Spatially organized fluorescent reporters for recording complex biological dynamics in cell population

Abstract

Biological signals, such as the dynamic concentrations of ions, levels of signaling molecules, and activities of protein kinases, interact in complex ways within cells, and can exhibit great cell-to-cell heterogeneity as a function of cell history and state. There is increasing desire to use multiple fluorescent reporters to simultaneously measure multiple biological signals in single cells across cell populations, such as those in the brain. However, due to the diffraction limit of optical imaging, the biological signals recorded from neurons in densely-labeled neural populations in vivo are often mixed with signals from closely passing axons and dendrites from other neurons, resulting in erroneous signaling events and artifactual correlations of measured neural activity. Also, it is not yet possible to simultaneously record any given set of biological signals in single cells, because there are limited sets of corresponding spectrally-orthogonal fluorescent reporters available to date. Even if the fluorescent reporters for all biological signals in all possible colors are developed in the future, the number of biological signals that can be simultaneously recorded are still limited by the number of available optical channels. In this thesis, I address these problems by developing two new technologies, soma-targeted fluorescent calcium indicators and spatially multiplexed imaging.

Soma-targeted fluorescent calcium indicators (or 'SomaGCaMPs', the first part of the thesis) are fluorescent reporters of calcium dynamics that are selectively localized at the soma, but not axons and dendrites, of neurons. In vivo optical imaging of SomaGCaMPs in dense neural circuits in mouse and zebrafish brains reported fewer artifactual spikes, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent reporters is a simple and powerful method for high-fidelity population imaging of neural activity in vivo.

Spatially multiplexed imaging (the second part of the thesis) enables simultaneous readout of multiple biological signals in single cells from fluorescent reporters regardless of their spectra. This is achieved by clustering reporters into spatially separated 'Signaling Reporter Islands' (or 'SiRIs') via self-assembling protein scaffolds or RNA scaffolds. Using the spatial dimension as an asset, SiRIs may open up the ability to simultaneously image nearly arbitrary numbers of signals within a physiological cascade.