High contrast fluorescence polarization microscopy through double tagged photoswitchable fluorescent proteins.

Area of Science:

• Cellular imaging
• Microscopy techniques
• Biophysics

Background:

• Fluorescence polarization microscopy (FPM) is limited by the rotational freedom of fluorescent probes.
• Enhancing contrast in FPM requires better control over fluorophore orientation.

Purpose of the Study:

• To develop a method for rigid anchoring of fluorescent proteins (FPs) in living cells.
• To improve contrast and orientational selectivity in FPM.
• To introduce a novel imaging scheme for enhanced polarization measurements.

Main Methods:

• Utilized double tagging of reversibly photoswitchable fluorescent proteins (dt-rsFPs) to cellular structures.
• Developed a camera frame-separated switching pulse scheme (FrExPAN) for polarization angle narrowing.
• Applied FrExPAN imaging to membranes of living HeLa cells and hippocampal neurons.

Main Results:

• Demonstrated significant contrast enhancement in FPM through rigid anchoring of dt-rsFPs.
• Successfully implemented FrExPAN for effective narrowing of the excitation angle range in living cells.
• Showcased selective imaging of cellular structures with distinct orientations.

Conclusions:

• Rigid anchoring of FPs via double tagging is a broadly applicable principle for enhancing FPM contrast.
• FrExPAN imaging enables precise orientational selection of cellular components.
• The method has potential implications for orientational contrast imaging and super-resolution techniques like SPoD.