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Expansion Microscopy Protocol on Neuronal Cells An investigation of performing the protein- retention expansion microscopy protocol on neuronal cells ELIZA LINDQVIST Department of Applied Physics KTH Royal Institute of Technology Supervisor: Hans Blom Examiner: Erik Lindahl Master’s Thesis Stockholm, Sweden 2018 TRITA-SCI-GRU 2018:317 Department of Applied Physics School of Engineering Sciences Royal Institute of Technology Stockholm SWEDEN Examensarbete som med tillstånd av Kungliga Tekniska Högskolan främlägges till offentlig granskning för avläggande av Civilingenjörsexamen i Teknisk Fysik 4 augusti 2018 i Seminarierum Becquerel på SciLifeLab, Tomtebodavägen 23, Solna. © Eliza Lindqvist, 4 augusti 2018 Abstract Expansion microscopy (ExM) enables imaging of preserved cellular or tissue specimens with nanoscale resolution on diffraction-limited instead of super-resolution microscopes. On broad terms ExM works by physically enlarging the specimen, after having labeled it with fluorescent probes anchored to a swellable gel. In this Master Thesis work I present an investigation of the protein retention Expansion Microscopy (proExM) protocol for expansion of cultured neuronal cells. The expansion of neurological networks enables for example the ability to pinpoint small topological protein changes inside the brain, which could take affect during the development of diseases like Alzheimer’s, epilepsy and Parkinson’s disease. To evaluate the protein retention protocol I stained neuronal cells with different antibodies and I compared images of samples imaged with confocal, STED and Expansion Microscopy. I quantified the expansion factor in neurons by measuring of the distance between fixed architectural Spectrin rings. To evaluate retained protein content I varied the digestion times and anchoring treatments to study how different treatments affected the imaged intensity. Here, I show that samples anchored with Acryloyl X – SE lose a significant amount of protein with increased enzymatic digestion times. Furthermore, I show that samples anchored with Acryloyl X – SE are further affected by the digestion times as the fluorescently labelled sample lose imaged intensity over time. This is in sharp contrast to expanded samples anchored with MA-NHS which shows no imaged intensity decrease with longer enzymatic digestion times. Preface This is a Master’s Thesis in Engineering Physics at the Department of Cellular physics, Royal Institute of Technology (KTH), Stockholm, Sweden. The presented laboratory work was performed at Science for Life Laboratory in the Cellular Biophysics group belonging to the Department of Applied Physics, Solna, Sweden. Acknowledgments I want to thank everyone who has helped me throughout this master’s thesis. Extra gratitude goes to my supervisor Hans Blom and examiner Erik Lindahl, for initial project idea, guidance and support. Furthermore, I would like to give my sincere gratitude to group members Steven Edwards and David Unnersjö-Jess, you have been the best and supported me tremendously, I cannot thank you enough. Huge thanks go also to Daniel Jans for general help and custom microscopy setup and William Björnstjerna for illustrating figures used in this report. Eliza Lindqvist 2018-06-10 Stockholm, Sweden CONTENTS LIST OF FIGURES .................................................................................................................. 1 1. INTRODUCTION ............................................................................................................. 4 2. BACKGROUND AND THEORY ..................................................................................... 6 FLUORESCENCE ........................................................................................................................ 6 CONFOCAL MICROSCOPY.......................................................................................................... 7 SUPER-RESOLUTION IMAGING ................................................................................................... 8 EXPANSION MICROSCOPY ....................................................................................................... 10 PROCEDURE ........................................................................................................................... 10 VARIANTS OF EXM ................................................................................................................ 13 APPLICATION OF EXM TO NEUROSCIENCE ............................................................................... 15 3. EXPERIMENTAL WORK ............................................................................................. 17 PROEXM PROTOCOL ............................................................................................................... 17 SAMPLE PREPARATION TECHNIQUES ........................................................................................ 18 IMAGING ................................................................................................................................ 20 DATA ANALYSIS AND REPRESENTATIONS................................................................................. 20 4. RESULTS ........................................................................................................................ 22 STAINING ............................................................................................................................... 22 COMPARISON BETWEEN CONFOCAL, STED AND EXPANSION MICROSCOPY .............................. 24 RESOLVING THE PERIODIC EXPRESSION OF SPECTRIN ............................................................... 26 VARIATION OF THE DIGESTION TIME ........................................................................................ 27 A COMPARISON BETWEEN ACRYLOYL X -SE AND MA-NHS .................................................... 30 5. DISCUSSION .................................................................................................................. 33 6. SUPPLEMENTARY ....................................................................................................... 35 7. APPENDICES ................................................................................................................. 37 MATLAB SCRIPT .................................................................................................................. 37 8. REFERENCES ................................................................................................................ 38 LIST OF FIGURES Figure 1. Jablonski diagram. A Jabionski diagram demonstrating the electron transition states of the fluorophore during absorption/emission processes. (A) The energy of an incoming photon (green) is absorbed by the fluorophore molecule and the fluorophore reaches the excited state. (B) Some of the energy is dissipated as heat or other processes. (C) The fluorophore returns to the ground state and a photon of lower energy and longer wavelength is emitted (red). .................... 6 Figure 2. Confocal microscope principle. Simple illustration of the principal light pathways in a confocal microscopy. Light emitted by the laser passes through a pinhole aperture, is reflected by a dichromatic mirror and scanned across the specimen in a defined focal plane. Light emitted from the specimen (fluorescence) emanating from the focal plane passes back through the dichromatic mirror and is focused as a confocal point at the detector pinhole aperture. ................ 7 Figure 3. Confocal versus STED imaging. Non-expanded neurons treated with Spectrin. (LEFT) Confocal image. (RIGHT) STED image. ...................................................................... 9 Figure 4. Schematic of polyelectrolyte network. (A) Schematic of a collapsed polyelectrolyte network, showing crosslinker (dot) and polymer chain (line). (B) Expanded network after H2O dialysis. ..................................................................................................................................... 12 Figure 5. Schematic of microtubules and polymer network. First, the specimen is fixed and treated with compounds that bind to the biomolecules called anchoring treatment. Next, a hydrogel of densely crosslinked monomers is polymerized throughout the cells/tissue called gelation. Then, the specimen-hydrogel composite is digested and then the specimen is finally ready for the expansion by dialysis in water. (A) Schematic of microtubules (green) and polymer network (blue). (B) A label that can be anchored to the gel at site of a biomolecule, is hybridized to the oligo-bearing secondary antibody top bound via the primary to microtubules (green lines) and is incorporated into the gel (blue lines) via the methacryloyl group (red dot). ...................... 12 Figure 6. Membrane expansion and ExFISH. (LEFT) Maximum intensity projection of confocal microscopy stack following expansion of membrane labeled Brainbow3.0 neurons [2]. (RIGHT) ExFISH image with delivered probes against six RNA targets in a cultured HeLa cell, Scale bar 20 µm [9]. .................................................................................................................. 13 Figure 7. The box used for staining. ....................................................................................... 19 Figure 8. Gel chamber schematic............................................................................................ 19 Figure 9. A petri dish with a lid. A petri dish with an added lid was used as image sample holder for the gel. The added screws on top were applied
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