Two-Photon Fluorescence Imaging of Single Fluorescent Proteins Inside Mammalian Cells

Abstract

Exciting progress has been made recently in biophysical techniques that allow optical imaging of single molecules in live cells. However, imaging of single fluorescent proteins in live cells is usually limited to bacteria that are small in size or proteins that are on the cell surface, where excitation through total internal reflection can be used to reduce fluorescence background and achieve single-molecule sensitivity. Eukaryotic cells are typically larger in size with higher autofluorescence background. To achieve single-molecule sensitivity deep inside a eukaryotic cell is thus challenging. We have used two-photon fluorescence excited by infrared lasers to reduce autofluorescence background. We show that single green fluorescent proteins can be imaged deep inside a mammalian cell using two-photon fluorescence. Discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution indicate that two-photon fluorescence is detectable at the single-molecule level deep inside a eukaryotic cell. Moreover, it is not necessary to use a pulsed laser for two-photon fluorescence excitation. A continuous-wave laser that is routinely used for optical tweezers can be used to excite two-photon fluorescence and achieve single-molecule sensitivity. These advantages could significantly benefit future application of this single-molecule technique in biological studies. We present applications of our technique, and demonstrate two-photon fluorescence imaging of eukaryotic cells at single-molecule level.