Method to Label Proteins Inside the Living Cell for Super-Resolution Fluorescence Microscopy

Super-resolution microscopy has gained increasing interest due to its ability to visualize single protein molecules and sub-diffraction limited cellular structure. However, studying the dynamics of intracellular proteins in living cells using super-resolution microscopy has remained difficult due to the poor permeability of fluorescent probes across the cell membrane. Thus, imaging of intracellular protein in a living cell is often accomplished by using fluorescent proteins, which comes with caveats such as over-expression of target protein and poor photostability. CPLC graduate student Kai Wen Teng and the Selvin group developed a method to deliver fluorescent probes efficiently up to the size of a full antibody for targeting intracellular proteins. The method uses a pore-forming toxin called Streptolysin O, which reversibly creates ~20-30 nm pores on the cell membrane without killing the cells. Using this method, virtually any fluorescent dye can be delivered and any protein targeted using affinity reagents such as fluorescent ligands, nanobodies, or antibodies. In addition, the live-cell friendly oxygen scavenger Oxyrase can be applied to prolong the photostability of fluorescent dyes. By combining the advantages of the delivery method with Oxyrase, the team tracked individual kinesin molecules by single-molecule fluorescence, imaged actin filament dynamics using super-resolution microscopy (dSTORM) over an hour, and measured various steps associated in cancer, all unprecedented in live-cell fluorescence imaging. Refer to: Labeling proteins inside living cells using external fluorophores for microscopy