Mechanical manipulation of bone and cartilage cells with 'optical tweezers'.

The single beam optical gradient trap (optical tweezers) uses a single beam of laser light to non-invasively manipulate microscopic particles. Optical tweezers exerting a force of approximately 7 pN were applied to single bone and cartilage derived cells in culture and changes in intracellular calcium levels were observed using Fluo-3 labelling. Human derived osteoblasts responded to optical tweezers with an immediate increase in [Ca2+]i that was inhibited by the addition of a calcium channel blocker nifedipine. Force applied to different regions of cells resulted in a variable response. [Ca2+]i elevation in response to load was lower in rat femur derived osteoblasts, and not apparent in primary chondrocytes and the osteocytic cell line (MLO Y4).     

单细胞机械性能检测方法与应用研究进展

细胞机械性能与细胞的生理状态与功能存在密切联系。早期对于细胞机械性能的研究受制于技术条件,只能获得细胞群的弹性或剪切模量,使得少量异质细胞的机械表型被淹没。近年来,单细胞机械性能检测技术得到了蓬勃发展。原子力显微镜、微吸管技术、光镊与光学拉伸、磁扭转流变仪与磁镊等单细胞机械性能检测技术展现出非常高的检测精度,但检测通量相对较低。新型微流控高通量检测方法的出现使检测通量呈几何式增长,有望解决大样本快速检测的需求。本文首先综述原子力显微镜、微吸管、光镊与光学拉伸和磁扭转流变仪与磁镊等单细胞机械性能检测技术。在此基础上,重点介绍细胞过孔、剪切诱导细胞变形和拉伸诱导细胞变形3种新兴微流控高通量检测技术的工作原理及最新研究进展,探讨各类方法的优缺点。最后,本文展望单细胞机械性能检测技术的未来发展方向。     

Optical tweezers in biology and medicine

Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion.     

Studies of plasma membrane mechanics and plasma membrane-cytoskeleton interactions using optical tweezers and fluorescence imaging

We use optical tweezers in conjunction with an optical position-sensing system, which spectrally filters signals generated by a trapped fluorescent microsphere to study plasma membrane (PM) mechanics and its interactions with cytoskeleton. We dynamically measure the PM tethering force on human embryonic kidney cells that are a standard cultured cell line. Recorded tethering force vs. PM displacement profiles, revealed the tether formation process, initiated with linear deformation of the PM, followed by a nonlinear regime and terminated with the local separation of PM. Tethering force vs. displacement profiles were used to estimate tether formation force and stiffness parameter of the PM. Integration of the force-displacement profiles yielded the work of tether formation, including linear and nonlinear components. Our results demonstrate that spectral filtering of the optically trapped fluorescent microsphere image formed on the position-sensing system overcomes the artifacts introduced by the transillumination imaging and allows accurate measures of PM mechanics before and during the initial stages of tether formation.     

The nonlinear mechanical response of the red blood cell

We measure the dynamical mechanical properties of human red blood cells. A single cell response is measured with optical tweezers. We investigate both the stress relaxation following a fast deformation and the effect of varying the strain rate. We find a power-law decay of the stress as a function of time, down to a plateau stress, and a power-law increase of the cell's elasticity as a function of the strain rate. Interestingly, the exponents of these quantities violate the linear superposition principle, indicating a nonlinear response. We propose that this is due to the breaking of a fraction of the crosslinks during the deformation process. The soft glassy rheology model accounts for the relation between the exponents we observe experimentally. This picture is consistent with recent models of bond remodeling in the red blood cell's molecular structure. Our results imply that the blood cell's mechanical behavior depends critically on the deformation process.     

Real-time detection of changes in the electrophoretic mobility of a single cell induced by hyperosmotic stress

Living cells survive environmentally stressful conditions by initiating a stress response. We monitored changes in the electrophoretic mobility (EPM) of single, optically trapped yeast cells under hyperosmotic stress conditions using optical tweezers combined with a position detector. We studied the dynamics of the EPM stress response for cells at different phases of the cell cycle.     

Estimation of cell Young's modulus of adherent cells probed by optical and magnetic tweezers: influence of cell thickness and bead immersion

A precise characterization of cell elastic properties is crucial for understanding the mechanisms by which cells sense mechanical stimuli and how these factors alter cellular functions. Optical and magnetic tweezers are micromanipulation techniques which are widely used for quantifying the stiffness of adherent cells from their response to an external force applied on a bead partially embedded within the cell cortex. However, the relationships between imposed external force and resulting bead translation or rotation obtained from these experimental techniques only characterize the apparent cell stiffness. Indeed, the value of the estimated apparent cell stiffness integrates the effect of different geometrical parameters, the most important being the bead embedding angle 2gamma, bead radius R, and cell height h. In this paper, a three-dimensional finite element analysis was used to compute the cell mechanical response to applied force in tweezer experiments and to explicit the correcting functions which have to be used in order to infer the intrinsic cell Young's modulus from the apparent elasticity modulus. Our analysis, performed for an extensive set of values of gamma, h, and R, shows that the most relevant parameters for computing the correcting functions are the embedding half angle gamma and the ratio h(u)/2R, where h(u) is the under bead cell thickness. This paper provides original analytical expressions of these correcting functions as well as the critical values of the cell thickness below which corrections of the apparent modulus are necessary to get an accurate value of cell Young's modulus. Moreover, considering these results and taking benefit of previous results obtained on the estimation of cell Young's modulus of adherent cells probed by magnetic twisting cytometry (MTC) (Ohayon, J., and Tracqui, P., 2005, Ann. Biomed. Eng., 33, pp. 131-141), we were able to clarify and to solve the still unexplained discrepancies reported between estimations of elasticity modulus performed on the same cell type and probed with MTC and optical tweezers (OT). More generally, this study may strengthen the applicability of optical and magnetic tweezers techniques by insuring a more precise estimation of the intrinsic cell Young's modulus (CYM).     

Deformation of Filamentous Escherichia coli Cells in a Microfluidic Device: A New Technique to Study Cell Mechanics

The mechanical properties of bacterial cells are determined by their stress-bearing elements. The size of typical bacterial cells, and the fact that different time and length scales govern their behavior, necessitate special experimental techniques in order to probe their mechanical properties under various spatiotemporal conditions. Here, we present such an experimental technique to study cell mechanics using hydrodynamic forces in a microfluidic device. We demonstrate the application of this technique by calculating the flexural rigidity of non-growing Escherichia coli cells. In addition, we compare the deformation of filamentous cells under growing and non-growing conditions during the deformation process. We show that, at low forces, the force needed to deform growing cells to the same extent as non-growing cells is approximately two times smaller. Following previous works, we interpret these results as the outcome of the difference between the elastic response of non-growing cells and the plastic-elastic response of growing cells. Finally, we observe some heterogeneity in the response of individual cells to the applied force. We suggest that this results from the individuality of different bacterial cells.     

Studying Single Red Blood Cells Under a Tunable External Force by Combining Passive Microrheology with Raman Spectroscopy

The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.     

Exploring mechanochemical processes in the cell with optical tweezers

Force and torque, stress and strain or work are examples of mechanical and elastic actions which are intimately linked to chemical reactions in the cell. Optical tweezers are a light-based method which allows the real-time manipulation of single molecules and cells to measure their interactions. We describe the technique, briefly reviewing the operating principles and the potential capabilities to the study of biological processes. Additional emphasis is given to the importance of fluctuations in biology and how single-molecule techniques allow access to them. We illustrate the applications by addressing experimental configurations and recent progresses in molecular and cell biology.     

Mechanical force characterization in manipulating live cells with optical tweezers

Laser trapping with optical tweezers is a noninvasive manipulation technique and has received increasing attentions in biological applications. Understanding forces exerted on live cells is essential to cell biomechanical characterizations. Traditional numerical or experimental force measurement assumes live cells as ideal objects, ignoring their complicated inner structures and rough membranes. In this paper, we propose a new experimental method to calibrate the trapping and drag forces acted on live cells. Binding a micro polystyrene sphere to a live cell and moving the mixture with optical tweezers, we can obtain the drag force on the cell by subtracting the drag force on the sphere from the total drag force on the mixture, under the condition of extremely low Reynolds number. The trapping force on the cell is then obtained from the drag force when the cell is in force equilibrium state. Experiments on numerous live cells demonstrate the effectiveness of the proposed force calibration approach.     

A new method to study shape recovery of red blood cells using multiple optical trapping.

In this new method for studying the shape recovery of deformed red blood cells, three optical traps ("optical tweezers") induce a parachute-shaped red cell deformation, which is comparable to the deformation in small capillaries. The shape recovery is recorded, and a relaxation time is obtained for each individual red blood cell. The sensitivity of this technique for the detection of differences in relaxation times is demonstrated on subpopulations of density-separated red blood cells: "young" cells have shorter (162 ms) and "old" cells have longer (353 ms) relaxation times compared with the total population (271 ms). The relaxation time is remarkably shorter (114 ms) when the plasma surrounding the cells is replaced by a phosphate-buffered saline solution. The main advantages of this technique are the relatively short measuring and preparation time and the physiological type of deformation and shape recovery in which all relevant cell properties play a role. Therefore, especially when automated further, the technique may be a powerful tool for the study of (sub)populations of pathological red blood cells.     

Using laser tweezers to measure twitching motility in Neisseria

Dynamic properties of type IV pili are essential for their function in bacterial infection, twitching motility and gene transfer. Laser tweezers are versatile tools to study the molecular mechanism underlying pilus dynamics at the single molecule level. Recently, these optical tweezers have been used to monitor pilus elongation and retraction in vivo at a resolution of several nanometers. The force generated by type IV pili exceeds 100 pN making pili the strongest linear motors characterized to date. The study of pilus dynamics at the single molecule level sheds light on kinetics, force generation, switching and mechanics of the Neisseria gonorrhoeae pilus motor.     

Compliance of bacterial polyhooks measured with optical tweezers.

In earlier work, a single-beam gradient force optical trap ("optical tweezers") was used to measure the torsional compliance of flagella in wild-type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180 degrees, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind-up of the flagellar hook, and the rigid phase to wind-up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small-angle compliance of these cells, as determined from the elastic rebound of the cell body after wind-up and release, was found to be the same as for wild-type cells. This confirms that the small-angle compliance of wild-type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.     

Tracking of Single Quantum Dot Labeled EcoRV Sliding along DNA Manipulated by Double Optical Tweezers

Fluorescence microscopy provides a powerful method to directly observe single enzymes moving along a DNA held in an extended conformation. In this work, we present results from single EcoRV enzymes labeled with quantum dots which interact with DNA manipulated by double optical tweezers. The application of quantum dots facilitated accurate enzyme tracking without photobleaching whereas the tweezers allowed us to precisely control the DNA extension. The labeling did not affect the biochemical activity of EcoRV checked by directly observing DNA digestion on the single molecule level. We used this system to demonstrate that during sliding, the enzyme stays in close contact with the DNA. Additionally, slight overstretching of the DNA resulted in a significant decrease of the 1D diffusion constant, which suggests that the deformation changes the energy landscape of the sliding interaction. Together with the simplicity of the setup, these results demonstrate that the combination of optical tweezers with fluorescence tracking is a powerful tool for the study of enzyme translocation along DNA.     

Spectrin-level modeling of the cytoskeleton and optical tweezers stretching of the erythrocyte

We present a three-dimensional computational study of whole-cell equilibrium shape and deformation of human red blood cell (RBC) using spectrin-level energetics. Random network models consisting of degree-2, 3, ..., 9 junction complexes and spectrin links are used to populate spherical and biconcave surfaces and intermediate shapes, and coarse-grained molecular dynamics simulations are then performed with spectrin connectivities fixed. A sphere is first filled with cytosol and gradually deflated while preserving its total surface area, until cytosol volume consistent with the real RBC is reached. The equilibrium shape is determined through energy minimization by assuming that the spectrin tetramer links satisfy the worm-like chain free-energy model. Subsequently, direct stretching by optical tweezers of the initial equilibrium shape is simulated to extract the variation of axial and transverse diameters with the stretch force. At persistence length p = 7.5 nm for the spectrin tetramer molecule and corresponding in-plane shear modulus mu(0) approximately 8.3 microN/m, our models show reasonable agreement with recent experimental measurements on the large deformation of RBC with optical tweezers. We find that the choice of the reference state used for the in-plane elastic energy is critical for determining the equilibrium shape. If a position-independent material reference state such as a full sphere is used in defining the in-plane energy, then the bending modulus kappa needs to be at least a decade larger than the widely accepted value of 2 x 10(-19) J to stabilize the biconcave shape against the cup shape. We demonstrate through detailed computations that this paradox can be avoided by invoking the physical hypothesis that the spectrin network undergoes constant remodeling to always relax the in-plane shear elastic energy to zero at any macroscopic shape, at some slow characteristic timescale. We have devised and implemented a liquefied network structure evolution algorithm that relaxes shear stress everywhere in the network and generates cytoskeleton structures that mimic experimental observations.     

New techniques for isolation of single prokaryotic cells

Since the 1960s, several new attempts have been made to improve the management of single prokaryotic cells using micromanipulator techniques. In order to facilitate the isolation of pure cultures we have recently developed an improved micromanipulation method for routine work. With the aid of this method single prokaryotic cells can be picked out of a mixed community under direct visual control. The isolated aerobic or anaerobic cells can be grown in pure culture or can be subjected to single cell PCR. Other powerful and completely new approaches are the applications of laser micromanipulation systems, such as optical tweezers or laser microdissection techniques. Of the latter two methods only optical tweezers have been successfully applied to cloning prokaryotic cells.     

应用光学微操作技术分选单条水稻染色体

报道了一种基于光镊技术的实用的单条染色体分选技术。具体介绍了用光镊与光刀结合、并辅以微吸管分选水稻单条染色体的过程。通过该方法得到的水稻单条染色体样品经过分子克隆,制备出了染色体特异的DNA片段并用于水稻基因组测序工作。还将光学微操作技术与现有的几种分选单条染色体的方法(如玻璃微针挑取、激光弹射以及流式细胞仪等)进行了比较。与这些方法相比,光学微操作方法具有液相环境中分离、操作简易、对染色体损伤小、选择性高、无污染等优点。     

Single cell analytics for nanobiology

Single cell analytics allows quantitative investigation of single biological cells from a structural, functional and proteomics point of view and opens possibilities to a novel unamplified cell analysis inherently insensitive to ensemble-averaging, cell-cycle or cell-population effects. We report on three different experimental methods and their application to cellular systems with single molecule sensitivity at the single cell level. Firstly, atomic force microscopy (AFM) can be used to elucidate the surface structure of living bacteria down to the nanometer scale where identification of irregular surface areas and 2D-arrays of regular protein s-layers is possible. Secondly, single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy allows quantitative identification of individual recognition events of membrane bound receptors. And thirdly, a novel, single cell analysis for protein fingerprinting in structured microfluidic device format will allow a future (label-free) on-chip electrophoretical protein separation of single cells without preamplification.     

Cell Visco-Elasticity Measured with AFM and Optical Trapping at Sub-Micrometer Deformations

The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30–600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components.