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

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3 Author(s)
Schrader, M. ; Department of Medical Physics, University of Turku, P.O. Box 123, FIN‐20521 Turku, Finland ; Hell, S.W. ; van der Voort, H.T.M.

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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. © 1996 American Institute of Physics.

Published in:

Applied Physics Letters  (Volume:69 ,  Issue: 24 )