15
Overview
1. Optical slice thickness
not definable
With a conventional microscope, unlike in con-
focal microscopy, sharply defined images of
“thick” biological specimens can only be
obtained if their Z dimension is not greater than
the wave-optical depth of field specified for the
objective used. Depending on specimen thick-
ness, object information from the focal plane is
mixed with blurred information from out-of-
focus object zones.
Optical sectioning is not possible; consequently,
no formula for optical slice thickness can be
given.
1. Optical slice thickness
1)
Corresponds to the FWHM of the intensity distri-
bution behind the pinhole (PSF
det
). The FWHM
results from the emission-side diffraction pattern
and the geometric-optical effect of the pinhole.
Here, PH is the variable object-side pinhole dia-
meter in µm.
1. Optical slice thickness
The term results as the FWHM of the total PSF –
the pinhole acts according to wave optics.
λ
stands for a mean wavelength – see the text
body above for the exact definition.
The factor 0.64 applies only to a fluorescent
point object.
Conventional microscopy
Confocal microscopy 1 AU < PH <
∞
Confocal microscopy PH < 0.25 AU
2. Axial resolution
(wave-optical depth of field)
Corresponds to the width of the emission-side
diffraction pattern at 80% of the maximum
intensity, referred to the object plane. In the
literature, the wave-optical depth of field in a
conventional microscope is sometimes termed
depth resolution. However, a clear distinction
should be made between the terms resolution
and depth resolution.
2. Axial resolution
FWHM of PSF
ill
(intensity distribution at the
focus of the microscope objective) in Z direction.
No influence by the pinhole.
2. Axial resolution
FWHM of total PSF in Z direction
As optical slice thickness and resolution are
identical in this case, depth resolution is often
used as a synonym.
3. For comparison: FWHM of PSF in the interme-
diate image (Z direction) – referred to the object
plane.
3. Approximation to 2. for NA < 0.5
3. Approximation to 2. for NA < 0.5
4. Lateral resolution
FWHM of the diffraction pattern in the interme-
diate image – referred to the object plane) in
X/Y direction.
4. Lateral resolution
FWHM of PSF
ill
(intensity distribution at the focus
of the microscope objective) in X/Y direction
plus contrast-enhancing effect of the pinhole
because of stray light suppression.
4. Lateral resolution
FWHM of total PSF in X/Y direction plus
contrast-enhancing effect of the pinhole be-
cause of stray light suppression.
All data in the table refer to quantities in the object space and apply to a fluorescent point object.
1) PH <
∞
is meant to express a pinhole diameter of < 4–5 AU.
0.88 .
em
n- n
2
-NA
2
+ 2
. n . PH
NA
2
2
0.64 .
(n- n
2
-NA
2
)
0.64 .
(n- n
2
-NA
2
)
0.88 .
exc
(n- n
2
-NA
2
)
n
.
em
NA
2
1.77
.
n
.
em
NA
2
1.77
.
n
.
exc
NA
2
1.28
.
n
.
NA
2
0.51
.
em
NA
0.51
.
em
NA
0,37
.
NA
Optical Image Formation
Part 1
337_Zeiss_Grundlagen_e 25.09.2003 16:16 Uhr Seite 18
