Development of Optical Trapping Techniques for in Vivo Investigations
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Development of optical trapping techniques for in vivo investigations Andrew C. Richardson Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Supervisor: Lene Oddershede Optical Tweezers Group The Niels Bohr Institute University of Copenhagen Denmark 31st of December 2008 I hereby declare that the content of this thesis is entirely my own work and contains no material which has been accepted for the award of any other degree or diploma at any University. The thesis comprises a discussion and summary of the work I carried out between September 2005 and September 2008 at the Niels Bohr institute as part of my PhD studies. To the best of my knowledge and belief, this dissertation contains no material previously published or written by another person, except where due reference has been made. —— The image on the front cover is of a ’Golden apple’ which is one of three such apples that levitate from the fountain at Gammeltorv in the centre of Copenhagen, on occasions such as the Queens birthday and Denmark’s constitutional day. Although the golden apples are not confined by optical forces like those used to trap gold nano-particles, it is a jovial and light hearted comparison to the microscopic equivalent. ii Contents Preface ix List of abbreviations xiii 1 Optical Trapping 1 1.1 Abriefhistoryofopticaltrapping . ..... 1 1.1.1 Opticaltweezing ............................. 2 1.2 Forcesinopticaltrapping . ... 3 1.2.1 Mieregime ................................ 3 1.2.2 Rayleighregime ............................. 6 1.2.3 Absorption ................................ 7 1.2.4 Intermediateregime ........................... 8 1.3 Calibration .................................... 8 1.3.1 Opticaltweezerssetup . 9 1.3.2 Calibration by power spectral analysis . ..... 10 1.3.3 Calibration by viscous drag force . 12 1.3.4 Comparison of power spectral analysis and drag force calibration . 12 1.4 Summary ..................................... 13 2 An investigation of optical trapping potential by the suppression of spher- ical aberrations 15 2.1 Overview ..................................... 15 2.1.1 Previousexperimentalwork . 16 2.1.2 Theoretical predictions . 19 2.2 Characterising the optical trapping potential . .......... 20 2.2.1 Experimentalsetup............................ 20 2.2.2 Compensation for spherical aberration . ..... 22 2.2.3 Experimentalprocedure . 23 2.2.4 Image optimisation and image analysis using LABview . ....... 27 2.2.5 Pixelcalibration ............................. 28 iii Contents 2.3 Resultsanddiscussion . .. .. .. .. .. .. .. .. 30 2.3.1 Increasedaxialconfinement . 30 2.3.2 Forcedisplacementprofile . 32 2.4 Sizedependency ................................. 35 2.4.1 Roleofthesidelobes .......................... 35 2.5 Summary ..................................... 36 3 Micro-injection of S. pombe yeast cells 39 3.1 Background.................................... 39 3.2 Overview ..................................... 40 3.3 Why use S. pombe ................................ 41 3.4 Micro-injection of S. pombe ........................... 42 3.4.1 Preparationofyeast ........................... 42 3.4.2 Selectionofaprobe ........................... 44 3.4.3 Micro-injection .............................. 45 3.4.4 Regeneration ............................... 49 3.5 Alternative methods of probe transfer to S.pombe ............... 50 3.5.1 Simple lipid assisted micro-injection (SLAM) . ....... 50 3.5.2 Vesiclefusion ............................... 50 3.6 Summary ..................................... 52 4 Calibration of optical tweezers in complex viscoelastic media 53 4.1 Overview ..................................... 53 4.2 Brief theoretical introduction . ...... 54 4.2.1 The Fluctuation dissipation theorem (FDT) calibration method . 54 4.2.2 Selecting the appropriate amplitude and frequency of stage driving . 56 4.3 Experimentalprocedure . 57 4.3.1 Passivemeasurements . .. .. .. .. .. .. .. 59 4.3.2 Activemeasurements. .. .. .. .. .. .. .. 60 4.3.3 Determining the positional calibration factor or beta value . 61 4.3.4 Pixelcalibration ............................. 62 4.3.5 Calibration of the channel delay from the input/output (data acqui- sition)card ................................ 62 4.4 Dataanalysis ................................... 63 4.5 Resultsanddiscussion . .. .. .. .. .. .. .. .. 63 4.5.1 CalibrationinWater........................... 63 4.5.2 Calibration in Entangled F-actin . 65 4.6 Summary ..................................... 67 iv Contents 5 Confocal-tweezers 69 5.1 Overview ..................................... 69 5.2 Confocalmicroscopy .............................. 70 5.3 Previous research in confocal tweezing . ....... 71 5.4 Integration of the tweezers . 73 5.4.1 2Dscanning................................ 73 5.4.2 Coupling the trapping laser into the optical path of the SP5 confocal system................................... 74 5.5 Experimentalmethod .............................. 75 5.6 Resultanddiscussion ............................. 76 5.7 Futurework.................................... 78 5.7.1 3Dscanning................................ 78 5.8 Summary ..................................... 79 6 Three dimensional optical control of individual quantum dots 81 6.1 Overview ..................................... 81 6.2 Whatarequantumdots? ............................ 82 6.3 Earlyexperiments ................................ 84 6.3.1 Trappingquantumdots .. .. .. .. .. .. .. 84 6.3.2 Visualisingquantumdots . 85 6.4 Improvements to the experimental setup and procedure . ......... 87 6.4.1 TheEMCCDalternative. 87 6.4.2 Bufferoptimisation............................ 89 6.4.3 Reducing the axial movement of the quantum dot by compensating forsphericalaberrations . 89 6.5 Experimentalprocedure . 89 6.5.1 Pressing a trapped quantum dot onto the lower glass surface of the sample, under high salt concentrations . 89 6.5.2 Using Streptavidin coated quantum dots for specific binding to a bi- otincoatedglasssurface. 91 6.6 Results....................................... 91 6.6.1 Trapping of individual quantum dots; a statistical argument . 91 6.6.2 Trapping of individual quantum dots; visual verification by blinking 92 6.6.3 Optical trap stiffness, κ,ofatrappedQD ............... 93 6.6.4 The inferred properties of a quantum dot . 95 6.7 Futureexperiments............................... 99 6.8 Summary ..................................... 100 Conclusions & outlook 101 v Contents Bibliography 101 vi CONTENTS List of Publications • A.C. Richardson, S.N.S. Reihani, and L.B. Oddershede. ”Combining confocal mi- croscopy with precise force-scope optical tweezers”. Proceedings of SPIE, 6326, pp 28.1-28.10, (2006). • A.C. Richardson, S.N.S. Reihani, and L.B. Oddershede. ”Extending the lateral trap- ping force of optical tweezers”. Proceedings of SPIE, 6644, pp 20.1-20.9, (2007). • M. Fischer, A.C. Richardson, S.N.S. Reihani, L.B. Oddershede, and K. Berg-Sørensen. ”Validation of FDT calibration method in complex media”. Proceeding of SPIE, 7038, pp 22.1-22.12, (2008). • A.C. Richardson, S.N.S. Reihani, and L.B. Oddershede. ”Non-harmonic potential of a single beam optical trap”. Optics Express, 16, pp 15709-15717, (2008). • L. Jauffred, A.C. Richardson, and L.B. Oddershede. ”Three-dimensional optical con- trol of individual quantum dots”. Nano Letters, 8, pp 3376-3380, (2008). • A.C. Richardson, M. Fischer, S.N.S. Reihani, K. Berg-Sørensen, and L.B. Oddershede. ”How to calibrate optical tweezers in viscoelastic media”. (Submitted). • A.C. Richardson, P.M. Hansen, and L.B. Oddershede. ”Micro-injection of S.pombe yeast cells”. (in progress). vii Preface From as early as Egyptian times, mankind has endeavoured to understand the things around it; the changing of the seasons and the phases of the moon are to name but a few of the early wanderings of our curious ancestors minds. Physics has played a dominant role in describing the physical laws and mechanisms by which our universe is governed. How- ever, understanding mankind and the processes ongoing within our own bodies had, until relatively recently, been left to biologists. The evolution of the field of biophysics has been rapid in recent years, largely because there was a need for it. Biophysicists approach biological problems from a very different perspective to that of biologists and this change in thinking has led to new insights into the mechanisms and processes occurring within cells [1]. There is no shortage of areas where long standing questions of biological function exist; cell rheology, cell motility, and cell mechanics are just a few of the areas where numerous questions remain unanswered, making biophysics an exciting area of research to work in. The interdisciplinary nature of biophysics brings together the experimental techniques of two quite different disciplines albeit often bound together by the same common tool, the microscope. Optical tweezers are one of the tools used to explore this new cellular environment and can be incorporated into a microscope with relative ease. Optical tweezers are a powerful tool with which to investigate single molecule interactions; the motion of molecular motors, the interaction of proteins and the dynamics of DNA are all areas where optical tweezers have made a significant contribution towards understanding how these biologically relevant molecules function. An interesting topic for biologists is cell mechanics. The polymerisation and de-ploymerisation of microtubules, as well as the role of actin in cell motility, have