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Publication

Photochromism-enabled quantitative biosensing using rsEGFP-based calcium indicators

Book - Dissertation

Fluorescence microscopy techniques are indispensable research tools that have enabled live cell imaging with low invasiveness, high sensitivity, and high spatiotemporal resolution. As a result, fluorescence microscopy has revolutionized our understanding of the inner workings of single cells and whole organisms. An integral part of the imaging performance is the fluorescent labels, as the quality of the labels influences the quality of the data. Therefore, researchers have continuously focused on the development of new and improved fluorophores. This is also true for fluorescent proteins (FPs), the genetically encoded labels that allow highly specific targeting, labeling and tracking in living cells. In the last decades, the toolbox of available FP variants has been expanded substantially, through the discovery of new FPs in various marine organisms, and by the engineering of existing FPs. An intriguing addition to this toolbox are the phototransformable FPs, which can undergo a photoinduced transformation that changes their spectral properties. These photophysically "smart" FPs are often relied upon for advanced imaging schemes, such as super-resolution fluorescence microscopy, single particle tracking or dynamic labeling. One example of phototransformable FPs are the reversibly switchable, or photochromic, FPs. Another impressive feat of FP technology is the development of FP-based biosensors, which change their fluorescence properties in response to a biological stimulus - such as enzymatic activity or changing ion concentrations. These molecules allow the dynamic observation of processes as they occur within the live cell, as has been done highly successfully for e.g. calcium (Ca2+) signaling. However, the combination of photophysically "smart" FPs and biosensors is an area of new and unexplored possibilities for super-resolution microscopy, optical highlighting and ratiometric imaging. The work presented here describes the development of novel and improved genetically encoded fluorescent reporters with expanded functionalities. Central to this work is the development of reversibly switchable genetically encoded calcium indicators (rsGECIs) by combining two concepts of FP technology: the photochromic behavior found in reversibly switchable FPs (RSFPs) and the FP-based calcium indicators used to visualize changing calcium concentrations. In the first chapter, I will present the research context and introduce the most important basic concepts and techniques regarding the work that follows. The next two chapters focus on my contributions to the improvement of well-known FPs for super-resolution fluorescence microscopy. In Chapter 2, I focus on the directed evolution of rsEGFP and describe the development and characterization of a family of improved-folding variants. These variants, the rsGreens, are applied in high-resolution imaging techniques such as pcSOFI and RESOLFT. In Chapter 3, I discuss how the non-phototransformable red fluorescent protein mCherry can be used to perform single-molecule localization microscopy via a chemically-induced blue-fluorescent state. Chapter 4 describes the first approach I used to create rsGECIs. Starting from two RSFPs with excellent photochromic behavior, rsGreen0.7 and Dronpa, I generated a series of structural variants of these FPs and integrated them into a GCaMP backbone. However, this strategy did not result in well-working probes. Chapter 5 discusses my efforts to develop new rsGECIs using a second strategy. Here, I started from the GCaMP family of calcium indicators and used rational mutagenesis to improve the photochromism in GCaMP. This resulted in the identification of a novel photochromic GCaMP mutant, which I used to determine the dynamically changing calcium concentrations in live cells. To achieve this, we also developed an advanced imaging scheme based on the calcium-dependent phototransformation of this new fluorescent indicator. This is followed by the final chapter, where I give the conclusions that could be drawn from this work and present future perspectives.
Publication year:2021
Accessibility:Open