Zeiss LSM 880 Operating Manual page 661

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