Zeiss LSM 880 Operating Manual page 205

LSM 880
A.4 Amplitude A
γ
= = ( −
A G ) 0 1
N
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
=1.000 (cylindrical),
C
γ
=0.500 (2-D Gaussian),
2DG
γ
=0.350 (3-D Gaussian),
3DG
γ
=0.076 (Gaussian-Lorentzian).
GL
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
N
=
c
⋅
V L
A
with V being the confocal volume and L
The volume V is calculated from equation
3
π ω ω
= ⋅ ⋅
2
2
V
r z
with ω axial focus radius and ω
z
calibration measurement using a dye with a high quantum yield and a known diffusion coefficient D from
the fitted diffusion time t
state.
10/2014 V_01
CHAPTER 1 - SYSTEM OPERATION
Left Tool Area and Hardware Control Tools
= 6.023 x 10
A
the lateral focus radius. The radii themselves have to be determined by a
r
and the structural parameter S employing a free diffusion model with triplet
d
000000-2071-464
23 -1
mol the Avogadro number.
ZEISS
(5a)
(5c)
(5d)
199