Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS)

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Total internal reflection epi-fluorescence (epi-TIRF) microscopy offers high axial confinement.
• Stimulated emission depletion (STED) nanoscopy provides superior lateral spatial discrimination.
• Imaging cell membranes at the macromolecular scale requires advanced microscopy techniques.

Purpose of the Study:

• To characterize a novel fluorescence microscope combining epi-TIRF and STED nanoscopy (TIRF-STED nanoscope).
• To evaluate the performance and limitations of the TIRF-STED nanoscope for imaging and spectroscopy.
• To demonstrate the capability of the TIRF-STED nanoscope for studying molecular dynamics.

Main Methods:

• Theoretical description of the sampling volume.
• Imaging of fluorescent beads to assess performance.
• Fluorescence correlation spectroscopy (FCS) on dye molecules and lipid analogs.
• Investigation of laser excitation polarization effects.

Main Results:

• Achieved lateral resolution down to 40-50 nm.
• Estimated axial confinement of approximately 55 nm.
• Demonstrated an all-optically induced measurement volume of less than 1 attoliter.
• Reduced out-of-focus background signal effectively.

Conclusions:

• The TIRF-STED nanoscope enables high-resolution imaging and spectroscopy of cell membranes.
• The combination of epi-TIRF and STED offers near-isotropic resolution.
• Limitations include the signal-to-background ratio inherent to the epi-TIRF scheme.