Integrating functional imaging with light sheet fluorescence microscopy

AGUSTÍN A. CORBAT; BRUNO MORETTI; HERNÁN E. GRECCO; MARTÍN HABIF

París

Congreso; Annual Meeting of the International Physics of Living Systems (iPoLS) Network; 2017

International Physics of Living Systems

The retooling of fluorescent proteins together with the development of confocal, two photon microscopy and, most recently, Light Sheet Fluorescence Microscopy (LSFM) allowed to observe whole embryos during development providing a more comprehensive understanding of the whole process. The low levels of photobleaching and phototoxicity that can be achieved by LSFM techniques makes them ideal for imaging living organisms during long times. While the observation of the phenotypic changes has provided an enormous insight into the development process, it is desirable to monitor the state of the complex signaling networks that regulate cellular function. In particular, cell death, mostly via apoptosis, is an important part of embryo development, tissue homeostasis and in the genesis of many pathological states. The core of the apoptotic machinery is a family of proteases known as caspases and are evolutionary conserved. Based on previously published caspase activity sensors, our group has recently developed homo-FRET based biosensors that allow us to monitor simultaneously the activity of 3 caspases (Caspase 3, 8 and 9) in single cells. Fluorescence anisotropy microscopy can be used to measure homo-FRET between fluorescent proteins of the same color. Unlike ratiometric FRET measurements, fluorescence anisotropy measurements can be done at a single emission wavelength band, and the emission spectra of the donor and acceptor fluorophores do not need to be well separated. By combining this sensors with LSFM, we aim to monitor the spatiotemporal activation of caspases of living embryos to understand organism shaping by apoptosis during development.