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High Force Magnetic Tweezers for Molecular Manipulation Inside Living Cells

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High Force Magnetic Tweezers for Molecular Manipulation Inside Living Cells HIGH FORCE MAGNETIC TWEEZERS FOR MOLECULAR MANIPULATION INSIDE LIVING CELLS Anthony de Vries De promotiecommissie: Prof. dr. ir. A. Bliek Universiteit Twente, voorzitter Prof. dr. J. Greve Universiteit Twente, promoter dr.ir. J.S. Kanger Universiteit Twente prof.dr. V. Subramaniam Universiteit Twente prof.dr. C.G. Figdor Universiteit Twente prof.dr. G. Reiss Universität Bielefeld prof.dr. M.J. Peters Universiteit Twente prof.dr. R. van Driel Universiteit van Amsterdam Acknowledgement This research was financially supported by NWO Earth and Life Sciences. It was performed in collaboration with the Swammerdam Institute for Life Sciences, University of Amsterdam. ISBN 90-9018799-5 Copyright © 2004 by A.H.B. de Vries HIGH FORCE MAGNETIC TWEEZERS FOR MOLECULAR MANIPULATION INSIDE LIVING CELLS PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit Twente, op gezag van de rector magnificus, prof. dr. F.A. van Vught, volgens besluit van het College voor Promoties in het openbaar te verdedigen op woensdag 10 november 2004 om 15.00 uur door Anthony Henk Bernard de Vries geboren op 26 mei 1972 te Gent, België Dit proefschrift is goedgekeurd door De promoter: prof. dr. J. Greve De assistent promotor: dr. ir. J.S. Kanger To my family Contents Contents 1. Introduction ...................................................................................................... 1 1.1. Biological background....................................................................... 1 1.2. Demands on in vivo nano-manipulation:........................................... 3 1.3. Exerting force on a probe................................................................... 4 1.4. Magnetic Tweezers ............................................................................ 5 1.5. Current status of cellular manipulation.............................................. 6 1.6. Goal of this thesis .............................................................................. 7 1.7. Outline ............................................................................................... 8 1.8. References.......................................................................................... 9 2. Designing the magnetic setup ........................................................................ 11 2.1. Introduction...................................................................................... 11 2.2. Theory on magnetism ...................................................................... 11 2.2.1. Forces on a magnetic particle ............................................ 11 2.2.2. Trapping a particle............................................................. 12 2.2.3. Magnetic saturation ........................................................... 13 2.3. Design of the magnetic tweezers ..................................................... 14 2.3.1. Design criteria.................................................................... 14 2.3.2. Functional Design.............................................................. 17 2.4. Flux generation ................................................................................ 17 2.5. Flux transport................................................................................... 18 2.6. Producing a gradient in the magnetic flux density........................... 19 2.6.1. Gradient produced by a single pole ................................... 19 2.6.2. Gradient produced by multiple poles................................. 21 2.6.3. Pole diameter ..................................................................... 31 2.6.4. 2D compared to 3D.......................................................... 32 2.6.5. Pole arrays ......................................................................... 34 i Contents 2.7. Conclusions...................................................................................... 36 2.8. References........................................................................................ 38 3. Magnetic Tweezers Implementation............................................................. 39 3.1. Introduction...................................................................................... 39 3.2. Fabrication of the pole tips .............................................................. 39 3.2.1. Design criteria.................................................................... 39 3.2.2. Laser cutting from thin sheet. ............................................ 41 3.2.3. Micromachining................................................................. 43 3.2.4. Magnetic Material for electroplating ................................. 44 3.2.5. ‘Through mask’ electroplating .......................................... 45 3.2.6. Electroplating using a Lift-off mask.................................. 50 3.2.7. Delayed lift-off electroplating ........................................... 53 3.2.8. Biological Aspects............................................................. 58 3.3. Optics............................................................................................... 59 3.3.1. Introduction ....................................................................... 59 3.3.2. Setup .................................................................................. 59 3.3.3. Fluorescence pathway........................................................ 60 3.3.4. Laser interference tracking pathway.................................. 61 3.3.5. Discussion.......................................................................... 62 3.4. Macroscopic yoke ............................................................................ 62 3.4.1. Magnetic circuits ............................................................... 63 3.4.2. First order approximation .................................................. 64 3.4.3. Model including saturation and flux leakage..................... 64 3.5. Electronics ....................................................................................... 66 3.5.1. Coils................................................................................... 66 3.5.2. Amplifier ........................................................................... 67 3.6. Conclusion ....................................................................................... 67 3.7. References........................................................................................ 68 ii Contents 4. Experimental results ...................................................................................... 69 4.1. Introduction...................................................................................... 69 4.2. Characterization of magnetic materials............................................ 69 4.2.1. Vibrating Sample Magnetometer....................................... 69 4.2.2. Characterisation of the cobalt layers.................................. 70 4.3. Force calibration .............................................................................. 72 4.3.1. Calibration techniques: ...................................................... 72 4.3.2. Force calibration using a micropipette .............................. 74 4.3.3. Force direction................................................................... 77 4.3.4. Force amplitude ................................................................. 78 4.4. Discussion and conclusions ............................................................. 82 4.5. References........................................................................................ 84 5. Application to Living Cells ............................................................................ 87 5.1. Introduction...................................................................................... 87 5.2. Viscoelastic behaviour of the cytoplasm ......................................... 89 5.3. Materials and methods ..................................................................... 90 5.3.1. Magnetic tweezers ............................................................. 90 5.3.2. Magnetic probes ................................................................ 90 5.3.3. Inserting beads into cells ................................................... 91 5.3.4. Positioning cells between poles......................................... 92 5.4. Results.............................................................................................. 93 5.4.1. Long range movement ....................................................... 94 5.4.2. Isotropic viscoelastic properties ........................................ 94 5.4.3. Anisotropic viscoelastic properties.................................... 98 5.4.4. Force dependence of the viscosity................................... 100 5.4.5. Manipulation using smaller beads ................................... 101 5.5. Discussion and Conclusions .......................................................... 103 5.6. References...................................................................................... 106 iii Contents 6. Preliminary results inside the cell nucleus ................................................. 109 6.1. Introduction.................................................................................... 109 6.2. Materials and Methods................................................................... 109 6.2.1. Magnetic tweezers and probes......................................... 109 6.2.2. Cell preparation ............................................................... 109 6.2.3. Bead Injection
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