Axial nanometer distances measured by fluorescence lifetime imaging microscopy

Area of Science:

• Biophysics
• Microscopy
• Nanotechnology

Background:

• Accurate measurement of molecular proximity to surfaces is crucial for understanding biological processes.
• Existing techniques often lack the precision or applicability for studying protein-ligand interactions near surfaces.

Purpose of the Study:

• To introduce a novel fluorescence lifetime imaging microscopy (FLIM) technique for precise distance measurements.
• To apply this technique to determine the size of proteins attached to microtubules.

Main Methods:

• Utilizing distance-dependent fluorescence lifetime modulations of fluorescent molecules near a metalized surface.
• Employing fluorescently labeled microtubules with attached unlabeled proteins as optical probes.
• Measuring fluorescence lifetimes to infer molecular positions with nanometer precision.

Main Results:

• Demonstrated the ability to measure absolute positions of fluorescent molecules within 100 nm of a metalized surface.
• Successfully determined the geometrical size of unlabeled proteins attached to microtubules.
• Achieved nanometer precision in size determination of the attached proteins.

Conclusions:

• The developed FLIM technique offers a powerful tool for high-precision distance measurements in nanometer-scale proximity.
• This method enables accurate characterization of protein dimensions in near-surface biological systems.
• The findings have implications for fields requiring precise nanoscale measurements, such as molecular biology and materials science.