The automation of a rapid and simple trapping-force calibration system is desired for optical tweezers to measure biological forces. One simple calibration method, the water-dragging-force method, is to calibrate the trapping force against a given water dragging force with an image-processing technique. However, the conventional image-processing technique is too slow because of the time it takes for image recording, transferring, storing, and retrieving. The pattern recognition technique of our automated calibration method is rapid and simple, because it directly processes the image signal without recording, storing, and retrieving any image. In an experiment, we have combined the dragging force method with the real-time pattern recognition technique to calibrate the relationship between the trapping force at a given laser power and a displacement of a bead. We demonstrate the calibration results of the trapping forces on two target beads 10 and 6.2 µm in diameter, separately, at two laser powers, 5 and 10 mW. Each calibration procedure is finished in 5 min at a pattern recognition rate of 10 Hz with a spatial resolution of 75 nm. We believe that this technique is reliable and rapid enough to be applied to biological force measurement.
Swimming activity of flagella is a main factor of the motility of bacteria. Flagella expressed on the surface of bacterial species serve as a primary means of motility including swimming. We propose to use optical tweezers to analyze the swimming activity of bacteria. The sample bacteria in the work is Pseudomonas aeruginosa, and it is a gram-negative bacterium and often causes leading to burn wound infections, urinary-tract infections, and pneumonia. The single polar flagellum of P. aeruginosa has been demonstrated to be important virulence and colonization factor of this opportunistic pathogen. We demonstrate a gene to regulate the bacterial swimming activity in P. aeruginosa PAO1 by biological method. However, the change of flagellar morphology was not observed by electron microscopy analysis, suggesting that the gene regulates the flagellar rotation that could not be detected by biological method. PFM exhibits a spatial resolution of a few nanometers to detect the relative position of the probe at an acquisition rate over 1 MHz. By binding a probe such as a bead or a quantum dot on the flagella, we expect the rotation of the probe due to the flagella could be detected. It is expected that the study of the swimming activity of P. aeruginosa provide potent method for the pathogenic role of the flagella in P. aeruginosa.
Integrin receptors serve as both mechanical links and signal transduction mediators between the cell and its environment. Experimental evidence demonstrates that conformational changes and lateral clustering of the integrin proteins may affect their binding to ligands and regulate downstream cellular responses; however, experimental links between the structural and functional correlations of the ligand-receptor interactions are not yet elucidated. In the present report, we utilized optical tweezers to measure the dynamic binding between the snake venom rhodostomin, coated on a microparticle and functioned as a ligand, and the membrane receptor integrin alpha(IIb)beta(3) expressed on a Chinese Hamster Ovary (CHO) cell. A progressive increase of total binding affinity was found between the bead and CHO cell in the first 300 sec following optical tweezers-guided contact. Further analysis of the cumulative data revealed the presence of "unit binding force" presumably exerted by a single rhodostomin-integrin pair. Interestingly, two such units were found. Among the measurements of less total binding forces, presumably taken at the early stage of ligand-receptor interactions, a unit of 4.15 pN per molecule pair was derived. This unit force dropped to 2.54 pN per molecule pair toward the later stage of interactions when the total binding forces were relatively large. This stepped change of single molecule pair binding affinity was not found when mutant rhodostomin proteins were used as ligands (a single unit of 1.81 pN per pair was found). These results were interpreted along with the current knowledge about the conformational changes of integrins during the "molecule activation" process.
The conformational change of integrin αIIbβ3 plays an important role in clot formation. However, the correlation between the structure and the function of integrin αIIbβ3 in interacting with its ligand is still not clear. In this report, we focus on the dynamic variation of the binding between integrin αIIbβ3 and its ligand, rhodostomin by using a photonic force microscopy (PFM). The PFM is used to trap a rhodostomin-coated bead and, then, shift it to bind a surrounding CHO αIIbβ3 cell. Meanwhile, it tracks, with a resolution of 1MHz, the Brownian fluctuations of the trapped bead. Theoretically, the smaller the amplitude of the Brownian fluctuations, the stronger the stiffness of the binding force between the rhodostomin and the CHO αIIbβ3 cell. Experimentally, a significant decrease of the Brownian fluctuations was observed during the interval between the 360th seconds and the 400th seconds after the trapped rhodostomin-coated bead contacted an integrin-expressed CHO αIIbβ3 cell. This observation reveals that it takes the rhodostomin 360 seconds to seek the correct position to bind to the integrin αIIbβ3. After 400 seconds, the rhodostomin has bound rigidly with the integrin αIIbβ3. We presume that the integrin αIIbβ3 has reached its final stage of conformational change.
Sample tracking with a high spatial sensitivity is highly desired in force measurement with optical tweezers. However, the trick that sample tracking via forward scattering pattern detection would provide a higher sensitivity than that via regular image detection has never been investigated. In this paper, we systematically study the influences of the position and the numerical aperture of the condenser on sample tracking via forward scattering pattern detection. In our experiment, a 60X condenser is used to form the forward scattering pattern of a sample bead upon a CCD camera. As the bead is transversely shifted at a step size of 30nm by a PZT XYZ stage, we measure the magnitude of the corresponding shift of the forward scattering pattern when the 60X condenser of different angular apertures is placed at various locations along the optical axis. Our result shows that the most sensitive forward scattering pattern occurs when the condenser collimates the forward scattering light from the sample bead. We also find that the larger the numerical aperture is, the higher the sensitivity of forward scattering pattern detection will be.
Optical tweezers is a newly developed instrument, which makes possible the manipulation of micro-optical particles under a microscope. In this paper, we present the automation of an optical tweezers which consists of a modified optical tweezers, equipped with two motorized actuators to deflect a 1 W argon laser beam, and a computer control system including a joystick. The trapping of a single bead and a group of lactoacidofilus was shown, separately. With the aid of the joystick and two auxiliary cursers superimposed on the real-time image of a trapped bead, we demonstrated the simple and convenient operation of the automated optical tweezers. By steering the joystick and then pressing a button on it, we assign a new location for the trapped bead to move to. The increment of the motion 0.04 (mu) m for a 20X objective, is negligible. With a fast computer for image processing, the manipulation of the trapped bead is smooth and accurate. The automation of the optical tweezers is also programmable. This technique may be applied to accelerate the DNA hybridization in a gene chip. The combination of the modified optical tweezers with the computer control system provides a tool for precise manipulation of micro particles in many scientific fields.
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