Point Spread Function and Resolution

Given the wave-like nature of light, there is a fixed limit to the ability to resolve objects that are very close to one another.  Thus, “resolution” depends on the wavelength of light and the light collecting ability of the objective lens, as expressed by the Numerical Aperture (NA).  Similarly, even with the highest magnification objective lenses and the smallest pixels sizes point sources smaller than the “diffraction limit of light microscopy” (~250nm in XY and ~500nm in Z) will appear larger than they really are depending upon these same constraints.  Thus the Point Spread Function (PSF) is used to express how a particular object smaller than the diffraction limit appears under specific imaging conditions.  The measurement of the apparent width of a fluorescent object at the point where its intensity is 50% of the maximum, the so-called “Full Width at Half Maximum (FWHM)” is a straightforward way to characterize the PSF, and is also often used as a short-hand measure of resolution.  So called “super-resolution” microscopy techniques, such as Single Molecule Localization Microscopy, Structured Illumination Microscopy and Stimulated Emission Depletion Microscopy, are able to “break” the diffraction limit through different innovative means.

See our chapter on super-resolution microscopy for more details.