Optical Tweezers 3d Photonic Force Spectroscopy

Since optical tweezers trapped microspheres can be used as an ultrasensitive force measurements technique, the knowledge of its theoretical description is of utmost importance. However, even the description of the incident electromagnetic fields under very tight focusing, typical of the optical trap, is not yet a closed problem. Therefore it is important to experimentally obtain whole accurate curves of the force as a function of wavelength, polarization and incident beam 3D position with respect to the center of the microsphere. Theoretical models for optical forces such as the Generalized Lorenz-Mie theory, can then be applied to the precisely evaluated experimental results. Using a dual trap in an upright standard optical microscope, one to keep the particle at the equilibrium position and the other to disturb it we have been able to obtain these force curves as a function of x, y and z position, incident beam polarization and also wavelength. Further investigation of optical forces was conducted for wavelengths in and out Mie resonances of the dielectric microspherical cavities for both TM and TE modes.6131Ashkin, A., Dziedzic, J.M., Bjorkholm, J.E., Chu, S., Observation of a single-beam gradient force trap for dielectric particles (1986) Opt. Lett., 11, pp. 288-290Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Grier, D.G., A revolution in optical manipulation (2003) Nature, 424, pp. 810-816Neuman, K.C., Block, S., Optical trapping (2004) Rev. Sci. Instrum., 75, pp. 2787-2809Lock, J.A., Calculation of the radiation trapping force for laser tweezers by use of generalized Lorenz-Mie theory. I. Localized model description of an on-axis tightly focused laser beam with spherical aberration (2004) Appl. Opt., 43, pp. 2532-2544Lock, J.A., Calculation of the radiation trapping force for laser tweezers by use of generalized Lorenz-Mie theory. II. On-axis trapping force (2004) Appl. Opt., 43, pp. 2545-2554Mazolli, A., Neto, P.A.M., Nussenzveig, H.M., Theory of trapping forces in optical tweezers (2003) Proc. Royal Soc. London Ser. A Math. Phys. Eng. Sci., 459, pp. 3021-3041Fontes, A., Neves, A.A.R., Moreira, W.L., De Thomaz, A.A., Barbosa, L.C., De Paula, A.M., Cesar, C.L., Double optical tweezers for ultrasensitive force spectroscopy in microsphere Mie scattering (2005) Appl. Phys. Lett., 87. , Art. No. 221109Ren, K.F., Gouesbet, G., Gréhan, G., Integral localized approximation in generalized Lorenz-Mie theory (1998) Appl. Opt., 37, pp. 4218-4225Lock, J.A., Excitation efficiency of a morphology-dependent resonance by a focused Gaussian beam (1998) J. Opt. Soc. Am. A, 15, pp. 2986-2994Davis, L.W., Theory of electromagnetic beams (1979) Phys. Rev. A, 19, pp. 1177-1779Ren, K.F., Gréhan, G., Gouesbet, G., Radiation pressure forces exerted on a particle arbitrarily located in a gaussian beam by using the generalized Lorenz-Mie theory and associated resonance effects (1994) Opt. Commun., 108, pp. 343-354Ren, K.F., Gréhan, G., Gouesbet, G., Symmetry relations in generalized Lorenz-Mie theory (1994) J. Opt. Soc. Am. A, 11, pp. 1812-181

Chemotaxis Study Using Optical Tweezers To Observe The Strength And Directionality Of Forces Of Leishmania Amazonensis

The displacements of a dielectric microspheres trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences. This system can measure forces on the 50 femto Newtons to 200 pico Newtons range, of the same order of magnitude of a typical forces induced by flagellar motion. The process in which living microorganisms search for food and run away from poison chemicals is known is chemotaxy. Optical tweezers can be used to obtain a better understanding of chemotaxy by observing the force response of the microorganism when placed in a gradient of attractors and or repelling chemicals. This report shows such observations for the protozoa Leishmania amazomenzis, responsible for the leishmaniasis, a serious tropical disease. We used a quadrant detector to monitor the movement of the protozoa for different chemicals gradient. This way we have been able to observe both the force strength and its directionality. The characterization of the chemotaxis of these parasites can help to understand the infection mechanics and improve the diagnosis and the treatments employed for this disease.6326Law, A.M.J., Aitken, M.D., Continuous-flow capillary assay for measuring bacterial chemotaxi (2005) Applied and Environmental Microbiology, 71 (6), pp. 3137-3143Khan, S., Jain, S., Reid, G.P., Trentham, D.R., The fast tumble signal in bacterial chemotaxis (2004) Biophysical Journal, 86 (6), pp. 4049-4058Neuman, K.C., Chadd, E.H., Liou, G.F., Bergman, K., Block, S.M., Characterization of photodamage to Escherichia coli in optical traps (1999) Biophysical Journal, 77 (5), pp. 2856-2863Who, World Health Organization, 2001Gontijo, B., Carvalho, M.L.R., Leishmaniose tegumentar Americana (2003) Revista da Sociedade Brasileira de Medicina Tropical, 36 (1), pp. 71-80Handman, E., Cell biology of Leishmania (2000) Advances in Parasitology, 44, pp. 1-39Rice, S.E., Purcell, T.J., Spudich, J.A., Building and using optical traps to study properties of molecular motors (2003) Biophotonics, PT B, 361, pp. 112-133Rohrbach, A., Stelzer, E.H.K., Three-dimensional position detection of optically trapped dielectric particles (2002) Journal of Applied Physics, 91 (8), pp. 5474-5488Gittes, F., Schmidt, C.F., Interference model for back-focal-plane displacement detection in optical tweezers (1998) Optics Letters, 23 (1), pp. 7-9Allersma, M.W., Gittes, F., DeCastro, M.J., Stewart, R.J., Schmidt, C.F., Two-dimensional tracking of ncd motiliry by back focal plane interferometry (1998) Biophysical Journal, 74 (2), pp. 1074-108

Force Spectroscopy And Two Photon Excited Luminescence In An Optical Tweezers System

Up to now optical spectroscopies have analyzed the scattered light or the heat generated by absorption as a function of the wavelength to get information about the samples. Among the light matter interaction phenomena one that has almost never been used for spectroscopy is the direct photon momenta transfer. Probably because the forces involved are very small, varying from hundreds of femto to tens of pico Newtons. However, the nowadays very popular Optical Tweezers can easily accomplish the task to measure the photon momenta transfer and may be the basis for the Optical Force Spectroscopy. We demonstrate its potential as such a tool by observing more than eight Mie resonance peaks of a single polystyrene microsphere, and showed the capability to selective couple the light to either the TE, TM or both microsphere modes depending of the beam size, the light polarization and the beam positioning. The Mie resonances can change the optical force values by 30-50%. Our results also clearly show how the beam polarization breaks the usually assumed azimuthal symmetry by Optical Tweezers theories. We also obtained the spectrum from the two photon excited luminescence using the Optical Tweezers to hold a single bead suspended and a femtosecond Ti:sapphire laser for the non-linear excitation. This spectrum shows the pair of peaks due to both TE and TM spherical cavity modes. We have been able to observe more than 14 Mie resonance peaks in the TPE luminescence. Our results are in good agreement with optical force calculations using Maxwell stress tensor and partial wave decomposition of the incident beam approximated to a 3th order gaussian beam.593017Ashkin, Dziedzic, J.M., Bjorkholm, J.E., Chu, S., Observation of a single-beam gradient force trap for dielectric particles (1986) Opt. Lett., 11, pp. 288-290Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Grier, D.G., A revolution in optical manipulation (2003) Nature, 424, pp. 810-816Felgner, H., Frank, R., Schliwa, M., Flexural rigidity of microtubules measured with the use of optical tweezers (1996) J. Cell Sci., 109, pp. 509-516Sakata-Sogawa, K., Kurachi, M., Sogawa, K., Fujii-Kuriyama, Y., Tashiro, H., Direct measurement of DNA molecular length in solution using optical tweezers: Detection of looping due to binding protein interactions (1998) Eur. Biophys. J., 27, pp. 55-61Berg, H.C., Berry, R.M., Absence of a barrier to backwards rotation of the bacterial flagellar motor demonstrated with optical tweezers (1997) Proc. Natl. Acad. Sci. U.S.A., 94, pp. 14433-14437Huruta, R.R., Mechanical properties of stored red blood cells using optical tweezers (1998) Blood, 92, pp. 2975-2977Brandão, M.M., Optical tweezers for measuring red blood cell elasticity: Application to the study of drug response in sickle cell disease (2003) Eur. J. Haematol., 70, pp. 207-211Ashkin, A., Dziedzic, J.M., (1977) Phys. Rev. Lett., 38, pp. 1351-1355Barber, P.W., Chang, R.K., (1988) Optical Effects Associated with Small Particles, , Word Scientific, SingaporeVan De Hulst, H.C., (1981) Light Scattering by Small Particles, , Dover, New YorkAshkin, A., Dziedzic, J.M., Observation of optical of dieletric particles by light-scattering (1981) Appl. Optics, 20, pp. 1803-1814Benner, R.E., Barber, P.W., Owe, J.F., Chang, R.K., Observation of structure resonances in the fluorescence spectra from microspheres (1980) Physical Review Letters, 44, pp. 475-478Leung, C.H., She, T.C., Lee, W.K., Positions of low order morphology dependent resonances determined by elastic light scattering (1995) J. Opt. Soc. Am. B, 12, pp. 1259-1266Schaschek, K., Popp, J., Kiefer, W., Observation of morphology dependent in- And output-resonances in time dependent Raman spectra of optically levitated microdroplets (1993) J. Raman Spectrosc., 24, pp. 69-75Ashkin, A., Dziedzic, J.M., Observation of resonances in radiation pressure on dieletric particles (1977) Phys. Rev. Lett., 38, pp. 1351-1354Bisht, P.B., Fukuda, K., Hirayama, S., Steady-state and time-resolved fluorescence study of some dyes in polymer microspheres showing morphology dependent resonances (1996) J. Chem. Phys., 105, pp. 9349-9361Arnold, S., Shift of whispering gallery modes in microspheres by protein adsorption (2003) Opt. Lett., 28, pp. 272-274Vollmer, F., Multiplexed DNA quantification by spectroscopic shift of two microsphere cavities (2003) Biophys. J., 85, pp. 1974-1979Ren, K.F., Gréhan, G., Gouesbet, G., Radiation pressure forces exerted on a particle arbitrarily located in a gaussian beam by using the generalized Lorenz-Mie theory and associated resonance effects (1994) Opt. Commun., 108, pp. 343-354Ren, K.F., Gréhan, G., Gouesbet, G., Symmetry relations in generalized Lorenz-Mie theory (1994) J. Opt. Soc. Am. A, 11, pp. 1812-1817Bohren, C.F., Huffman, D.R., (1983) Absorption and Scattering of Light by Small Particles, , Wiley, New YorkVan De Hulst, H.C., (1981) Light Scattering by Small Particles, , Dover, New YorkDavis, L.W., Theory of electromagnetic beams (1979) Phys. Rev. A, 19, pp. 1177-1779Richards, B., Wolf, E., Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system (1959) Proc. R. Soc. London A, 253, pp. 358-379Ren, K.F., Gouesbet, G., Gréhan, G., Integral localized approximation in generalized Lorenz-Mie theory (1998) Appl. Opt., 37, pp. 4218-4225Lock, J.A., Excitation efficiency of a morphology-dependent resonance by a focused Gaussian beam (1998) J. Opt. Soc. Am. A, 15, pp. 2986-2994Bateman, J.B., Weneck, E.J., Eshler, D.C., Determination of Particle Size and Concentration from Spectrophotometric Transmission (1959) J. Colloid Sci., 14, pp. 308-32

Measuring Electrical And Mechanical Properties Of Red Blood Cells With A Double Optical Tweezers

The fluid lipid bilayer viscoelastic membrane of red blood cells (RBC) contains antigen glycolproteins and proteins which can interact with antibodies to cause cell agglutination. This is the basis of most of the immunohematologic tests in blood banks and the identification of the antibodies against the erythrocyte antigens is of fundamental importance for transfusional routines. The negative charges of the RBCs creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. The first counterions cloud strongly binded moving together with the RBC is called the compact layer. This report proposes the use of a double optical tweezers for a new procedure for measuring: (1) the apparent membrane viscosity, (2) the cell adhesion, (3) the zeta potential and (4) the compact layer's size of the charges formed around the cell in the electrolytic solution. To measure the membrane viscosity we trapped silica beads strongly attached to agglutinated RBCs and measured the force to slide one RBC over the other as a function of the relative velocity. The RBC adhesion was measured by slowly displacing two RBCs apart until the disagglutination happens. The compact layer's size was measured using the force on the silica bead attached to a single RBC in response to an applied voltage and the zeta potential was obtained by measuring the terminal velocity after releasing the RBC from the optical trap at the last applied voltage. We believe that the methodology here proposed can improve the methods of diagnosis in blood banks.6326Eylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., The contribution of sialic acid to the surface charge of the erythrocyte (1962) J. Biol. Chem., 237, pp. 1992-2000Pollack, W., Reckel, R.P., A reappraisal of the forces involved in Hemagglutination (1977) Int Archs Allergy Appl. Immun., 54, pp. 29-42Ashkin, A., Dziedzic, J.M., Bjorkholm, J.E., Chu, S., Observation of a single-beam gradient force trap for dielectric particles (1986) Opt. Lett., 11, pp. 288-290Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Grier, D.G., A revolution in optical manipulation (2003) Nature, 424, pp. 810-816Zhu, C., Bao, G., Wang, N., Cell Mechanics: Mechanical response, cell adhesion, and molecular deformation (2000) Annu. Rev. Biomed. Eng., 2, pp. 189-226Neuman, K.C., Block, S., Optical trapping (2004) Rev. Sci. Instrum., 75, pp. 2787-2809Saffman, P.G., Delbruck, M., Brownian motion in biological membranes (1975) Proc. Nat. Acad. Sci. USA, 72, pp. 3111-3113Dimova, R., Danov, K., Pouligny, B., Ivanov, I.B., Drag of a solid particle trapped in a thin film or at an interface: Influence of surface viscosity and elasticity (2000) J. Colloid and Interface Science, 226, pp. 35-43Hochmuth, R., Worthy, P., Evans, E., Red cell extensional recovery and the determination of membrane viscosity (1979) Biophys. J., 26, pp. 101-114Sze, A., Erickson, D., Ren, L., Li, D., Zeta-potential measurement using the Smoluchowski equation and the slope of the current-time relationship in electroosmotic flow (2003) J. Colloid and Interface Science, 261, pp. 402-410Hunter, R.J., (1981) Zeta Potential in Colloid Science, , Academic Press, New YorkPollack, W., Hager, H.J., Reckel, R., Toren, D.A., Singher, H.O., A study of the forces involved in the second stage of hemaggltination (1965) Transfusion, 5, pp. 158-183Chelidze, T., Dielectric spectroscopy of blood (2002) J. Non-crystalline Solids, 305, pp. 285-294Hymer, W.C., Barlow, G.H., Blaisdell, S.J., Continuous flow electrophoretic separation of proteins and cells from mammalian tissues (1987) Cell Biophys., 10, pp. 61-85Hashimoto, N., Fujita, S., Yokoyama, T., Cell electrophoretic mobility and glycerol lysis of human erythrocytes in various diseases (1998) Electrophoresis, 19, pp. 1227-123

Mechanical Properties Of Stored Red Blood Cells Using Optical Tweezers

We have developed a method for measuring the red blood cell (RBC) membrane overall elasticity μ by measuring the deformation of the cells when dragged at a constant velocity through a plasma fluid by an optical tweezers. The deformability of erythrocytes is a critical determinant of blood flow in the microcirculation. We tested our method and hydrodynamic models, which included the presence of two walls, by measuring the RBC deformation as a function of drag velocity and of the distance to the walls. The capability and sensitivity of this method can be evaluated by its application to a variety of studies, such as, the measurement of RBC elasticity of sickle cell anemia patients comparing homozygous (HbSS), including patients taking hydroxyrea (HU) and heterozygous (HbAS) with normal donors and the RBC elasticity measurement of gamma irradiated stored blood for transfusion to immunosupressed patients as a function of time and dose. These studies show that the technique has the sensitivity to discriminate heterozygous and homozygous sickle cell anemia patients from normal donors and even follow the course of HU treatment of Homozygous patients. The gamma irradiation studies show that there is no significant change in RBC elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not, but there was a huge change in the measured elasticity for the RBC units stored for more than 21 days after irradiation. These finds are important for the assessment of stored irradiated RBC viability for transfusion purposes because the present protocol consider 28 storage days after irradiation as the limit for the RBC usage.593016Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Barjas-Castro, M.L., Brandão, M.M., Fontes, A., Costa, F.F., Cesar, C.L., Saad, S.T.O., Elastic properties of irradiated red blood cell units measured by optical tweezer (2002) Transfusion, 42, pp. 1196-1199Brandão, M.M., Fontes, A., Barjas-Castro, M.L., Barbosa, L.C., Costa, F.F., Cesar, C.L., Saad, S.T.O., Optical tweezers for measuring red blood cell elasticity: Application to the study of drug response in sickle cell disease (2003) European Journal of Haematology, 70, pp. 207-211Williamson, L.M., Warwick, R.M., Transfusion-associated graft-versus-host disease and its prevention (1995) Blood Rev., 9, pp. 251-261Button, L.N., Dewolf, W.C., Newburger, P.E., The effecr of irradiation on blood components (1981) Transfusion, 21, pp. 419-426Platt, O.S., The sickle syndrome (1995) Blood: Principles and Practice of Hematology, , R. I Hadlin, S. E. Lux, T. P. Stossel, J. B. Lippincott, PhiladelphiaBallas, S.K., Dover, G.J., Charache, S., Effect of hydroxyurea on the rheological properties of sickle erythrocytes in vivo (1989) Am. J. Hematol, 32, pp. 104-111Groner, W., Mohandas, N., Bessis, M., New optical technique for measuring erythrocyte deformability with the ektacytometer (1980) Clin. Chem., 26, pp. 1435-1442De Franceschi, L., Bachir, D., Galacteros, F., Tchernia, G., Cynober, T., Alper, S., Platt, O., Brugnara, C., Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease (1997) J Clin Invest, 100, pp. 1847-1852Hochmuth, R.M., Worthy, P.R., Evans, E.A., Red cell extensional recovery and the determination of membrane viscosity (1979) Biophys. J., 26, pp. 101-114Evans, E.A., La Celle, P.L., Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation (1975) Blood, 45, pp. 29-43Itoh, T., Chien, S., Usami, S., Effects of hemoglobin concentration on deformability of individual sickle cells after deoxygenation (1995) Blood, 85, pp. 2245-2253Evans, E.A., Mohandas, N., Membrane-associated sickle hemoglobin: A major determinant of sickle erythrocyte rigidity (1987) Blood, 70, pp. 1443-1449Dong, C., Chadwick, R.S., Schechter, A.N., Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation (1992) Biophys. J., 63, pp. 774-783Suzuki, Y., Tateishi, N., Cicha, I., Decreased deformability of the X-ray irradiated red blood cells stored in manitol-adenine-phosphate medium (2000) Clin. Hemorheol. Micro-cire., 22, pp. 131-14

Determination Of Fluid Viscosity And Femto Newton Forces Of Leishmania Amazonensis Using Optical Tweezers

The displacements of a polystyrene microsphere trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences such as the measurement of forces of living microorganisms or the viscosity of local fluids. The technique we used allowed us to measure forces on the 200 femto Newtons to 4 pico Newtons range of the protozoa Leishmania amazonensis, responsible for a serious tropical disease. These observations can be used to understand the infection mechanism and chemotaxis of these parasites. The same technique was used to measure viscosities of few microliters sample with agreement with known samples better than 5%. To calibrate the force as a function of the microsphere displacement we first dragged the microsphere in a fluid at known velocity for a broad range of different optical and hydrodynamical parameters. The hydrodynamical model took into account the presence of two walls and the force depends on drag velocity, fluid viscosity and walls proximities, while the optical model in the geometric optics regime depends on the particle and fluid refractive indexes and laser power. To measure the high numerical (NA) aperture laser beam power after the objective we used an integration sphere to avoid the systematic errors of usual power meters for high NA beams. After this careful laser power measurement we obtained an almost 45 degrees straight line for the plot of the optical force (calculated by the particle horizontal displacement) versus hydrodynamic force (calculated by the drag velocity) under variation of all the parameters described below. This means that hydrodynamic models can be used to calibrate optical forces, as we have done for the parasite force measurement, or vice-versa, as we did for the viscosity measurements.593017Ashkin, A., Dziedzic, J.M., Bjorkholm, J.E., Chu, S., Observation of a single-beam gradient force optical trap for dieletric particles (1986) Optics Letters, 11, pp. 288-290Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Greulich, K.O., (1999) Micromanipulation by Light in Biology and Medicine, , Basel, Boston, Berlin: BirkhäuserSakata-Sogawa, K., Direct measurement of DNA molecular length in solution using optical tweezers: Detection of looping due to binding protein interactions (1998) Eur. Biophys. J., 27, pp. 55-61Konig, K., Determination of motility forces of human spermatozoa using an 800 nm optical trap (1996) Cell. Mol. Biol., 42, pp. 501-509Barjas-Castro, M.L., Elastic properties of irradiated RBCs measured by optical tweezers (2002) Transfusion, 42, pp. 1196-1199Huruta, R.R., Mechanical properties of stored red blood cells using optical tweezers (1998) Blood, 92, pp. 2975-2977Felgner, H., Muller, O., Schliwa, M., Calibration of light forces in optical tweezers (1995) Appl. Optics, 34, pp. 977-982Svoboda, K., Block, S., Biological applications of optical forces (1994) Annu. Rev. Biophys. Biomolec. Struct., 23, pp. 247-285Henon, S., Lenormand, G., Richert, A., Gallet, F., A new determination of the shear modulus of the human erythrocyte membrane using optical tweezers (1999) Biophys. J., 76, pp. 1145-1151Konig, K., Determination of motility forces of human spermatozoa using an 800nm optical trap (1996) Cell. Mol. Biol., 42, pp. 501-509Who, Whorld Health Organization, 2001Herwaldt, B.L., Leishmanias (1999) Lancet, 354, pp. 1191-1199Killick-Kendrick, R., The life-cycle of Leishmania in the sandfly with special reference to the form infective to the vertebrate host (1990) Ann. Parasitol. Hum. Comp., 65 (1 SUPPL.), pp. 37-42Handman, E., Cell biology of Leishmania (2000) Adv. Parasitol, 44, pp. 1-39Happel, J., Brenner, H., (1991) Low Reynolds Number Hydrodynamics with Special Applications to Particulate Media, , Klumer, Dordrecht(1971) Handbook of Chemistry and Physics, , Chemical Rubber, ClevelandPurcell, E.M., Life at low Reynolds number (1977) Am. J. Phys., 45, pp. 124-13

Studying Red Blood Cell Agglutination By Measuring Membrane Viscosity With Optical Tweezers

The red blood cell (RBC) viscoelastic membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that are responsible for cell agglutination. Manipulating RBCs rouleaux with a double optical tweezers, we observed that the cells slide easily one over the others but are strongly connected by their edges. An explanation for this behavior could be the fact that when the cells slide one over the others, proteins are dragged through the membrane. It confers to the movement a viscous characteristic that is dependent of the velocity between the RBCs and justifies why is so easy to slide them apart. Therefore, in a first step of this work, by measuring the force as a function of the relative velocity between two cells, we confirmed this assumption and used this viscous characteristic of the RBC rouleaux to determine the apparent membrane viscosity of the cell. As this behavior is related to the proteins interactions, we can use the apparent membrane viscosity to obtain a better understanding about cell agglutination. Methods related to cell agglutination induced by antigen-antibody interactions are the basis of most of tests used in transfusion centers. Then, in a second step of this work, we measured the apparent membrane viscosity using antibodies. We observed that this methodology is sensitive to different kinds of bindings between RBCs. Better comprehension of the forces and bindings between RBCs could improve the sensibility and specificity of the hemagglutination reactions and also guides the development of new potentiator substances.6644Fontes, A., Fernandes, H.P., Barjas-Castro, M.L., Thomaz, A.A., Pozzo, L., Barbosa, L.C., Cesar, C.L., Red blood cell membrane viscoelasticity, agglutination and zeta potential measurements with double optical tweezers (2006) Proceedings of SPIE, 6088, pp. 296-305Eylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., The contribution of sialic acid to the surface charge of the erythrocyte (1962) J. Biol. Chem, 237, pp. 1992-2000Pollack, W., Reckel, R.P., A reappraisal of the forces involved in Hemagglutination (1977) Int Archs Allergy Appl. Immun, 54, pp. 29-42Fontes, A., Giorgio, S., de Castro Jr., A.B., Neto, V.M., Pozzo, L.Y., Marques, G.P., Barbosa, L.C., Cesar, C.L., Determination of femto Newton forces and fluid viscosity using optical tweezers: Application to Leishmania amazonensis (2005) Proceedings of SPIE, 5699, pp. 419-425Saffman, P.G., Delbruck, M., Brownian motion in biological membranes (1975) Proc. Nat. Acad. Sci. USA, 72, pp. 3111-3113Dimova, R., Danov, K., Pouligny, B., Ivanov, I.B., Drag of a solid particle trapped in a thin film or at an interface: Influence of surface viscosity and elasticity (2000) J. Colloid and Interface Science, 226, pp. 35-4

Evidence Of Chemotaxis By Quantitative Measurement Of The Force Vectors Of Trypanossoma Cruzi In The Vicinity Of The Rhodnius Prolixus Midgut Wall Cell

In this work we used a methodology to study chemotaxis of Trypanossoma cruzi (T. Cruzi) in real time using an Optical Tweezers system. Trapped beads were used as a force transducer for measuring forces of the same order of magnitude as typical forces induced by flagellar motion. Optical Tweezers allowed real time measurements of the force vectors, strength and direction, of living parasites under chemical or other kinds of gradients. This seems to be the ideal tool to perform observations of taxis response of cells and microorganisms with high sensitivity to capture instantaneous responses to a given stimulus. We applied this methodology to investigate the T. cruzi under distinct situations: the parasite alone and in the presence of its insect-vector Rhodnius prolixus (R. prolixus). © 2009 SPIE.7400http://www.who.int/tdr/diseases/chagas/diseaseinfo.htmlAnna, B., Carole, A.P., Eukaryotic chemotaxis at a glance (2008) J. Cell Science, 121, pp. 2621-2624Laszlo, K., Chemotaxis: The proper physiological response to evaluate phylogeny of signal molecules (1999) Acta Biol Hung, 50, pp. 375-394Law, A.M.J., Aitken, M.D., Continuous-flow capillary assay for measuring bacterial chemotaxis (2005) Appl. Environ. Microbiol., 71, pp. 3137-3143Khan, S., Jain, S., Reid, G.P., Trentham, D.R., The fast tumble signal in bacterial chemotaxis (2004) Biophys. J., 86, pp. 4049-4058Neuman, K.C., Chadd, E.H., Liou, G.F., Bergman, K., Block, S.M., Characterization of photodamage to escherichia coli in optical traps (1999) Biophys. J., 77, pp. 2856-2863Bleul, C.C., Farzan, M., Choe, H., Parolin, C., Clark-Lewis, I., Sodroski, J., Springer, T.A., The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry (1996) Nature, 382 (6594), pp. 829-833. , DOI 10.1038/382829a0Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S., Kitamura, Y., Yoshida, N., Kishimoto, T., Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF- 1 (1996) Nature, 382, pp. 635-638Nelson, R.D., Quie, P.G., Simmons, R.L., Spontaneous migration of human polymorphonuclear leukocytes and monocytes, chemotaxis under agarose-new and simple method for measuring chemotaxis and (1975) J. Immunol., 115, pp. 1650-1656Blair, D.F., How bacteria sense and swim (1999) Annu. Rev. Microbiol, 49, pp. 489-522Rao, C.V., Glekas, G.D., Ordal, G.W., The three adaptation systems of bacillus subtilis chemotaxis (2008) Trends Microbiol, 16, pp. 480-487Barros, V.C., Oliveira, J.S., Melo, M.N., Gontijo, N.F., Leishmania amazonensis: Chemotaxic and osmotaxic responses in promastigotes and their probable role in development in the phlebotomine gut (2006) Exp. Parasitol., 112, pp. 152-157Pfeffer, W., (1888) Unters. Botan. Inst., 2, pp. 582-661. , TubingenAdler, J., A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by escherichia coli (1973) J. Gen. Microbiol., 74, pp. 77-91Alves, C.R., Albuquerque-Cunha, J.M., Mello, C.B., Nogueira, E.S.G.D.N.F., Bourguingnon, S.C., Souza, W.D., Azambuja, P., Gonzalez, M.S., Trypanosoma cruzi: Attachment to perimicrovillar membrane glycoproteins of rhodnius prolixus (2007) Experimental Parasitology, 116, pp. 44-52Fontes, A., Giorgio, S., De Castro Jr., A.B., Neto, V.M., De Pozzo, L.Y., Marques, G.P., Barbosa, L.C., Cesar, C.L., Determination of Femto Newton forces and fluid viscosity using optical tweezers - Application to Leishmania amazonensis (2005) Progress in Biomedical Optics and Imaging - Proceedings of SPIE, 5699, pp. 419-425. , DOI 10.1117/12.586427, 59, Imaging, Manipulation, and Analysis of Biomolecules and Cells: Fundamentals and Applications II

Optical Tweezers And Multiphoton Microscopies Integrated Photonic Tool For Mechanical And Biochemical Cell Processes Studies

The research in biomedical photonics is clearly evolving in the direction of the understanding of biological processes at the cell level. The spatial resolution to accomplish this task practically requires photonics tools. However, an integration of different photonic tools and a multimodal and functional approach will be necessary to access the mechanical and biochemical cell processes. This way we can observe mechanicaly triggered biochemical events or biochemicaly triggered mechanical events, or even observe simultaneously mechanical and biochemical events triggered by other means, e.g. electricaly. One great advantage of the photonic tools is its easiness for integration. Therefore, we developed such integrated tool by incorporating single and double Optical Tweezers with Confocal Single and Multiphoton Microscopies. This system can perform 2-photon excited fluorescence and Second Harmonic Generation microscopies together with optical manipulations. It also can acquire Fluorescence and SHG spectra of specific spots. Force, elasticity and viscosity measurements of stretched membranes can be followed by real time confocal microscopies. Also opticaly trapped living protozoas, such as leishmania amazonensis. Integration with CARS microscopy is under way. We will show several examples of the use of such integrated instrument and its potential to observe mechanical and biochemical processes at cell level.6644Denk, W., Strickler, J.H., Webb, W.W., (1990) Science, 248, p. 73Xu, C., (1996) Proc. Natl. Acad. Sci. USA, 93, pp. 10-763Minami, T., Hirayama, S., (1990) J. Photochem. Photobiol. A - Chem, 53 (1), p. 11Lakowicz, J.R., Berndt, K.W., (1991) Rev. Scientific Instrum, 62 (7), p. 1727Becker, W., (2004) Microscopy Res. Technique, 63 (1), p. 58Ha, T., (1996) PNAS, 93 (13), p. 6264Gordon, G.W., (1998) Biophysical J, 74 (5), p. 2702Szavo, G., (1992) Biophysical J, 61 (3), p. 661Campagnola, P.G., (1999) Biophysical J, 77 (6), p. 3341J. X. Cheng JX and X. S. Xie, J. Physical Chem. B 108 (3): 827 (2004)M. Muller et al, J. Microscopy 197, 150 Part 2 (2000)Goksör, M., Enger, J., Hanstorp, D., Optical manipulation in combination with multiphoton microscopy for single-cell studies (2004) Applied Optics, 43 (25), p. 4831Ajito, K., Morita, M., (1999) Surf. Science, 428, p. 141Jess, P.R.T., Garces-Chavez, V., Smith, D., Mazilu, M., Paterson, L., Riches, A., Herrington, C.S., Dholakia, K., (2006) Opt. Express, 14 (12), p. 5779Fontes, A., Ajito, K., De Paula, A.M., Neves, A.R., Moreira, W.L., Barbosa, L.C., Cesar, C.L., (2003) Microsc. Microanal, 9 (SUPPL. 2), pp. 164-165A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. Thomaz, L. C. Barbosa, A. M. de Paula and C. L. Cesar, Phys. Rev. E. 72, 012903 (1-4) (2005)Fontes, A., Neves, A.A.R., Moreira, W.L., de Thomaz, A.A., Barbosa, L.C., de Paula, A.M., Cesar, C.L., (2005) Appl. Phys. Lett, 87, p. 22110