Rethinking resolution estimation in fluorescence microscopy: from theoretical resolution criteria to super-resolution...

Area of Science:

• Optical Microscopy
• Biophotonics
• Imaging Science

Background:

• Optical microscope resolution is traditionally limited by numerical aperture and light wavelength.
• Advanced fluorescence microscopy, including super-resolution techniques, has introduced new resolution estimation methods, causing confusion.
• Accurate resolution quantification is crucial for interpreting fluorescence microscopy data.

Purpose of the Study:

• To summarize historical concepts of optical resolution limits.
• To discuss factors affecting practical fluorescence microscope resolution.
• To provide practical methods for measuring fluorescence microscope resolution from images.

Main Methods:

• Review of classical resolution criteria (e.g., Abbe, Rayleigh).
• Analysis of factors degrading resolution in fluorescence microscopy (e.g., aberrations, scattering, photobleaching).
• Demonstration of image-based resolution measurement techniques.

Main Results:

• A comprehensive overview of theoretical resolution limits and practical influencing factors.
• Clear methodologies for experimentally determining fluorescence microscope resolution.
• Examples illustrating the application of resolution measurement techniques.

Conclusions:

• Resolution estimation in fluorescence microscopy requires understanding both theoretical limits and practical challenges.
• Standardized methods for resolution measurement are essential for reliable data interpretation.
• This work provides a guide for researchers to accurately quantify fluorescence microscope resolution.