Zeiss LSM 880, Operating Manual

Page: 394 / 690

CHAPTER 1 - SYSTEM OPERATION
ZEISS Left Tool Area and Hardware Control Tools LSM 880
388 000000-2071-464 10/2014 V_01
5.4.21.5 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. 546).
•
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:
𝐵 =
𝜎
2
−𝜎
0
2
(
𝑘
)
−𝑜𝑜𝑜𝑠𝑠𝑠
=
𝑆
2
𝜀
2
𝑛+𝑆
2
𝜀𝑛
𝑆𝜀𝑛
= 𝑆 ( 𝜀 + 1)
𝑁 =
(( 𝑘
)
−𝑜𝑜𝑜𝑠𝑠𝑠 )
2
𝜎
2
−𝜎
0
2
=
𝜀𝑛
𝜀+1
Fig. 546 NandB Method Parameters tool