Tools and Techniques for Single Cell Applications in Development and Neurobiology

Abstract

Fluorescence microscopy of genetically encoded fluorescent proteins (FPs) and biosensors has transformed modern biological research - a phenomenon often referred to as the "fluorescent protein revolution". From the very beginning of this revolution, the discovery and engineering of new protein probes and the development of new microscopy modalities have mutually enabled each other. Prominently, breaking the fundamental diffraction limit of light in various super-resolution modalities was enabled by the discovery and engineering of photomodulatable FPs such as the photoactivatable paGFP or the photoconvertible FPs Dendra2 or EosFP. While photoconvertible FPs (pcFPs) are formidable markers for super-resolution microscopy and highlighting of structures or cells for pulse-chase-type experiments, they suffer from some key drawbacks for applications in complex, sensitive biological tissues. The near-ultraviolet (UV) light needed for efficient photoconversion cannot be axially confined inside tissue volumes, has limited tissue penetration and is phototoxic to light sensitive samples such as developing embryos. Primed conversion elegantly overcomes this requirement for near-UV light and can be axially confined to single cells in three dimensional tissues. However, this process has been limited to a single FP and remained mechanistically elusive, preventing protein engineering of probes as well as many biological applications. Following the introduction, in the second part of this thesis I describe my efforts to advance both the infrastructure and protein probes for primed conversion. I provide a detailed protocol for the simple, safe, and reproducible implementation of primed conversion into a commercial confocal laser scanning microscope (CLSM). Further, I uncover the mechanistic details underlying primed conversion and use this knowledge to engineer a host of pcFPs with improved characteristics for various primed conversion applications. I establish the first ever multi-color photoactvation localization microscopy (PALM)- scheme using two spectrally identical variants of mEos2, separating them solely based on their mode of photoconversion. Further, I provide a semiautomatic platform to use primed conversion of single blastomeres in a developing mouse embryo to computationally reorient the embryo and correct for the rapid movement and rotation, that would otherwise lead to the exclusion of the respective embryo from analysis. In the third part of this thesis I talk about a new class of genetically encoded Calcium ion (Ca2+) indicators GECIs, that we developed for two-photon (2P) imaging using cheap and powerful alternatives to the commonly used Titanium Sapphire (Ti::Sapph) lasers. We optimize several variants of the best in class GECIs of the GCaMP-series to excite efficiently at the center wavelength of non-tunable 1030nm Ytterbium-doped optical fiber lasers (YbFLs). This spectral shift allows for traditional 2P imaging using lasers of drastically reduced costs as well as kHz frame-rate imaging of Ca2+-dynamics using swept line angular projection microscopy (SLAPMi). Furthermore, I propose these new GECIs to enable simultaneous dual-color Ca2+-imaging together with existing red GECIs such as jRGECO1a.