The New Test Tube of the Millennium, the Live Cell - R. Truant

This section of the course is focused on the use of Aequorea Fluorescent Proteins (AFPs) in live cell imaging. The current palette of AFPs ranges numbers of 18-20, and in colors from 440 to 650 nm, but the properties of these fluorophores means that some are more practical than others for live cell imaging. The properties of many of these proteins will be summarized and discussed, as well as methods used to derived new fluorescent proteins with desired properties. A brief overview of fluorescent microscope design and setup to optimize for AFP visualization and live cell imaging will be discussed, as well as practical conditions of cell growth and maintenance for live cell imaging over short and long time periods. Paired fluorophores will be discussed for techniques such as co-localization of two proteins and Forster resonant energy transfer (FRET), and lifetime imaging. Location and dynamics of proteins in live cells will be discussed with an overview of qualitative fluorescent recovery after photo bleaching (FRAP) and fluorescence loss induced by photo bleaching (FLIP) with multiple fluorophores. Newer AFPS and their use in fluorescent photo activation will also be summarized.
A second revolution from the use of AFPs is in the design and use of AFPs as biosensors, either as individual protein markers responding to local nano environments, as location sensors to detect enzymatic activity, or as conformational switching sensors to detect a conformational change in a protein in response to a protein-protein or protein-factor interaction. Finally, we will conclude with an overview of detecting protein complex interactions in vivo by the use of bimolecular fluorescent complementation, or BiFC, with newly developed vectors.