Differential aberration correction (DAC) microscopy: a new molecular ruler.

Area of Science:

• Biophysics
• Optical Microscopy
• Molecular Imaging

Background:

• Measuring molecular distances is crucial in bio-molecular sciences.
• Existing fluorescence microscopy methods have limitations in specific distance ranges (1-10 nm and >300 nm).
• Bridging the resolution gap in microscopy is an ongoing challenge.

Purpose of the Study:

• To introduce a simple experimental technique for measuring molecular distances between 10 and 300 nm.
• To achieve a resolution of approximately 20 nm in both 2D and 3D.
• To provide a method that complements existing techniques like Förster energy transfer microscopy and diffraction-limited microscopy.

Main Methods:

• Development of a novel fluorescence microscopy technique.
• Utilizing a computational approach to correct optical aberrations across the entire field of view.
• Employing a differential method where sample probes and reference probes are affected identically by aberrations.

Main Results:

• Successful bridging of the resolution gap in fluorescence microscopy.
• Achieved a resolution of approximately 20 nm for molecular distance measurements.
• Demonstrated the technique's applicability in both 2D and 3D.

Conclusions:

• The developed technique offers a significant advancement for molecular distance measurements in the intermediate range.
• This method provides a valuable tool for investigating structural and functional questions in bio-molecular sciences.
• The computational aberration correction ensures accurate measurements over the whole field of view.