study the effect on a single cell quantitatively in real-time with a spatial resolution ~ 1 mum within a single cell.

Probing the dynamic differential stiffness of dsDNA interacting with RecA in the enthalpic regime

RecA plays a central role in homologous recombination of DNA. When RecA combines with dsDNA to form RecA-dsDNA nucleofilament, it unwinds dsDNA and changes its structure. The unwinding length extension of a DNA segment interacting with RecA has been studied by various techniques, but the dynamic differential stiffness of dsDNA conjugating with RecA has not been well characterized. We applied oscillatory optical tweezers to measure the differential stiffness of dsDNA molecules, interacting with RecA, as a function of time at a constant stretching force of 33.6pN. The values of the differential stiffness of DNA (for stretching force in the range of 20.0pN to 33.6pN) measured by oscillatory optical tweezers, both before and after its interaction with RecA, are consistent with those measured by stationary optical tweezers. In the dynamic measurement, we have shown that the association (or binding) rate increases with higher concentration of RecA; besides, we have also monitored in real-time the dissociation of RecA from the stretched RecA-dsDNA filament as ATPgammaS was washed off from the sample chamber. Finally, we verified that RecA (I26C), a form of RecA mutant, does not affect the differential stiffness of the stretched DNA sample. It implies that mutant RecA (I26C) does not bind to the DNA, which is consistent with the result obtained by conventional biochemical approach.

Mapping of three-dimensional optical force field on a micro-particle trapped in a fiber-optical dual-beam trap

We report the first direct experimental mapping of three-dimensional optical force field on a silica micro-particle trapped in a counter-propagating dual-beam trap. We tracked the three-dimensional Brownian motion of the trapped particle (by optical position sensing) and analyzed the particle position distribution to obtain the force constant of the optical force field on the particle along each direction. The trapping beams scattered by the trapped particle along two directions (mutually orthogonal to each other and also to the trapping beams) were projected on a pair of quadrant photo-detectors (QPDs) to facilitate high-speed (20 KHz) three-dimensional position tracking. Position tracking over two mutually orthogonal planes intrinsically provides one set of redundant data for a self-consistency check. At optical wavelength λ = 532nm, the force constants of the three-dimensional optical force field on a silica micro-particle (diameter = 2.58μm) were determined to be kx = 1.61×10-1 pN/μm, ky = 1.49×10-1 pN/μm, and kz = 4.43×10-2 pN/μm when the total trapping power was about 21mW and the distance between the two fiber end-faces was 125μm. The set of force constants (kx, ky, and kz) completely defines the optical force field E(x, y, z) = [kxx2 + kyy2 + kzz2]/2 (in the parabolic potential approximation) on the trapped particle.

Myosin Cortical Assembly and Spindle Oscillations During Hela Cell Division

The cortical actomyosin layer below the membrane of dividing cells and the microtubules of the mitotic spindle are coupled through internal feedback mechanisms. Identifying the factors that control molecular motor distribution is essential for understanding cell division. The equatorial contractile ring forms by myosin assembly in the middle and disassembly in the flanking regions. To better understand the mechanical and biochemical mechanisms of contractile ring assembly, we imaged HeLa cells expressing MRLC-GFP by confocal microscopy at high spatial and temporal resolution. MRLC-GFP also marks spindle poles; this allowed us to study the correlation between mitotic spindle movements and cortical myosin distribution. We confirmed that the onset of myosin assembly in the contractile ring occurs in anaphase. We observed that the mitotic spindle exhibited oscillations upon entry into anaphase, similar to oscillations observed during the asymmetric division of C. elegans embryos by other groups. We used particle tracking software to follow spindle position and JFilament to track cortical myosin intensity distribution during these oscillations. We further exerted controlled and localized forces while following the cellular response using fluorescence imaging. Anti-integrin coated micro-beads were attached to HeLa cells expressing MRLC-GFP. We applied external forces to a bead by an optical tweezers and imaged the dynamics of myosin and mitotic spindle during cell division. Experimental results showed that the spindle appeared to move to an asymmetric post perturbation position. This observation is consistent with the expected coupling between force-dependent cortical flow and spindle position and will be useful in more detailed studies of the response of cytoskeleton to external or internal forces.

The interaction of lipopolysaccharide-coated polystyrene particle with membrane receptor proteins on macrophage measured by optical tweezers

Lipopolysaccharide (LPS) is one of the cell wall components of Gram-positive bacteria recognized by and interacted with receptor proteins such as CD14 on macrophage cells. Such a process plays an important role in our innate immune system. In this paper, we report the application of optical tweezers (λ = 1064nm Gaussian beam focused by a water-immersed objective lens with N.A. = 1.0) to the study of the dynamics of the binding of a LPS-coated polystyrene particle (diameter = 1.5μm) onto the plasma membrane of a macrophage cell. We demonstrated that the binding rate increased significantly when the macrophage cell was pre-treated with the extract of Reishi polysaccharides (EORP) which has been shown to enhance the cell surface expression of CD14 (receptor of LPS) on macrophage cells.