Potential of confocal microscopes to resolve in the 50–100 nm range

Abstract

We determine the resolution of high-performance confocal microscopes by measuring the three-dimensional point–spread function (3D-PSF) of an optimized confocal setup. The 3D-PSF is standardized by recording the scattered light of pointlike objects. For a wavelength of 543 nm and a specified numerical aperture of 1.4 (oil), we find an axial and lateral focal full width at half-maximum (FWHM) of 460±20 and 145±10 nm, respectively. A high signal-to-noise ratio is obtained by using recording times comparable to those of near-field scanning optical microscopy. We further reduce the effective PSF extent by means of a three-dimensional deconvolution technique exploiting the information gained from the measurement of the focus. We show that it is possible to obtain an axial and lateral FWHM of the far-field effective PSF after deconvolution of 80 and 40 nm, respectively.