Nandb Analysis - Zeiss LSM 880 Operating Manual

ZEISS
Fig. 546 NandB Method Parameters tool
For example:
EGFP (about 20nM) in buffer ph7.4: D=90 µm
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:
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CHAPTER 1 - SYSTEM OPERATION
Left Tool Area and Hardware Control Tools
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5.4.21.5

NandB Analysis

• The
function
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
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".
2 /s -> Set the Pixel dwell time to 25-32 µs/pixel and the
NandB (Number&Brightness)
acquired along the following
10/2014 V_01
LSM 880