Zeiss LSM 880, Operating Manual

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Fig. 7 Geometric-optical (a) and wave-optical confocality (c) [XZ view].
The pinhole diameter decreases from (a) to (c).
Accordingly, PSFdet (green) shrinks until it approaches the order of magnitude of PSFill (red).
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).
The smaller the pinhole diameter, the more PSF
det
ap-
proaches the order of magnitude of PSF
ill
. In the limit case
(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
ill
and PSF
det
at
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.
a) PH~3.0 AU
PSF
det
> PSF
ill
b)
PSF
det
>= PSF
ill
c)
Geometric-optical
confocality Wave-optical
confocality
PH~1 AU PH~0,25 AU
FWHM
det, axial
FWHM
ill, lateral
FWHM
ill, axial
PSF
det
>> PSF
ill
FWHM
det, lateral