Zeiss LSM 880, Руководство по эксплуатации

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Resolution
Resolution, in case of large pinhole diameters (PH >1AU),
is meant to express the separate visibility, both laterally
and axially, of points during the scanning process. Imagine
an object consisting of individual points: all points spaced
closer than the extension of PSF
ill
are blurred (spread), i.e.
they are not resolved.
Quantitatively, resolution results from the axial and lateral
extension of the scanning laser spot, or the elliptical half-
intensity area of PSF
ill
. On the assumption of homogeneous
pupil illumination, the following equations apply:
11
At first glance, equations (2a) and (3) are not different from
those known for conventional imaging (see Beyer, H., [1]).
It is striking, however, that the resolving power in the con-
focal microscope depends only on the wavelength of the
illuminating light, rather than exclusively on the emission
wavelength as in the conventional case.
Compared to the conventional fluorescence microscope,
confocal fluorescence with large pinhole diameters leads to
a gain in resolution by the factor (
λ
em
/
λ
exc
) via the stokes
shift.
If NA < 0.5, equation (2) can be approximated by:
seite 7
PSF
tot
(x,y,z) = PSF
ill
(x,y,z)
.
PSF
det
(x,y,z)
seite 9
FWHM
ill,axial
=
seite 11
FWHM
det,axial
=
0.88 .
em
n- n
2
-NA
2
+
2 . n . PH
NA
2 2
0.88 .
exc
(n- n
2
-NA
2
)
5
1.77 . n .
exc
NA 2
FWHM
ill,lateral
= 0.51
exc
NA
seite 12
PSF
tot
(x,y,z) =
(
PSF
ill
(x,y,z)
) 2
em
.
exc
2
exc
+
2
em
5
2
FWHM
tot,axial
=
0.64 .
(n- n
2 -NA 2 )
5
1.28 . n .
NA
2
FWHM
tot,lateral
= 0.37
NA
(2a)
Lateral:
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PSF
tot
(x,y,z) = PSF
ill
(x,y,z)
.
PSF
det
(x,y,z)
seite 9
FWHM
ill,axial
=
seite 11
FWHM
det,axial
=
0.88 .
em
n- n 2 -NA 2
+
2 . n . PH
NA
2 2
0.88 .
exc
(n- n 2 -NA 2 )
5
1.77 . n .
exc
NA
2
FWHM
ill,lateral
= 0.51
exc
NA
seite 12
PSF
tot
(x,y,z) =
(
PSF
ill
(x,y,z)
)
2
em
.
exc
2
exc
+ 2
em
5
2
FWHM
tot,axial
=
0.64 .
(n- n
2 -NA 2 )
5
1.28 . n .
NA 2
FWHM
tot,lateral
= 0.37
NA
(3)
Axial:
seite 7
PSF
tot
(x,y,z) = PSF
ill
(x,y,z)
.
PSF
det
(x,y,z)
seite 9
FWHM
ill,axial
=
seite 11
FWHM
det,axial
=
0.88 .
em
n- n
2
-NA
2
+
2 . n . PH
NA
2 2
0.88 .
exc
(n- n 2 -NA 2 )
5
1.77 . n .
exc
NA
2
FWHM
ill,lateral
= 0.51
exc
NA
seite 12
PSF
tot
(x,y,z) =
(
PSF
ill
(x,y,z)
)
2
em
.
exc
2
exc
+ 2
em
5
2
FWHM
tot,axial
=
0.64 .
(n- n
2 -NA 2 )
5
1.28 . n .
NA 2
FWHM
tot,lateral
= 0.37
NA
(2)
n
= refractive index of immersion liquid
NA = numerical aperture of the objective
λ
exc
= wavelength of the excitation light
OpticalImageFormation PART 1