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

Area of Science:

• Cellular and Molecular Imaging
• Biophysics
• Microscopy Techniques

Background:

• Imaging single fluorescent proteins in live cells is hindered by strong cellular autofluorescence.
• Total internal reflection fluorescence (TIRF) microscopy reduces excitation volume but is limited to cell surface or membrane proteins.
• Detecting proteins deep within eukaryotic cells requires a microscopy technique with minimal excitation volume and low autofluorescence.

Purpose of the Study:

• To report the detection of single fluorescent proteins deep inside eukaryotic cells using two-photon fluorescence (TPF) microscopy.
• To demonstrate the capability of TPF microscopy for single-molecule imaging within cells.
• To compare the brightness and photobleaching characteristics of enhanced green fluorescent proteins (EGFP) and monomeric teal fluorescent proteins (mTFP1.0) in vivo.

Main Methods:

• Utilized two-photon fluorescence (TPF) microscopy with a sub-femtoliter excitation volume.
• Imaged single enhanced green fluorescent proteins (EGFP) and single monomeric teal fluorescent proteins (mTFP1.0) within eukaryotic cells.
• Analyzed single-molecule fluorescence trajectories, including photobleaching events and fluorescence on-time.

Main Results:

• TPF microscopy successfully detected single EGFP and mTFP1.0 molecules several microns deep inside cells with low autofluorescence.
• Discrete stepwise photobleaching was observed for both EGFP and mTFP1.0 under TPF excitation.
• mTFP1.0 was found to be approximately twofold brighter than EGFP, but its fluorescence on-time before bleaching was about tenfold shorter.

Conclusions:

• TPF microscopy offers high sensitivity for single-molecule imaging within eukaryotic cells, overcoming depth limitations of other techniques.
• The findings highlight TPF's utility for quantitative measurements of protein properties, such as brightness and stability, in vivo.
• This technique is valuable for determining protein stoichiometry and dynamics deep within cells.