Detection of single fluorescent proteins inside eukaryotic cells using two-photon fluorescence

Imaging single fluorescent proteins in a live cell is a challenging task because of the strong cellular autofluorescence. Autofluorescence can be minimized by reducing fluorescence excitation volume. Total internal reflection fluorescence (TIRF) microscopy has been routinely used to reduce excitation volume and detect single protein molecules in or close to cell membrane. However, the limited penetration depth of evanescent field excludes imaging of single fluorescent proteins that reside deep inside a eukaryotic cell. Here we report detection of single fluorescent proteins inside eukaryotic cells by two-photon fluorescence (TPF) microscopy. TPF has an excitation volume less than 0.1 femtoliter (fL). Cell autofluorescence under TPF is low and thus enables us to detect single enhanced green fluorescent proteins (EGFP) and single monomeric teal fluorescent proteins (mTFP1.0) that reside several microns deep inside the cell. Discrete stepwise photobleaching of TPF was observed for both proteins inside the cell. Quantitative analysis of single-molecule fluorescence trajectories show that mTFP1.0 is about twofold brighter than EGFP, while its fluorescence on-time before bleaching is about 10 fold shorter. These findings demonstrate the sensitivity of TPF for imaging of eukaryotic cells at single-molecule level and will be useful for measurement of protein stoichiometry inside the cell.