Theses and Dissertations at Montana State University (MSU)
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Item Improving the two-photon absorption properties of fluorescent proteins for neuroscience(Montana State University - Bozeman, College of Letters & Science, 2020) Molina, Rosana Sophia; Chairperson, Graduate Committee: Thomas Hughes; Yong Qian, Jiahui Wu, Yi Shen, Robert E. Campbell, Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Understanding the fluorescence change in red genetically encoded calcium ion indicators' in the journal 'Biophysical Journal' which is contained within this dissertation.; Tam M. Tran, Robert E. Campbell, Gerard G. Lambert, Anya Salih, Nathan C. Shaner, Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Blue-shifted green fluorescent protein homologues are brighter than enhanced green fluorescent protein under two-photon excitation' in the journal 'The Journal of physical chemistry letters' which is contained within this dissertation.; Jonathan King, Jacob Franklin, Nathan Clack, Christopher McRaven, Vasily Goncharov, Daniel Flickinger, Karel Svoboda, Mikhail Drobizhev, Thomas E. Hughes were co-authors of the article, 'An instrument to optimize fluorescent proteins for two-photon excitation' which is contained within this dissertation.Untangling the intricacies of the brain requires innovative tools that power basic research. Fluorescent proteins, first discovered in jellyfish, provide a genetically encodable way to light up the brains of animal models such as mice and fruit flies. They have been made into biosensors that change fluorescence in response to markers of neural activity such as calcium ions (Ca 2+). To visualize them, neuroscientists take advantage of two-photon excitation microscopy, a specialized type of imaging that can reveal crisp fluorescence images deep in the brain. Fluorescent proteins behave differently under twophoton excitation compared to one-photon excitation. Their inherent two-photon properties, namely brightness and peak absorption wavelength, limit the scope of possible experiments to investigate the brain. This work aims to understand and improve these properties through three projects: characterizing a set of red fluorescent protein-based Ca 2+ indicators; finding two-photon brighter green fluorescent proteins; and developing an instrument to screen for improved fluorescent proteins for two-photon microscopy. Analyzing nine red Ca 2+ indicators shows that they can be separated into three classes based on how their properties change in a Ca 2+-dependent manner. In one of these classes, the relative changes in one-photon properties are different from the changes in two-photon properties. In addition to characterizing, identifying and directly improving fluorescent proteins for enhanced two-photon properties is important. Presented here is a physical model of the light-absorbing molecule within the green fluorescent protein (the chromophore). The model predicts that green fluorescent proteins absorbing at higher energy wavelengths will be brighter under two-photon excitation. This proves to be the case for 12 blueshifted green fluorescent proteins, which are up to 2.5 times brighter than the commonly used Enhanced Green Fluorescent Protein. A way to directly improve fluorescent proteins is through directed evolution, but screening under two-photon excitation is a challenge. An instrument, called the GIZMO, solves this challenge and can evolve fluorescent proteins expressed in E. coli colonies under two-photon excitation. These results pave the way for better two-photon fluorescent protein-based tools for neuroscience.Item If you build it, they will come: engineering the next generation of optical tools to image neural activity deep within the living brain(Montana State University - Bozeman, College of Letters & Science, 2017) Barnett, Lauren Marie; Chairperson, Graduate Committee: Thomas Hughes; Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Deciphering the molecular mechanism responsible for GCAMP6M's Ca 2+ dependent change in fluorescence' in the journal 'PLoSONE' which is contained within this thesis.; Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Making pKa-altering mutations in GCAMP6M changes the Ca 2+-dependent fluorescence response' submitted to the journal 'PLoSONE' which is contained within this thesis.; Jelena Platisa, Marko Popovic, Vincent A. Pieribone and Thomas Hughes were co-authors of the article, 'A fluorescent, genetically-encoded voltage probe capable of resolving action potentials' in the journal 'PLoSONE' which is contained within this thesis.; Lauren M. Barnett, Mikhail Drobizhev, Geoffrey Wicks, Alexander Mikhaylov, Thomas E. Hughes and Aleksander Rebane were co-authors of the article, 'Two-photon directed evolution of green fluorescent proteins' in the journal 'Nature Scientific Reports' which is contained within this thesis.To see the activity of large, integrated neural circuits functioning in real-time inside of a living brain, neuroscientists will need multiple genetically-encoded fluorescent activity sensors that can be individually targeted to specific cell types, are fast enough to resolve multiple action potentials, can be distinguished from one another and imaged deep within the brain. The goal of this work is to better understand and improve upon the most recent generations of genetically-encoded Ca 2+ and voltage sensors, and to expand biosensor utility in two-photon excitation, which will be necessary to image neural activity deep within the brain. Genetically-encoded Ca 2+ sensors measure the intracellular Ca 2+ release that occurs downstream of an action potential. The GCaMP6 series are the best Ca 2+ sensors available, however little is known about how they work. Measurements of four different states in GCaMP6m reveal that its large Ca 2+-dependent change in 470 nm excited fluorescence is due to a redistribution of the chromophore protonation state, from a neutral form excited at ~400 nm to an anionic form excited at ~470 nm, via a change in pK a. Making pK a-altering mutations in GCaMP6m changes the Ca 2+-dependent fluorescence response. This highlights the importance of Delta pK a and identifies key amino acid positions that will be important for improving GCaMP6m and GCaMP-like biosensors. A direct readout of an action potential would be ideal for capturing complex signal transduction in the brain. This will require a bright, fast voltage sensor. ElectricPk is the first genetically-encoded voltage sensor with a fluorescence response fast enough to resolve multiple action potentials in mammalian neurons. This design indicates it is possible to couple a fluorescence change with a very fast (~1 ms) voltage-dependent movement in the Ciona intestinalis voltage-sensitive phosphatase protein. Whether imaging a downstream Ca 2+ signal or a direct change in membrane potential, to image neuronal activity in deep brain tissue biosensors will need to be brightly fluorescent in two-photon excitation. The two-photon directed evolution of green fluorescent proteins presented here is a proof-of-principle design that shows a high-throughput screen focused on improving the two-photon properties of a fluorescent protein is possible.