Evolutionary engineering of green fluorescent protein calcium biosensors

Neurobiology continues to be one of the great frontiers in biological sciences. The number of neurons in the brain, and the complex neuronal circuits they constitute, will keep scientists trying to decipher them challenged for years to come. In the last decade, the use of genetically encoded calcium indicators (GECIs) to monitor and visualize neuronal activity has greatly advanced. Calcium imaging using GECIs has become a principal modality to elucidate neuronal coding and signaling processes. GECIs provide clear advantages over synthetic calcium dyes by enabling long-term expression and chronic imaging in targeted neurons in vivo. Whilst most improvements of GECIs have been primarily focusing on faster kinetics, calcium sensitivity, brightness and signal strength; less attention has been on GECIs’ likely impact on cellular environments via calcium buffering. Studies have shown that long-term expression of GECIs at high intracellular concentrations can lead to pathological changes and reduced responsiveness in cells. The objective of this dissertation was to design a new family of GECIs suitable for long-term monitoring of neuronal calcium activity. In contrast to previous optimization strategies, here a new species of calcium binding protein, troponin C from Opsanus tau, was used as a basis for the development of a minimal calcium-binding domain. The minimal domain was fused to brighter fluorescent proteins to generate novel GECIs with improved properties. Consequently, the novel GECIs were optimized through iterative rounds of directed molecular evolution and screening, resulting in the Twitch-family of GECIs. In Chapter 2, we describe the structure-function relationships of a previously published FRET-based calcium indicator, the TN-XXL. The structure-function relationship in FRET- based GECIs is largely uncharacterized due to the artificial and multi-modular composition. By utilizing a combination of protein engineering, spectroscopic and biophysical analyses, we show that two of the four calcium binding sites dominate the FRET output. Furthermore, we found that local conformational changes of these sites match the kinetics of FRET change. We show that TN-XXL changes from a flexible elongated structure to a rigid globular shape upon binding calcium. The insights gained from this work formed the basis for the engineering of the FRET-based GECIs described in this work. In Chapter 3, a newly developed minimal domain FRET-based GECI, Twitch-1CD, was introduced into auto-antigen-specific and non–auto-antigen-specific CD4+ T cells. We demonstrated for the first time in vivo how a GECI is fully expressed in T cells, and thus allowing for detailed recording and visualization of calcium signaling during T cell antigen- recognition. In Chapter 4, we orchestrated the evolution of the Twitch-family of GECIs, with better signal- to-noise ratios (SNR), greater dynamic range (∆R/R) and calcium kinetics. These indicators underwent rational design and directed molecular evolution, followed by bacterial plate screening and a fluorescent imaging screening assay in hippocampal neurons. The novel GECIs were subsequently applied in a series of studies, emphasizing their improvements to previous FRET-based GECIs

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Narrow line width operation of a 980 nm gain guided tapered diode laser bar

We demonstrate two different schemes for the spectral narrowing of a 12 emitter 980 nm gain guided tapered diode laser bar. In the first scheme, a reflective grating has been used in a Littman Metcalf configuration and the wavelength of the laser emission could be narrowed down from more than 5.5 nm in the free running mode to 0.04 nm (FWHM) at an operating current of 30 A with an output power of 8 W. The spectrum was found to be tunable within a range of 16 nm. In the second scheme, a volume Bragg grating has been used to narrow the wavelength of the laser bar from over 5 nm to less than 0.2 nm with an output of 5 W at 20 A. To our knowledge, this is the first time spectral narrowing has been performed on a gain guided tapered diode laser bar. In the Littman Metcalf configuration, the spectral brightness has been increased by 86 times and in the volume Bragg grating cavity the spectral brightness has been improved over 18 times when compared to the free running operation. These schemes could be also extended for other wavelengths of interest in the future