Microfluidic cryofixation for time-correlated live- imaging, cryo-fluorescence microscopy and electron microscopy of Caenorhabditis elegans Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program International Max Planck Research School “Physics of Biological and Complex Systems” of the Georg-August University School of Science (GAUSS) submitted by Giovanni Marco Nocera from Naples, Italy Göttingen, 2018 Thesis Committee Thomas Burg, PhD Max Planck Institute for Biophysical Chemistry, Göttingen Biological Micro- and Nanotechnology Prof. Dr. Sarah Köster Georg-August-University, Göttingen Institute for X-Ray Physics, Research Group Cellular Biophysics Prof. Dr. Stefan Hell Max Planck Institute for Biophysical Chemistry, Göttingen Dept. of NanoBiophotonics Members of the Examination Board Referee: Thomas Burg, PhD Max Planck Institute for Biophysical Chemistry, Göttingen Group of Biological Micro- and Nanotechnology 2nd Referee: Prof. Dr. Sarah Köster Georg-August-University, Göttingen Institute for X-Ray Physics, Group of Cellular Biophysics 3rd referee: Prof. Dr. Stefan Hell Max Planck Institute for Biophysical Chemistry, Göttingen Department of NanoBiophotonics Further members of the Examination Board Dr. Henrik Bringmann Max Planck Institute for Biophysical Chemistry, Göttingen Group of Sleep and Waking Prof. Dr. Silvio Rizzoli University Medical Center, Göttingen Department of Neuro- and Sensory Physiology Prof. Dr. Jörg Enderlein Georg-August-University, Göttingen Group of Single Molecule Spectroscopy and Imaging for Biophysics and Complex Systems Date of oral examination: October 15th, 2018 To Carmela, Giovanni and Maria Table of Content Abstract ................................................................................................................................ 1 Chapter 1 Introduction ........................................................................................................ 2 1. Motivation ...................................................................................................................................... 3 2. Light microscopy ........................................................................................................................... 3 2.1. Fluorescence microscopy at room temperature ................................................................... 3 2.2. Fluorescence microscopy at cryogenic temperature ............................................................ 4 3. Electron microscopy ..................................................................................................................... 7 4. Correlative microscopy ................................................................................................................. 8 4.1. Chemical fixation of biological samples ................................................................................ 9 4.2. Low temperature for the fixation of biological samples ..................................................... 9 4.3. Current technological limitations of cryofixation technology .......................................... 15 5. Microfluidics for cryofixation .................................................................................................... 15 6. Structure of the thesis ................................................................................................................. 18 Chapter 2 System design & Methods .................................................................................... 20 1. Microfluidic device for cryofixation ......................................................................................... 21 2. Microfluidic chip fabrication procedure .................................................................................. 27 3. Main heater fabrication procedure ............................................................................................ 30 3.1. Main heater with temperature sensors fabrication procedure .......................................... 33 4. Heat transfer maximization ....................................................................................................... 34 5. Assembly setup procedure ......................................................................................................... 37 6. Electronic controls of the setup for in situ cryofixation ........................................................ 38 7. Rhodamine B temperature dependence ................................................................................... 39 8. Rhodamine B bead measurements ........................................................................................... 39 9. Main heater calibration procedure ............................................................................................ 39 10. C. elegans sample preparation ................................................................................................. 40 11. Post-cryofixation sample transfer ......................................................................................... 40 12. Sample preparation and imaging for CLEM ...................................................................... 41 13. Cryoimmersion light microscopy of cryofixed C. elegans .................................................. 41 Chapter 3 Cryofixation of Caenorhabditis elegans within the field of view of a light microscope ......................................................................................................................... 46 1. C. elegans confinement in microfluidic channel ....................................................................... 47 2. Temperature control of the microfluidic channel enables cryofixation of living samples 49 3. Integration of cryofixation with live imaging .......................................................................... 51 4. Extent of ice formation analyzed by brightfield and DIC microscopy ............................... 52 Chapter 4 Cryofluorescence microscopy of Caenorhabditis elegans prepared by microfluidic cryofixation ................................................................................................... 57 1. Cryofixation preserves GCaMP fluorescence ......................................................................... 59 2. Immersion microscopy at cryogenic temperature .................................................................. 64 2.1. C. elegans shape is preserved after sample transfer ............................................................. 64 2.2. Image quality in confocal cryoimmersion microscopy ...................................................... 67 3. STED effects on cryofixed GCaMP and gut granules .......................................................... 69 Chapter 5 Electron microscopy of microfluidic cryofixed samples .................................. 74 1. Cryofixed samples are not thawed during chip recovery ...................................................... 75 2. Time-resolved light-electron microscopy workflow .............................................................. 77 3. Quality of the cryopreservation ................................................................................................. 79 4. Preserving spatial orientation of in situ cryofixed samples .................................................... 83 Chapter 6 Temperature measurement at the microscale ........................................................ 88 1. Design of resistive heater with built-in electronic temperature sensors ............................. 89 2. Visualization of temperature gradients within microfluidic channel volume ..................... 93 2.1. Effect of the solid matrix on the temperature coefficient of fluorescence of RhB- loaded melamine beads ........................................................................................................................ 96 Chapter 7 Conclusions and Outlook ................................................................................. 97 1. Limitations of cryofixation in microfluidic systems ............................................................... 98 2. Limitations of cryoimmersion setup....................................................................................... 101 Acknowledgements .......................................................................................................... 105 Recurring Abbreviations................................................................................................... 108 Bibliography ..................................................................................................................... 109 Appendix ........................................................................................................................... 114 Appendix I: Brief summary of experimental parameters ............................................................. 115 Abstract Light and electron microscopy are complementary methods to study biological systems at the cellular and sub-cellular scale. Light microscopy is compatible with live cells, allowing features of interest to be selectively marked with fluorescent molecules and followed in real time. Electron microscopy, in contrast, requires cells to be fixed. Cryofixation is a preferred method of fixation, since rapid freezing can preserve hydrated samples in a near-native state. However, state-of-the- art cryofixation systems require a transfer step that imposes a time lapse between the images