The theoretical resolution of a conventional fluorescence microscope can be calculated by a formula originally described by Ernst Abbe in 1873.

With:
D : resolution.
l : wavelength of the emission light.
NA : numerical aperture of the objective lens.
n : refractive index of the immersion medium.
α : half-angular aperture of the objective lens.

The theoretical resolution of a STED microscope can be calculated by a modified Abbe formula.

With:
D : resolution.
l : wavelength of the emission light.
n : refractive index of the immersion medium.
α : half-angular aperture.
I : peak intensity of the STED laser.
I_sat : saturation intensity.

Whereas I is proportional to the laser power of the STED beam, the parameter I_sat is dependent on the fluorophore: The lower the saturation intensity of a given fluorophore, the easier it is to switch molecules from the excited state (ON state) to the ground state (OFF state). I_sat is the characteristic saturation intensity of a fluorophore defined as the laser intensity at which 50% of the molecules can be switched off. The resolution can therefore be enhanced by carefully selecting the optimal fluorophores.

In addition, the resolution can be improved by increasing the STED laser power (I) (typical STED lasers have power ratings of 1 W power output or higher). Tuning of the resolution in STED images is usually achieved by modulation of the STED laser power after selecting optimal dyes for STED microscopy.

By shrinking the volume in which fluorophores are allowed to emit photons the signal-to-noise (S/N) ratio is decreased as a consequence. This leads to noisier images if no measures are taken to increase the number of photons emitted by the reduced volume, e.g. by using accumulation of multiple images of the same region or simply by increasing the laser power for excitation.

Resolution enhancement as a function of the STED laser power. Fluorescent beads were used to demonstrate the resolution enhancement of STED (a) over classical confocal microscopy (b). The resolution in the STED image can be tuned by changing the laser power of the STED beam and thereby altering the power density in the donut (c-g and h).
Adapted with permission from The Optical Society (OSA). (Benjamin Harke, Jan Keller, Chaitanya K. Ullal, Volker Westphal, Andreas Schönle, and Stefan W. Hell, 'Resolution scaling in STED microscopy' Opt. Express 16, 4154-4162 (2008))