Zeiss LSM 880, Operating Manual

Page: 205 / 690

CHAPTER 1 - SYSTEM OPERATION
LSM 880 Left Tool Area and Hardware Control Tools ZEISS
10/2014 V_01 000000-2071-464 199
A.4 Amplitude A
1 ) 0 (
− = =
G
N
A
γ
(5a)
where
γ
is the geometric factor accounting for the point spread function (PSF) and N the mean number of
particles.
In the FCS software
γ
can be a fit or a predefined fixed value. In case
γ
is a fit value N must be fixed in the
fit procedure. N is normally a fit parameter.
Please note that
γ
takes different values for different fitting models depending on the assumed intensity
distribution of the point spread function (PSF):
γ
C
=1.000 (cylindrical),
γ
2DG
=0.500 (2-D Gaussian),
γ
3DG
=0.350 (3-D Gaussian),
γ
GL
=0.076 (Gaussian-Lorentzian).
γ
can also be calibrated, if a known concentration c of a dye is measured. In this case N can be fixed and
γ
fitted. The obtained number can be entered as the calibrated fixed number. N can be calculated from
equation
A
L V
N
c
⋅
=
(5c)
with V being the confocal volume and L
A
= 6.023 x 10
23
mol
-1
the Avogadro number.
The volume V is calculated from equation
z r
V
ω ω π ⋅ ⋅ =
2
2
3
(5d)
with
ω
z
axial focus radius and
ω
r
the lateral focus radius. The radii themselves have to be determined by a
calibration measurement using a dye with a high quantum yield and a known diffusion coefficient D from
the fitted diffusion time
t
d
and the structural parameter S employing a free diffusion model with triplet
state.