Cell–cell and cell–matrix adhesions are essential mechanisms in both tissue and organs in a living body. Cell adhesion strength is modulated in processes such as inflammation, mammalian development, protection of infection, wound healing, and tumor cell metastasis by controlling the expression of cell adhesion molecules. However, quantifying adhesion strength at the level of a single cell is difficult.
One potentially useful technique is applying a local impulsive force to cells using an IR femtosecond laser pulse. When the pulse is focused on (cultured) cells through a high numerical-aperture objective lens, a local explosion induced at the laser focal point generates a stress wave. A short packet of the stress waves affects the cells as an impulsive force. When the pulse energy is tuned to be at the generation threshold (i.e., just enough energy to generate an explosion), the impulsive force has a strong effect only in the vicinity of the laser focal point (a few tens of micrometers in diameter). Other methods of generating a stress wave—such as using gunpowder, electric sparks, or optical breakdown via nanosecond and picosecond lasers—make it too difficult to localize the stress wave sufficiently. Measuring cell adhesion strength requires us to quantify the local impulsive force by the IR femtosecond laser pulse, but this has not previously been done.
Figure 1. Local force measurement for femtosecond laser impulse. (a) Femtosecond laser (120fs, 800nm) was focused in the vicinity of an atomic force microscopy (AFM) cantilever. The impulsive force generated at the laser focal point was loaded on the cantilever. (b) The bending movement induced by the force loading was monitored as a shift of the cantilever (green line) and analyzed by a model of transient vibration (blue line). QPD: Quadrant photodiode. x0, z0: Distances between the laser focal point and the top of the AFM cantilever along x- and z-axis, respectively. They are tuned to be a few tens of micrometers.
To overcome this issue, we developed a new local force measurement system using atomic force microscopy (AFM).1–4 The principle is shown in Figure 1(a). An AFM cantilever is placed in the vicinity of the laser focal point, and the impulsive force generated by the femtosecond laser is loaded on the cantilever. The cantilever's deflection and vibration is detected by a linked quadrant photodiode. A representative result is shown in Figure 1(b). We succeeded in detecting the impulsive force via the cantilever's (natural) frequency. The initial amplitude of the signal indicates the magnitude of the impulsive force, whereas the decay of the vibration is caused by viscous drag. Using this technique, we were able to quantify the impulsive force.
Figure 2. Representative estimate of adhesion strength by the femtosecond laser impulse. (a) Femtosecond laser was focused at the ventral (i.e., left) side of the leukocyte attached to endothelial cell layer. (b) The leukocyte moved with the laser irradiation, indicating that the intercellular adhesion between the leukocyte and endothelial cell was partially broken. The impulse to break the adhesion was estimated to be 0.3×10-12Ns.
We used the method to evaluate intercellular adhesion. In inflammation, circulating leukocytes (white blood cells that fights infection) and endothelial cells (in the walls of blood vessels) critically interact. We estimated this interaction by measuring the intercellular adhesion strength. We prepared a co-culture of leukocytes on an endothelial cell monolayer on a cover slip, and placed the entire set-up on a glass-bottomed dish: see Figure 2(a). When a femtosecond laser pulse was focused through a 40× objective lens at the ventral (i.e., left) side of an adherent leukocyte, the leukocyte moved toward the opposite (right) side of the laser focal point: see Figure 2(b). We estimated the impulse to induce the slipping movement of an adherent leukocyte on an endothelial cell layer from our previous laser AFM measurement (0.3×10−12Ns).
This method enables us to estimate the adhesion of various types of cells observed by optical microscopes. As well as the example in Figure 2, we have successfully estimated the intercellular adhesion strength of endothelial cells, and others including those known as NIH3T3 (fibroblast) and PC12 (pheochromocytoma) cells. Although this work has focused on cell biology, potential applications include quantifying the binding strength and/or fragility of heterojunctions of complex micro-objects, such as components in micro-electromechanical systems, biocompatible materials, optical elements integrated with glasses and polymers, micrometer-sized protein crystals in capillary tubes, and so on. In this work, we estimated the adhesion as an impulse. However, strictly speaking, the adhesion strength should be evaluated as the energy gap between the bonded and free conditions. We are now considering a method to estimate the energy gap from our impulse experimental results.
Yoichiroh Hosokawa, Takanori Iino
Graduate School of Materials Science
Nara Institute of Science and Technology (NAIST)
Yoichiroh Hosokawa received a PhD in applied physics from the Osaka University in 2000. He is now an associate professor at NAIST.
Man Hagiyama, Akihiko Ito
Faculty of Medicine
1. T. Iino, Y. Hosokawa, Direct measurement of femtosecond laser impulse in water by atomic force microscopy, Appl. Phys. Express
3, p. 107002, 2010. doi:10.1143/APEX.3.107002
2. Y. Hosokawa, M. Hagiyama, T. Iino, Y. Murakami, A. Ito, Noncontact etimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy, Proc. Nat'l Acad. Sci. USA
108, p. 1777-1782, 2011. doi:10.1073/pnas.1006847108
3. Y. Hosokawa, Non-contact quantification of laser micro-impulse in water by atomic force microscopy and its application for bio-mechanics, Proc. SPIE
8204, p. 82041D, 2011. doi:10.1117/12.903239
4. T. Iino, Y. Hosokawa, Controllability of femtosecond laser-induced impulse in water evaluated by local force measurement system using atomic force microscopy, J. Appl. Phys.
112, p. 66106, 2012. doi:10.1063/1.4754815