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Measuring Fluid Properties on a Microscopic Scale Using Optically Trapped Microprobes Mark Cronin-Golomb Biomedical Engineering Tufts University With the help of: Boaz Nemet Yossef Shabtai Lisa Goel at Tufts University Tayyaba Hasan Paal Selbo at Wellman Laboratories of Photomedicine, MGH Scanning Probe Microfluidic Analysis Viscosity is an important indicator of biopolymer concentration. Flow analysis is important in development of microfluidic devices. Method: Confocal phase sensitive detection of optical tweezer beam reflected from a trapped probe bead set in sinusoidal oscillation by the tweezer beam enables micrometer scale spatially resolved viscosity measurements at 10kHz data acquisition rates. AcoustoOptic deflector Scanning Mirrors Tweezers principle Electric Field - + Confocal microscope principle Sample Stage Detector Raster display Scanning Mirror Pinhole Laser Beam Expander Prior methods to measure viscoelasticity Video microscopy of magnetically induced fluctuations Schmidt F.G., Ziemann,F. & Sackmann,E. Eur. Biophys. J. 24, 348 (1996). Positional and temporal statistics of trapped bead trap strength and viscosity A. Pralle, E.L. Florin, E.H.K. Stelzer & J.K.H. Horber, Appl. Phys. A-Mat. Sci. & Proc. 66, S71 (1998). Viscosity measurement using position sensing detector M.T. Valentine, L.E. Dewalt & H.D. OuYang, “Forces on a colloidal particle in a polymer solution: A study using optical tweezers.” Journal of PhysicsCondensed Matter 8, 9477-9482 (1996). Experiment Details Ti-Sapphire Laser # P APD BS AOD * OL * # CCD Camera DM Lock-in Amplifier # SM * AOD Driver At large oscillation amplitudes the potential well splits •As the tweezer beam is moved back and forth, the probe bead lags behind. •The bead is bright when the tweezer beam illuminates it. •The confocal signal is highest when the tweezer beam is centered on the probe bead. Theoretical Background dx x a sin( t ) L(t ) dt x: trap position : viscous drag : tweezer spring constant a: amplitude of trap oscillation : frequency of trap oscillation L(t): Brownian forcing function du u a cos t dt u x a sin t I (t ) 1 u (t ) 2 Experimental Results 0.30 80 0.20 70 60 0.15 50 40 0.10 30 20 0.05 10 0.00 0 0 1 2 3 4 5 t 6 7 8 9 nd j2 2 Harmonic phase (deg) 90 0.25 nd A2 2 Harmonic amplitude (a.u) 100 Signal to Noise Ratio Relative Position Detection 0 2 c a SNR 2 0 c2 k BT 1/ 2 8 Absolute Position Detection 0c c a SNR 2 0 c2 k BT 1/ 2 8 30 25 Confocal -1/2 ~f 20 15 SNR 10 Valentine-3/2 ~f 5 0 0 1000 2000 3000 frequency (Hz) 4000 Viscosity Image Viscosity distribution around A. pullulans imaged by raster scanning an optically trapped probe bead. This blastospore has a halo of the polysaccharide pullulan around it. Note the viscosity gradient. Flow field measurement An optically trapped microsphere is used as a probe for two-dimensional velocity field imaging using scanning optics. A fluid viscosity map may be obtained simultaneously. Calibration is based on a single length measurement only. Applications are anticipated in the design of microfluidic devices. Microfluidics New microfluidic devices are being constantly developed. Their fluid dynamics need to be understood. After A. D. Stroock, S. K. W. Dertinger, A. Ajdari, I. Mezi, H. A. Stone, G. M. Whitesides, Science 295, 647 (2002) Flow Off Probe Bead Flow On Probe Bead r a Oscillating Laser Trap a Oscillating Laser Trap Experimental velocity from tweezers ( m m/sec) Comparison of tweezer and video velocity measurement 40 35 30 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 video velocity m m /sec ) Note offset induced by Brownian motion of probe bead Flow Measurement 17mm Flow scale bar mm / sec Force Measurement Flow measurement is one example of force measurement. We can use tweezers to apply forces to probe beads and measure their response. Bead stuck on pullulan around blastospore: Use To Study Effects Of Photodynamic Therapy On Adhesive Properties Of Cancer Cells Tayyaba Hassan and Paal Selbo, Wellman Lab MGH Photodynamic Therapy (PDT) is frequently extremely effective in controlling the primary malignancy, but have also been associated with an increase in distant metastasis. PDT, used as clinical cancer therapy worldwide, is a method in which photosensitizers (PS) are administered to tumor cells and are activated by light at the appropriate wavelength, where a combination of light, oxygen, and PS are toxic to tumors. E-Cadherin/Catenin Complex Overview Van Aken, E. et al., Virchows Arch., 2001 Conclusions Our scanning confocal tweezers microscope can measure velocity and viscosity simultaneously. Viscosity can be measured rapidly with microspheres on microscopic scale. Absolute measurements are obtained in real time for the flow velocity with minimal calibration. Results from the measurements of the flow shear in z suggest that this technique has the potential of mapping the full 3-D distribution of fluid flow and viscosity.