Durham E-Theses Development of enhanced multi-spot structured illumination microscopy with uorescence dierence WARD, EDWARD,NICHOLAS How to cite: WARD, EDWARD,NICHOLAS (2019) Development of enhanced multi-spot structured illumination microscopy with uorescence dierence, Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/13233/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk 2 Development of enhanced multi- spot structured illumination microscopy with fluorescence difference Edward N. Ward ii Abstract This project sets out to design and construct a novel super-resolution technique for biological imaging: enhanced multi-focal structured illumination microscopy; merging the fields of structured-illumination and point-spread function engineering. Computer simulations demonstrate the theoretical potential of this technique and suggest at least 1.4 times increase over existing structured illumination methods. Building on this, new pattern projection techniques based on holography are developed to project the required illumination patterns over an extended field of view. In addition, new techniques based on graphical- processor unit programming are developed for the post-processing and reconstruction of multi-focal structured illumination data. Finally, these techniques are tested in the imaging of a range of biological structures in both living and fixed cells. While the holographic projection and post-processing techniques proved successful, the gains achieved with enhanced multi-focal structured illumination microscopy were limited. While there is a measured gain in resolution – and a potential improvement in depth sectioning – these advantages are not apparent on all structures. Finally, the relative merits of the techniques over existing methods are discussed and potential future directions are suggested. iii Declaration The work described herein was undertaken at the Department of Chemistry, Durham University, between September 2015 and November 2018. All of the work is my own, except where specifically stated otherwise. No part has previously been submitted for a degree at this or any other university. Statement of copyright The copyright of this thesis rests with the author. No quotation from it should be published without the author’s prior written consent and information derived from it should be acknowledged. iv Contents Abstract ....................................................................................................................... ii Declaration ................................................................................................................. iii Statement of copyright .............................................................................................. iii List of figures and tables ......................................................................................... vii Abbreviations used ..................................................................................................... x Acknowledgements ................................................................................................... xii 1. Optical microscopy................................................................................................. 7 1.1 Introduction ..................................................................................................... 7 1.1.1 Fluorescence microscopy.............................................................................. 8 1.2 Breaking the diffraction barrier ..................................................................... 18 1.2.1 STED microscopy ...................................................................................... 19 1.2.2 Single-molecule imaging ............................................................................ 23 1.3 Motivation for this work ................................................................................ 27 2. Structured-illumination microscopy .................................................................. 28 2.1 Introduction ................................................................................................... 28 2.2 Image reconstruction for structured illumination microscopy ...................... 30 2.3 Advances in structured illumination microscopy .......................................... 31 2.3.1 Increasing axial resolution in structured illumination microscopy ............ 31 2.3.2 Increasing lateral resolution in structured illumination microscopy .......... 33 2.4 Image scanning microscopy .......................................................................... 36 2.5 Multi-focal Structured Illumination Microscopy .......................................... 38 2.5.1 Image reconstruction in multi-spot structured-illumination microscopy ... 39 2.5.2 Pattern-illuminated Fourier Ptychography ................................................. 43 2.5.3 Joint Richardson-Lucy Deconvolution ....................................................... 44 2.6 Summary ........................................................................................................ 45 3. Enhanced multi-spot structured illumination microscopy ............................... 46 3.1 Introduction ................................................................................................... 46 3.1.1 Point-spread function engineering .............................................................. 46 3.1.2 Difference microscopy ............................................................................... 49 3.1.3 Theory of enhanced multi-spot structured illumination microscopy ......... 50 3.2 Simulated eMSIM imaging ........................................................................... 52 v 3.2.1 Defining the point-spread function ............................................................. 52 3.2.2 Building excitation patterns ........................................................................ 53 3.2.3 Building model data ................................................................................... 53 3.2.4 Recovering super-resolution image ............................................................ 54 3.3 Results ........................................................................................................... 57 3.3.1 Reconstruction techniques .......................................................................... 59 3.3.2 Resolution improvement in eMSIM ........................................................... 60 3.4 Summary ........................................................................................................ 66 4. Holography ........................................................................................................... 68 4.1 Introduction ................................................................................................... 68 4.2 Hologram calculation .................................................................................... 70 4.2.1 Superposition of gratings and lenses .......................................................... 70 4.2.2 Iterative Fourier transform algorithms ....................................................... 72 4.2.3 Direct search algorithms ............................................................................. 77 4.3 Aberration correction ..................................................................................... 80 4.3.1 Background ................................................................................................. 80 4.3.2 Global phase correction .............................................................................. 82 4.4 Location-dependent correction of aberrations ............................................... 85 4.5 Holography in MSIM and eMSIM ................................................................ 90 4.6 Summary ........................................................................................................ 91 5. Optical methods in holographic projection microscopy ................................... 92 5.1 Pattern projection ........................................................................................... 92 5.1.1 Spatial light modulators .............................................................................. 92 5.1.2 Amplitude modulation ................................................................................ 92 5.1.3 Phase modulation ........................................................................................ 95 5.2 Optical setup .................................................................................................. 98 5.2.1 Excitation light source ................................................................................ 98 5.2.2 Holographic projection optics .................................................................. 100 5.2.3