Zeiss LSM 880 Operating Manual page 664

FWHM
ill,axial
seite 9
FWHM
FWHM
ill,lateral
Wave-opticalconfocality
If the pinhole is closed down to a diameter of <0.25 AU (vir- FWHM
seite 11
tually "infinitely small"), the character of the image changes.
Additional diffraction effects at the pinhole have to be taken
into account, and PSFdet (optical slice thickness) shrinks
FWHM
det,axial
to the order of magnitude of PSF
figure 7c). seite 11
In order to achieve simple formulae for the range of smallest
FWHM
pinhole diameters, it is practical to regard the limit of PH = 0
FWHM
at first, even though it is of no practical use. In this case,
PSF and PSF are identical. The total PSF can be written as:
seite 12
det ill
PSF (x,y,z) =
FWHM
tot
seite 12
In fluorescence applications it is furthermore necessary to
.
consider both the excitation wavelength λ
PSF
em
2
tot
2 +
sion wavelength λ
em
exc
wavelength :
1
2
FWHM =
tot,axial
FWHM
tot,axial
FWHM = 0.37
tot,lateral
FWHM
tot,lateral
1 For rough estimates, the expression
14
0.88 .
seite 11
exc
=
2 2
(n- n -NA )
FWHM =
det,axial
0.88 .
exc
=
1.77 . n .
ill,axial
(n- n 2 -NA exc 2 )
2
NA
1.77 . n .
exc exc
= 0.51
seite 12
NA NA 2
(
PSF (x,y,z) = PSF (x,y,z)
tot ill
exc
= 0.51
ill,lateral
NA
.
0.88 .
2
em exc
2
= em +
2 + 2
(Z resolution) (see also
n- n 2 -NA 2
exc em
ill
0.88 .
=
0.64 .
det,axial
=
2
n- n -NA
tot,axial
2 2
(n- n -NA
2
( )
PSF (x,y,z)
= 0.37
ill
tot,lateral
NA
( ) 2
and the emis-
(x,y,z) = PSF (x,y,z)
exc
exc
ill
2
. This is done by specifying a mean
em
.
em exc
0.64 .
2 2
+
exc em
2 2
(n- n -NA )
0.64 .
=
(n- n 2 -NA 2 )
NA
= 0.37
NA
λ
 ≈ √λ ·λ suffices.
em exc
0.88 .
2
2 . n . PH
em +
NA
n- n 2 -NA 2
Thus, equations (2) and (3) for the widths of the axial and
2
)
lateral half-intensity areas are transformed into:
Axial:
FWHM
2
2 . n . PH
NA
If NA < 0.5, equation (2) can be approximated by:
2 2
2 . n . PH
em + FWHM
FWHM
NA
2
)
Lateral:
FWHM
(5)
(6)
1.28 . n .
NA
1.28 . n .
Note:
Withtheobjectbeingamirror,thefactorinequa-
tion7is0.45(insteadof0.64),and0.88(insteadof
1.28)inequation7a.Forafluorescentplaneoffinite
thickness,afactorof0.7canbeusedinequation7.
Thisunderlinesthatapartfromtheparameters(NA,
λ  ,n)influencingtheopticalslicethickness,thetype
ofspecimenalsoaffectsthemeasurementresult.
seite 11
2 FWHM =
det,axial
seite 12
(
PSF (x,y,z) = PSF (x,y,z)
tot ill
seite 12
.
em exc
2
(
PSF 2 (x,y,z) = + 2 PSF (x,y,z)
exc em
tot ill
.
0.64 .
em exc
=
2
tot,axial
2 + 2 2 2
(n- n -NA )
exc em
0.64 .
=
1.28 . n . = 0.37
tot,axial
tot,lateral NA
(n- n 2 -NA 2 )
NA 2
= 0.37
tot,lateral NA
2
NA 2
0.88 .
2
2 . n . PH
em +
NA
2 2
n- n -NA
2
)
2
)
(7)
(7a)
(8)
2
1.28 .
NA
1.28 .
NA