Zeiss LSM 880, Operating Manual

Page: 667 / 690

17
Overview
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.
Conventional microscopy
1. Optical slice thickness
not definable
With a conventional microscope, unlike in confo-
cal 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 thickness, object infor-
mation 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 thickness1)
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 text for
the exact definition.
The factor 0.64 applies only to a fluorescent
point object.
2. Axial resolution
(wave-optical depth of field)
Corresponds to the width of the emission-side dif-
fraction 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 of field.
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 iden-
tical in this case, depth resolution is often used
as a synonym.
3. For comparison: FWHM of PSF in the inter-
mediate 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 intermedi-
ate 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.
Confocal microscopy 1 AU < PH <∞ Confocal microscopy PH < 0.25 AU
OpticalImageFormation PART 1
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA
2
1.28
.
n
.
NA 2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA 2
1.28
.
n
.
NA
2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA
2
1.28
.
n
.
NA 2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA 2
1.28
.
n
.
NA
2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA
2
1.28
.
n
.
NA 2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA
2
1.28
.
n
.
NA 2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA 2
1.28
.
n
.
NA
2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA 2
1.28
.
n
.
NA
2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA 2
1.28
.
n
.
NA 2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA 2
1.28
.
n
.
NA
2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥
seite 15
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
0.51
.
em
NA
1.77
.
n
.
exc
NA 2
1.28
.
n
.
NA
2
0,37
.
NA
0.51
.
exc
NA
seite 19
system constant
number of pixels
.
zoomfactor
.
magnification
obj
d
pix
=
3.92
.
NA
.
system constant
number of pixels
.
magnification
obj
.
exc
Z
≥