Imaging and tracking of single GFP molecules in solution

Area of Science:

• Biophysics
• Optical Microscopy
• Cell Biology

Background:

• Single-molecule visualization and tracking are crucial for understanding cellular processes.
• Previous methods were limited to thin interfacial layers, hindering in-situ studies.
• Detecting single molecules in 3D systems like living cells remained a challenge.

Purpose of the Study:

• To demonstrate the visualization and tracking of single protein molecules in 3D samples.
• To overcome the limitations of previous interfacial-layer-confined observations.
• To establish a foundation for single-molecule studies within living cells.

Main Methods:

• Utilized a wide-field fluorescence microscope with an Ar(+)-laser and a low-light-level CCD camera.
• Detected single green fluorescent protein (GFP) molecules in gels and viscous solutions up to 10 micrometers deep.
• Employed a high-speed framing mode for 5 ms time resolution and achieved 30 nm localization accuracy.

Main Results:

• Successfully visualized and tracked single GFP molecules in 3D samples at room temperature.
• Determined photon emission before photodestruction (< or = 4 * 10^5 photons).
• Tracked freely diffusing GFP molecules with a frame rate of approximately 80 Hz, enabling diffusion coefficient derivation.

Conclusions:

• Single protein molecule visualization and tracking in 3D samples is achievable.
• The developed method provides accurate localization and tracking of diffusing molecules.
• This technique holds significant potential for future studies of molecular dynamics within living cells.