1 2 Fluorescent protein-based tools Outline for neuroscience An animatd primer on biosensor development Fluorescent proteins (FPs) Robert E. Campbell Department of Chemistry Other fluorophore technologies Single FP-based biosensors
Imaging Structure & Function in the Nervous System Cold Spring Harbor, July 31, 2019.
Lots of structural Lots of structural information information & Transmitted light Fluorescence microscopy of fluorescent color microscopy of live cells live cells No molecular provides molecular information information (more colors = more information) 5 6 Fluorescence microscopy requires fluorophores Non-natural fluorophores for protein labelling Trends Bioch. Sci., 1984, 9, 88-91.
O O O N O N 495 nm 519 nm 557 nm 576 nm
- - CO2 CO2 S O O O N O N C N N C S ϕ = quantum yield - - ε = extinction coefficient CO2 CO2 ϕ Brightness ~ * ε S C i.e., for fluorescein N Proteins of interest ϕ = 0.92 N Fluorescein Tetramethylrhodamine ε = 73,000 M-1cm-1 C S (FITC) (TRITC) A non-natural fluorophore must be chemically linked to Non-natural fluorophores made by chemical synthesis a protein of interest…