Two-dimensional tracking of ncd motility by back focal plane interferometry

Area of Science:

• Biophysics
• Microscopy techniques
• Motor protein dynamics

Background:

• Optical traps are crucial for studying molecular motors.
• High-resolution detection of probe displacement is essential for accurate dynamic analysis.
• Existing methods may lack the necessary spatial or temporal resolution.

Purpose of the Study:

• To introduce and validate a novel far-field interference technique for detecting micron-sized optically trapped probe displacement.
• To theoretically explain the principles of back-focal-plane detection.
• To apply this method to investigate the motility of the ncd motor protein.

Main Methods:

• Utilized far-field interference to detect laser intensity shifts in the back-focal plane.
• Employed a quadrant photodiode for two-dimensional displacement measurement.
• Performed spectral analysis on bead motion data for dynamic parameter extraction.
• Used an in vitro bead assay with ncd-coated silica beads on microtubules.

Main Results:

• Achieved approximately 10-microsecond time resolution and high spatial resolution.
• Developed and validated a quantitative theory for back-focal-plane detection.
• Measured the ATP-dependent motility of ncd motor protein.
• Observed an average axial velocity of 230 +/- 30 nm/s for ncd-coated beads.
• Detected increased viscous drag near the surface and found motor constraints to be small.

Conclusions:

• The developed far-field interference technique offers high spatial and temporal resolution for probe displacement detection.
• This method allows for precise measurement of motor protein dynamics, including velocity and mechanical properties.
• The study provides new insights into the biophysical behavior of the ncd motor protein during motility.