Zeiss LSM 880 Operating Manual page 660

Resolution and Confocality
Wherever quantitative data on the resolving power and
depth discrimination of a confocal LSM are specified, it is
necessary to distinguish clearly whether the objects they
refer to are point-like or extended, and whether they are
reflective or fluorescent. These differences involve distinctly
varying imaging properties. Fine structures in real biological
specimens are mainly of a filiform or point-like fluorescent
type, so that the explanations below are limited to point-like
fluorescent objects. The statements made for this case are
well applicable to practical assignments.
As already mentioned, the pinhole diameter plays a decisive
role in resolution and depth discrimination. With a pinhole
diameter greater than 1 AU (AU = Airy unit – see Details
"Optical Coordinates"), the depth discriminating properties
under consideration are essentially based on the law of
geometric optics (geometric-optical confocality).
Fig. 7 Geometric-optical (a) and wave-optical confocality (c) [XZ view].
The pinhole diameter decreases from (a) to (c).
Accordingly, PSF det (green) shrinks until it approaches the order of magnitude of PSF ill (red).
a) PH~3.0 AU
FWHM
ill, lateral
FWHM
det, lateral
PSF >> PSF
det
Geometric-optical
confocality
10
b)
PSF
ill
The smaller the pinhole diameter, the more PSF
proaches the order of magnitude of PSF
(PH < 0.25 AU), both PSFs are approximately equal in size,
and wave-optical image formation laws clearly dominate
(wave-optical confocality).
Figure 7 illustrates these concepts. It is a schematic repre-
sentation of the half-intensity areas of PSF
selected pinhole diameters.
Depending on the pinhole diameter, the data and computa-
tion methods for resolution and depth discrimination will
differ. A comparison with image formation in conventional
microscopes is interesting as well. The following sections
deal with this in detail.
PH~1 AU c)
> PSF
det ill
ap-
det
. In the limit case
ill
and PSF at
ill det
PH~0,25 AU
PSF >= PSF
det ill
Wave-optical
confocality