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ELYRA 7
Left Tool Area and Hardware Control Tools
ZEISS
03/2019 V_02
000000-2262-999
257
5.3.23.5
NandB Analysis
•
The function
NandB
(Number&Brightness)
Analysis
calculates the image content in two
ways:
−
It counts the photons per second per
molecule referred to as the
Brightness
.
−
It counts the number of molecules referred
to as
Number
.
•
Click
NandB Analysis
in the
ICS
tool. The
Method Parameters
tool shows the input
options for this function (Fig. 435).
•
Select the desired image in the image container,
then click
Select
to define this image as the
Input
image.
•
The tool works with time series images which
should be acquired along the following
guidelines:
−
It is necessary to oversample the images in
XY at least by a factor of 3 but better by up
to a factor of 10. A pixel size of about 30 nm
is beneficial as the method needs to be able
to detect the fluctuation of the molecules
within the confocal volume.
−
The imaging speed needs to be adapted to
the speed of the molecules movement. For
most experiments a speed of 6 or 7 (around
1 frame per second) has proven to be a fairly
good setting. The scanner needs to be fast
enough to "walk along" with the molecule:
Too slow, and the molecule "escapes", too
fast, and the molecule "stays behind".
For example:
EGFP (about 20nM) in buffer ph7.4: D=90 µm
2
/s -> Set the
Pixel dwell time to 25-32 µs/pixel and the
Pixel size to 80-50nm
•
Within the
Method Parameters
tool the following choices can be made:
−
Result Images
Number N, brightness B (measured);
This method calculates the apparent number
of molecules N and apparent brightness of molecules B according to the following formula:
)
1
(
)
(
2
2
2
2
0
2
+
=
+
=
−
−
=
ε
ε
ε
ε
σ
σ
S
n
S
n
S
n
S
offset
k
B
1
)
)
((
2
0
2
2
+
=
−
−
=
ε
ε
σ
σ
n
offset
k
N
Fig. 435
NandB Method Parameters tool