Cell palpation with an optically trapped particle
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We have developed a cell palpation system to investigate cell stiffness from the reaction force generated on a particle that is fixed on a cell. In this method, a particle is used as a probe, and is manipulated towards a cell using optical tweezers. Using this method, we can obtain information of local stiffness of a cell. We investigate focal adhesion formation of a cell probed by a particle and we report different particle coating utilized for attaching certain protein in cell membrane. Also we discuss the effects of endocytosis.
KeywordsOptical tweezers Cell stiffness Micromanipulation
The cell’s cytoskeleton, which is consists of proteins, provides the mechanical stiffness of the cell . In general, cytoskeleton is related with cell maturation and disorder, and is easily affected by any defect in a cell. Cytological diagnosis is one of the clinical procedures that can identify certain cell conditions by studying its morphology affected by cytoskeletal changes, which may be caused by a disease. Thus, we believe that the cytoskeletal structure and also cell stiffness can become important parameters to identify different cell conditions. We developed a cell palpation system to investigate cell stiffness from reaction force generated on a particle fixed on the cell, by moving the particle using optical tweezers [2, 3]. Optical tweezers is a technique to trap and to manipulate a micron-sized object by radiation pressure force exerted by a focused laser beam [4, 5]. Optical tweezers has been utilized to measure weak force, such as molecular-molecular interactions [6–8], to investigate mechanism of force generation of kinesin on microtubule [9, 10], and study the mechanical property of DNA . We use this technique to apply force on a particle attached on a cell and to measure the reaction force on the particle. The advantage of this method is that we can investigate the local mechanical property of the cell within the vicinity of the attached particle.
For the measurement of cell stiffness in local, the magnetic beads methods have been proposed and performed [12, 13]. However, there are some difficulties associated with this method; the position where the magnetic bead is to be attached cannot be controlled, and the bead is ingested into the cell while the bead is being oscillated for stiffness measurement. Also atomic force microscope (AFM) method has been performed by several researchers , although positioning of AFM probe and probe-to-cell attachment are not easily conducted. There are some other methods for investigating the mechanical properties of cells, such as micro fluidic cell method [15, 16] and a microtool method [17, 18]. However, these methods are for cell level observation, and inappropriate for local mechanical property measurement.
In contrast optical tweezers method is suitable to investigate local mechanical property because this method enables us to place the particle onto a cell under optical microscope and specify measuring point in local. And we can measure the reaction force shortly after attachment to avoid endocytosis. The optical tweezers method has been performed also by other groups [19, 20]. Our cell palpation system has significance on quantitative measurement capability and sensitivity to apply small force and detect weak reaction force.
In this work we investigate the effects of cell adhesion formation and effects of different surface coatings of particles to cell palpation with optical tweezers. First we describe the principle of cell stiffness measurement with optical tweezers, and explain the actual measurement scheme in the cell palpation system. Then we report the results on the measurement of cell adhesion formation using the cell palpation system and discuss suitable timing of cell stiffness measurement.
2 Principle of cell palpation
3 Instrumentation of cell palpation system
We use a high numerical aperture water immersion objective (Nikon, NA = 1.20, ×60) for the optical tweezers. The image of the particle is magnified with relay lens (×4) and imaged on a 1/3 inch CCD camera. A pixel size of CCD is equivalent to 30 nm under the microscope. Due to the use of sub-pixel resolution capability of position estimation and reduction of unevenness of illumination, we achieved 4 nm of noise equivalent position fluctuation. Using the developed setup, we can measure the precise position of trapped particle.
4 Cell measurement
Although in Fig. 4, the decrease of oscillation amplitude in four cycles of movement is unclear, the oscillation amplitude will gradually decrease as it continuously oscillates the particle. That is because an adhesion site (focal adhesion, FA) maturates thus increasing the cell stiffness, and the number of collagen molecules the bind to integrin increases as particle rotate on cell surface.
By considering the attachment of particle on the cell surface, there are two possible measurement modes for cell palpation. One mode is to put and oscillate a particle on a cell surface and measure the particle movement continuously (mode 1). The other mode is to put the particle and wait a certain time, then move it back and forth once and measure the particle position (mode 2). These modes will give different types of cell property on mechanics. In mode 1, new bond formation and bond rapture always occur during oscillation, so we can investigate one bond formation and rapture as stepwise change in oscillation amplitude. From this data, we can estimate one bond contribution on total adhesion phenomena. Also, we induce mechanical stimuli on the cell through the oscillating movement of particle. From this, we can identify the cellular mechanical response as a result of the mechanical stimuli [22, 23]. In mode 2, we can investigate natural mechanical properties of cells because no reactive response is induced on the cell. We need to select suitable measurement mode in actual situation. In this work, we aim to investigate cell adhesion formation and effect of surface coating of particles to cell palpation. For this reason, we used mode 2 measurement in following experiments. Also once a particle attached on cell surface this particle hardly removed from the cell, because of bond formation between surfaces of cell and particle. Thus we usually do not reuse a particle after one measurement, but utilize a new particle floating around cells as a probe for next measurement every time.
5 Cell adhesion formation measurement
A cell forms complex adhesion structure through contact with other cells or substrates. The adhesion structure is called focal adhesion (FA). FA is important to stabilize a cell at a certain location and it plays an important role on cell migration and cancer metastasis. We employed the mode 2 scheme to investigate the time course of focal adhesion formation by changing the elapsed time t to a single back-and-forth movement after putting a particle on the cell surface. For each elapsed time, we measured 20 cells. Cells are mouse fibroblast cell (Balb 3 T3) and mouse osteosarcoma cell (LM8). For the particles, we have employed 2-micron diameter polystyrene beads coated with collagen. The particles were moved in 400 nm from a contact point by use of optical tweezers, which had a spring constant k of 4mN/m, and measured the magnitude of reaction force from the cell. In this measurement resolution and accuracy of reaction force measurement are estimated as 2 pN and 16 pN, respectively.
To ensure strong binding between collagen and particle surface, we used carboxyl polystyrene particle (Spherotech Inc., CP-20-10) and formed covalent bond between first amino group of collagen and carboxyl group by use of cross-linker. Cells are dispersed on glass bottom dish 24 to 36 hours before experiment. The glass bottom dish is coated with collagen to bind tightly. Finally the cells are incubated after putting the particles on the cell surface in 37 °C, 5 % CO2 atmosphere.
The increase in cell stiffness after 75 min may be due to the ingestion of the particle through endocytosis. According to this, we should better choose 60 min after particle contact to the cell surface can avoid initial adhesion formation and endocytosis. In addition, since this condition is expected to depend on cell types, we may choose the suitable condition for the duration of adhesion maturation.
6 Effect of surface coating on particle
Although in previous experiment we have used a collagen coated particle for cell palpation, we expect different coating materials that bind to different target sites can provide additional information on cell stiffness. Therefore, we have also done experiments using particles with different coatings. We have chosen cadherin as the target site, which exists widely on cell membrane . Cadherin is not a force sensitive protein, as opposed to integrin, which is a force sensing protein and may cause reaction against applied force.
To bind a particle to cadherin we used anti-cadherin antibody fixed on particles. Similar to previous experiments, we have used cross-linkers to form covalent bond between antibody and particle surface. The particles used are 2-micron diameter polystyrene and cells are mouse osteosarcoma cell LM8.
We developed the cell palpation system, which is applicable to adhesive cells. In current system, we can measure cell stiffness and investigate the maturation of cell adhesion processes. Advantages of using optical tweezers are the ease of probe particle manipulation, flexibility in specifying which cell location to place the particle, and immediate measurement of force reaction.
We believe cell stiffness is an important parameter on monitoring cell activity and malignancy of cancer cells or other diseases related to cells. We expect the cell palpation system to be utilized as a useful tool for future diagnostics in the medical field.
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