Abstract
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.
Highlights
Fluorescence imaging at single-molecule level has become increasingly important in biophysical and cell biological studies [1]
We demonstrate that two-photon fluorescence (TPF) can be used to detect single fluorescent proteins (FP) molecules inside mammalian cells, which adds TPF to the growing list of techniques that can resolve single-molecule fluorescence in the context of mammalian cells
Due to the intrinsic 3D resolution of TPF [9] and deep sample penetration [20,23], this imaging method is not limited to the surface of the cell, but can image proteins that are deep inside the cell
Summary
Fluorescence imaging at single-molecule level has become increasingly important in biophysical and cell biological studies [1]. The ability to visualize single molecules in real time allows direct observation of intermediate states that are often hidden in ensemble experiments [2]. The detection of these intermediates is often crucial in understanding these processes at molecular level [3]. To visualize single molecules in live cells, specific fluorophore labeling is required. TIRF detection of single molecules has been widely used to measure stoichiometry of proteins in plasma membrane [12,13,14,15,16]. To achieve single-molecule sensitivity inside eukaryotic cells that have larger cell body compared to bacterium, either light sheet microscopy or TPF is needed to suppress autofluorescence background. To the best of our knowledge, TPF imaging of mammalian cells at single-molecule level has not been reported
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