Probing rotation dynamics of biomolecules using polarization based fluorescence microscopy

Area of Science:

• Biophysics
• Microscopy
• Optical Imaging

Background:

• Fluorescence polarization (FP) and fluorescence anisotropy (FA) are established techniques for studying biomolecular dynamics.
• Understanding molecular rotation and interactions is crucial in various biological and chemical applications.

Purpose of the Study:

• To develop and demonstrate a two-photon fluorescence polarization microscopy technique.
• To assess the capability of FA microscopy in probing molecular rotational mobility and interactions.
• To investigate the axial resolution limitations of the developed FA microscopy setup.

Main Methods:

• Utilized a two-photon fluorescence microscope excited by a linearly polarized femtosecond laser.
• Separated emitted fluorescence into parallel and perpendicular polarization components using two detectors.
• Derived fluorescence anisotropy (FA) images from combinations of excitation and emission polarization states.

Main Results:

• Demonstrated that FA microscopy can probe the rotational mobility of fluorescent molecules and their environmental interactions.
• Observed that FA imaging exhibits lower axial resolution compared to standard fluorescence intensity images.
• Identified that the reduced axial resolution is an intrinsic characteristic of the current experimental setup.

Conclusions:

• FA microscopy is a viable tool for investigating molecular rotation and interactions.
• The intrinsic axial resolution limitation necessitates careful consideration in experimental design.
• Further optimization of the experimental setup may be required to improve axial resolution in FA imaging.