Zeiss LSM 880 Operating Manual page 659

The total PSF (PSF ) of a confocal microscope behind the
tot
pinhole is composed of the PSFs of the illuminating beam
path (PSF ; point illumination) and the detection beam path
ill
(PSF ; point detection). Mathematically, this relationship
det
can be described as follows:
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.
PSF (x,y,z) = PSF (x,y,z) PSF
tot ill
PSF corresponds to the light distribution of the laser spot
ill
that scans the object. It's size is mainly a function of the
laser wavelength and the numerical aperture of the micro-
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scope objective lens. It is also influenced by diffraction at
the objective lens pupil (as a function of pupil illumination,
0.88 .
see details) and the aberrations of all optical components
exc
FWHM =
ill,axial
2 2
integrated in the system. [Note: In general, these aberra- (n- n -NA )
tions are low, having been minimized during system design].
Moreover, PSF may get deformed if the laser focus enters
ill
thick and light-scattering specimens, especially if the refrac-
1.77 . n .
exc
tive indices of immersion liquid and mounting medium are
2
NA
not matched and/or if the laser focus is at a great depth
below the specimen surface (see Hell, S., et al., [7]).
exc
FWHM = 0.51
ill,lateral
NA
Fig. 6a Section through the 3D-PSF in Z direction as generated
by a diffraction limited objective lens – left, and in XY-direction
– right (computed; dimensionless representation); the central,
elliptical maximum is distinctly visible.
X
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0.88 .
Z
FWHM =
det,axial
n- n
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2
( )
PSF (x,y,z) = PSF (x,y,z)
tot ill
.
em exc
2
2 + 2
exc em
0.64 .
(x,y,z)
(1)
det
2 2
2 . n . PH
em +
NA
2 2
-NA
1.28 . n .
OpticalImageFormation
PSF is also influenced by all these factors and, addition-
det
ally, by the pinhole size. For reasons of beam path efficiency
(see Part 2), the pinhole is never truly a point of infinitely
small size and thus PSF is never smaller in dimension than
det
PSF . It is evident that the imaging properties of a confocal
ill
LSM are determined by the interaction between PSF
PSF . As a consequence of the interaction process, it can
det
be written: PSF ≤ PSF .
tot ill
With the pinhole diameter being variable, the effects ob-
tained with small and big pinhole diameters must be ex-
pected to differ. In the following sections, various system
states are treated in quantitative terms.
From the explanations made so far, it can also be derived
that the optical slice is not a sharply delimited body. It does
not start abruptly at a certain Z position, nor does it end
abruptly at another. Because of the intensity distribution
along the optical axis, there is a continuous transition from
object information suppressed and such made visible.
Accordingly, the out-of-focus object information actually
suppressed by the pinhole also depends on the correct set-
ting of the image processing parameters (PMT high voltage,
contrast setting). Signal overdrive or excessive offset should
be avoided.
Fig. 6b The central maximum in this illustration is called Airy
disk and is contained in the 3D-PSF as the greatest core diameter in
lateral direction.
X
Y
PART 1
and
ill
9