Question:

Resolution of optical microscopy is limited by wave optics to about 200 to 400 nm. However, diffraction patterns generated by individual fluorescent particles (point spread functions, PSF) contain location information with much higher precision. Here, we describe a user-friendly approach for single fluorescence particle 3D localization and tracking with nanometer precision in a standard fluorescence microscope.

Methods:

Images of fluorescent particles were obtained over a large range (about 7 µm) of vertical positions and analysed by whole pattern (WP) comparison with a PSF recorded for the specific setup and measurement of the outermost ring radius (ORR).

Results:

For a vertical range of 4.5 µm, standard deviation (SD) from the predicted value, indicating validity, was 15.6 and 18.5 nm for WP and ORR. The precision, determined as SD of the distance between two particles, ranged from 3 to 6 nm. Local viscosity may be determined from random particle movement with a precision of about 5% of the measured value.

Conclusion:

With a validity in the range of 20 nm and a precision of about 4-5 nm obtained by standard fluorescent microscopy, the PSF approach offers a valuable tool for a variety of experimental investigations of particle/structure localizations and local viscosity, e.g. in studies of the endothelial surface layer.