Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
ZEISS Left Tool Area and Hardware Control Tools LSM 880
198 000000-2071-464 10/2014 V_01
A. Amplitudes
The Amplitude of the correlation function is influenced by the offset, background and the number of
particles in dependence of the geometric factor. The amplitude is also influenced by the process of
correlation.
A.1 The " 1 "
In a normal correlation, the curve converges to 1, in case intensities I are correlated as is the case with the
FCS software. Note that in other cases, if fluctuations
d
I are correlated, the correlation function converges
to 0, if no bleaching occurs.
) ( 1 ) (
t t
I I
G G
+ =
∂
(2)
You can therefore easily convert G I (
t
) to G
d
I (t) values by adding a fixed offset of –1.
A.2 Offset d
c d
=
(3)
c is any rational number that can be negative or positive.
In the FCS software the offset can be a fit parameter or a fixed value.
In some cases, especially if very slow or immobile components are present, there can be a positive offset
from 1. This offset can be taken into account by fitting to d. On the other hand, if the offset is known, it
can be fixed. The offset will be added to each correlation value.
A.3 Background B
2
) 1 (
t
b
I
I
B
− =
(4)
where I
b
is the background intensity and I
t
is the total intensity.
The FCS software treats the background always as a fixed value and never as a fit parameter. Thus the
user has to define the background.
Note that the background in this case refers to a non-correlating background. If there is no background
intensity, B = 1, otherwise B < 1. The background can be determined by measuring an unlabeled solution
or cell at the same settings than the real experiment and recording the count rate I
b
. The real experiment
with the labeled species will give I
t
. A non-correlating background will result in a lower amplitude and
hence overestimation of molecule numbers, if not corrected for. Note the squared correction term.