Micro-optics for Opto-genetic Neuro- stimulation with Micro-LED Arrays Lionel Chaudet Photonics Group Department of Physics Imperial College London Supervised by Pr. Mark Neil 2014 Thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) Imperial College of Science, Technology and Medicine Abstract The breakthrough discovery of a nanoscale optically gated ion channel protein, Channelrhodopsin 2 (ChR2), in combination with a genetically expressed optically activated ion pump, Halorhodopsin, allowed the direct stimulation and inhibition of individual action potentials with light alone. This thesis describes the development of optics and micro-optics which when used with micro-led array sources, collects and projects light efficiently and uniformly onto such opto- genetically modified specimens. When used with enhanced light gated ion channels and pumps these systems allow us to further our understanding of both brain and visual systems. Micro-LED arrays (MLA) permit spatio-temporal control of neuron stimulation on sub-millisecond timescales. However, micro-led arrays are disadvantaged by the broad-angular spread of their light emission and their low spatial fill factor. We present the design of macro and micro-optics systems for use with a micro-LED  arrays  consisting  of  a  matrix  of  25μm  diameter  micro-LEDs with 150 or 80μm centre-to-centre spacing. On one system, the micro-LED array is imaged onto off-the-shelf micro-optics using macro-optics and in the other system; micro-LED array and custom micro-optics are optimised and integrated together. The two systems are designed to improve the fill-factor from 2% to more than 78% by capturing a larger fraction of the LED emission and directing it correctly to the sample plane. This approach allows low fill factor arrays to be used effectively, which in turn has benefits in terms of thermal management and electrical drive from CMOS backplane electronics. These systems were implemented as an independent set that could be connected to a variety of different microscopes available for patch-clamp and multi-electrode measurements. In addition, the feasibility of an eye prosthesis was tested using virtual reality optics and a fake eye to stimulate ganglion cells and a system including an integrated fundus camera was constructed for in-vivo stimulation of the genetically modified retina of a mouse. 1 Acknowledgement This thesis has only been possible with the support of many people whom I individually thank here. I would like to thank my supervisor, Professor Mark Neil, for his patience, encouragement and time given in preparation of this thesis. I wish to thank the people I worked with directly on this project, Dr Patrick Degeenar, Dr Rolando Berlinguer-Palmini, Dr Kamyar Mehran, John Barret and Na Dong from Newcastle Universty, Pleun Maaskant from Tyndall, Dr Christian Bamann from MPI, Dasha Nelidova and Daniel Hillier from FMI, and Dr Peter Lanigan and Angelos Loukidis from Scientifica, for their ideas and will to reach a common goal by using their skills and keeping an open mind to others’ ideas. I am also grateful to everyone who helped develop my ideas, discussed, commented on and read my work. I thank my research group at Imperial College London. Special thanks are due to all my fellow colleagues for giving me their valuable time and resources: Lionel Fafchamps, James Clegg, Douglass Kelly, Sean Warren, Hugh Sparks, Sunil Kumar, Hugo Sinclair, Vincent Maioli. This work could also not have been completed without the competences of the mechanical and optical workshop, Simon Johnson, Martin Kehoe and Melvyn Patmore. Thanks also to my family and Isabel for their love, and particularly Isabel for patiently reading and correcting my thesis. Anyone I may have missed in the acknowledgement is also thanked. 2 Author declaration All the work presented in this thesis is my own with the following exceptions: (cid:120) The micro-LED arrays described in chapters 2 to 7 were developed and fabricated by Tyndall in Ireland. (cid:120) The development of the micro-LED array CMOS backplane, electronic associated with it and software to drive the micro-LED array was undertaken in the Department of Electrical Engineering, Newcastle University (Chapter 5 and 8). (cid:120) One set of opto-mechanics was designed in conjunction with Scientifica (Chapter 6). (cid:120) The biological experiments were carried out in conjunction with the Max-Plank Institut (MPI) for Biophysics, Frankfurt (Germany), Department of Electrical Engineering, Newcastle University (United Kingdom) and at Friedrich Miller Institute (FMI), Basel (Switzerland). ‘The  copyright  of  this  thesis  rests  with  the  author  and  is  made  available  under  a  Creative  Commons   Attribution Non-Commercial No Derivative licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must clear to others the licence terms of  this  work’   3 To my wife and to my brother 4 Table of Contents Abstract ................................................................................................................................. 1 Acknowledgement ....................................................................................................................... 2 Author declaration ....................................................................................................................... 3 Table of Contents ......................................................................................................................... 5 List of figures ............................................................................................................................... 8 List of tables .............................................................................................................................. 14 List of abbreviations ................................................................................................................... 15 Chapter 1: Introduction ............................................................................................................ 16 Thesis outline .................................................................................................................................... 18 Chapter 2: Optogenetics .......................................................................................................... 20 2.1 Introduction ............................................................................................................................. 20 2.2 Neurons ................................................................................................................................... 20 2.3 The visual sensory system ....................................................................................................... 23 2.4 Control tools ............................................................................................................................ 24 2.5 Targeting the cells of interest.................................................................................................. 32 2.6 Light delivery ........................................................................................................................... 33 2.7 Reading the outcome .............................................................................................................. 37 2.8 Conclusions .............................................................................................................................. 41 Chapter 3: Technology and principle ........................................................................................ 42 3.1 Principle of the project Optoneuro ......................................................................................... 42 3.2 Micro-LED arrays (MLA) .......................................................................................................... 44 3.3 Micro-optics ............................................................................................................................ 64 3.4 Conclusions .............................................................................................................................. 69 Chapter 4: Projection optics with off-the-shelf micro-optics: Basic design principle ................... 70 4.1 Projection optics principle with off-the-shelf MO................................................................... 70 4.2 Relay optics (ROs): Theory and proof of principle................................................................... 74 5 4.3 Conclusion ............................................................................................................................... 89 Chapter 5: Design  and  implementation  of  the  basic  design:  “PO  1” ........................................... 91 5.1 Off the shelf RO design ............................................................................................................ 91 5.2 In-house optical design of ROs ................................................................................................ 94 5.3 Testing  of  “PO  1” ................................................................................................................... 110 5.4 Conclusion ............................................................................................................................. 116 Chapter 6: Advanced  design  and  implementation:  “PO  2” ....................................................... 117 6.1 Design .................................................................................................................................... 117 6.2 Focus and magnification adjustment .................................................................................... 125 6.3 Testing ................................................................................................................................... 129 6.4 Opto-mechanical design ........................................................................................................ 132 6.5 Conclusion ............................................................................................................................. 136 Chapter 7: Custom micro-optics development for direct integration onto micro-LED arrays ..... 138 7.1 Principle and Requirements .................................................................................................. 138 7.2 Micro-lens array fabrication .................................................................................................. 143 7.3 Modelling............................................................................................................................... 144 7.4 Integration onto the MLA ...................................................................................................... 170 7.5 Conclusion ............................................................................................................................. 171 Chapter 8: Biological experiments .......................................................................................... 173 8.1 MPI experiments: Stimulating cultured cells in a conventional microscope ........................ 174 8.2 Newcastle University: MEA measurements and virtual reality (VR) optics .......................... 179 8.3 In-vivo stimulation of a living mouse retina .......................................................................... 186 8.4 Conclusion ............................................................................................................................. 195 Chapter 9: Conclusion and future work................................................................................... 196 Publications/ Presentations ..................................................................................................... 202 Bibliography ............................................................................................................................ 203 Appendices 213 A.1 Optical set-ups estimation spreadsheet ............................................................................... 213 6 A.2 Micro-lens arrays parameters ............................................................................................... 219 A.3 Cost of the RO studied and developed ................................................................................. 221 A.4 Abbe Diagram n -v ............................................................................................................... 221 d d A.5 Demonstration of the aplanatic condition ............................................................................ 222 A.6 Projection optics PO 2 detailed parameters ......................................................................... 223 A.7 LED Material characteristics .................................................................................................. 224 A.8 MO characteristics for different magnifications and tube lenses used for microscopes with 150  and  80μm  diameter ................................................................................................. 225 A.9 Equation to calculate the optimum thickness for each material for the MLA/MO system .. 226 A.10 Dispersion diagrams for PET and D263 (Schott) ................................................................. 229 A.11 Modelling  results  for  the  80μm  pitch  GaN  MLA  and  MO ................................................... 229 A.12 Modelling  results  for  the  80μm  pitch  GaN/Al O MLA and MO ......................................... 231 2 3 A.13 Track length and pupil shift calculation study .................................................................... 233 A.14 Summary of permission for third party copyright works ................................................... 236 A.15 Permissions for third party copyright works ...................................................................... 239 7 List of figures Figure 2-1: Neuron (nerve cell) schematic ............................................................................................ 21 Figure 2-2: Description of the important steps of an action potential happening at the membrane of a neuron ................................................................................................................................................ 22 Figure 2-3: Schematic of the eye and of a cross section of the retina .................................................. 23 Figure 2-4: Degeneration of the retina in a rat model of retinitis pigmentosa[17] .............................. 24 Figure 2-5: Optogenetic tools for modulating membrane potentials .................................................. 26 Figure 2-6: Activation cycle of a G-protein by a G-protein-coupled receptor receiving a signaling molecule [27] ........................................................................................................................................ 28 Figure 2-7: Photocurrent amplitude depends on the wavelength of light[38]. ................................... 31 Figure 2-8: Patterned illumination strategies [47]. ............................................................................... 34 Figure 2-9: Patch-clamping  recording  configurations  schematics  [30]: ............................................... 38 Figure 3-1: Illumination with no projection optics ............................................................................... 42 Figure 3-2: Illumination principle with micro-optics ............................................................................. 43 Figure 3-3: Inner working of a LED showing circuit ((a) and (b)) and band diagram (c) when under bias .............................................................................................................................................................. 45 Figure 3-4: Band gap and lattice constant of some compound semiconductors [81]. ......................... 46 Figure 3-5: LED structures[30] .............................................................................................................. 49 Figure 3-6: Polar plot using Lambert's law and the Snell's/Descartes Law for a flat emitter ............... 50 Figure 3-7: Schematics of Tyndall National institute LED ..................................................................... 51 Figure 3-8: Characteristics of on-sample illumination [60] ................................................................... 53 Figure 3-9: Dendritic excitation [60] ..................................................................................................... 53 Figure 3-10: Spectrally weighted irradiances and their relationship with thermal and photochemical safety margins. ...................................................................................................................................... 54 Figure 3-11: 16 by 16 MLA .................................................................................................................... 55 Figure 3-12: MLA emitter design schematics ........................................................................................ 56 Figure 3-13: MLA designs with 16 by 16 LEDs and 150μm pitch .......................................................... 57 Figure 3-14: 90 by 90 MLA with 80μm pitch ......................................................................................... 57 Figure 3-15: Geometric diagram of 1/ a 25μm diameter single emitter, 2/ a 90μm diameter single emitter and 3/ a cluster of 14 emitters 20μm each .............................................................................. 58 Figure 3-16: Variation of the collection efficiency with the magnification of the microscope objective and two types of light emission for 4x (0.16NA), 10x (0.3NA), 20x (0.4NA), 40x (0.8NA) and 60x (1NA) microscope objectives .......................................................................................................................... 59 8 Figure 3-17: Schematic of the MLA coupled with the MO by relay optics ........................................... 60 Figure 3-18: Variation of the collection efficiency with the size of the emitters and the type of emission ................................................................................................................................................ 62 Figure 3-19: Variation of the collection efficiency with micro-optics for Tyndall's MLAs for 4x (0.16NA), 10x (0.3NA), 20x (0.4NA), 40x (0.8NA) and 60x (1NA) microscope objectives..................... 63 Figure 3-20: The micro-lens techniques and their development timeline[102] ................................... 66 Figure 3-21: Micro-lens array manufactured by multi-step photolithographic process. ..................... 67 Figure 3-22: Micro-lens arrays made using a replication process at Glyndwr Innovations [108] ((a)) and at RPC Photonics [109] ((b)) ........................................................................................................... 67 Figure 4-1: Photography of micro-lens arrays with a microscope under white light: .......................... 71 Figure 4-2: Projection optics principle .................................................................................................. 72 Figure 4-3: Light spot on the cell body.................................................................................................. 73 Figure 4-4: Schematic of a simple optical telecentric relay connected to a microscope ..................... 74 Figure 4-5: Proof of principle: Experimental set-up.............................................................................. 75 Figure 4-6: Proof of principle: imaging ................................................................................................. 76 Figure 4-7: Proof of principle: Collection efficiencies measurement ................................................... 77 Figure 4-8: Technique to measure the quality of the imaging of the MLA with ROs ........................... 79 Figure 4-9: Transverse ray fan plots of the RO used within the proof of principle set-up ................... 80 Figure 4-10: MTF plot for a perfect circular lens .................................................................................. 81 Figure 4-11: MTF of the relay optics used within the proof of principle set-up ................................... 82 Figure 4-12: Schematic of the angular full field of view ....................................................................... 84 Figure 4-13: Map showing design types commonly used for various combinations of aperture and the field of view [128] ................................................................................................................................. 85 Figure 4-14: Aplanatic meniscus lens with index of refraction n .......................................................... 87 Figure 5-1: Sill Optics Relay Solution Modelling Results: Spot Diagram ............................................... 92 Figure 5-2: Sill Optics Relay Solution Modelling Results: Ray Fan ........................................................ 93 Figure 5-3: Sill Optics Relay Solution Modelling Results: MTF .............................................................. 93 Figure 5-4: Sill Optics Relay Solution Modelling Results: Chromatic Focal Shift................................... 94 Figure 5-5: Schematic of an achromatic doublet (f = 60mm) without and with an aplanatic-normal meniscus lens ........................................................................................................................................ 96 Figure 5-6: Ray diagrams for the f = 60mm achromatic doublet at 0, 1.2 and 1.7mm from the center .............................................................................................................................................................. 96 Figure 5-7: Ray diagrams of the f = 60mm achromatic doublet coupled with an aplanatic-normal meniscus lens at 0, 1.2 and 1.7mm from the center ............................................................................ 97 9