Axial:
n
= refractive index of immersion liquid,
NA = numerical aperture of the microscope objective,
λ
exc
= wavelength of the excitation light
If NA < 0.5, equation (2) can be approximated by:
Lateral:
At first glance, equations (2a) and (3) are not dif-
ferent from those known for conventional imaging
(see Beyer, H., [3]). It is striking, however, that the
resolving power in the confocal microscope
depends only on the wavelength of the illumina-
ting light, rather than exclusively on the emission
wavelength as in the conventional case.
Compared to the conventional fluorescence
microscope, confocal fluorescence with large pin-
hole diameters leads to a gain in resolution by the
factor (
λ
em
/
λ
exc
) via the Stokes shift.
9
Resolution
Resolution, in case of large pinhole diameters
(PH >1 AU), is meant to express the separate visi-
bility, both laterally and axially, of points during
the scanning process. Imagine an object consisting
of individual points: all points spaced closer than
the extension of PSF
ill
are blurred (spread), i.e. they
are not resolved.
Quantitatively, resolution results from the axial and
lateral extension of the scanning laser spot, or the
elliptical half-intensity area of PSF
ill
. On the
assumption of homogeneous pupil illumination,
the following equations apply:
FWHM
ill,axial
=
0.88 .
exc
(n- n
2
-NA
2
)
≈
1.77 . n .
exc
NA
2
FWHM
ill,lateral
= 0.51
exc
NA
Optical Image Formation
Part 1
(2)
(2a)
(3)
337_Zeiss_Grundlagen_e 25.09.2003 16:16 Uhr Seite 12
