Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Left Tool Area and Hardware Control Tools ZEISS
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−
Result Images:
Number b, brightness e (molecular);
This method calculates the number of
molecules n and their brightness e according to the following formula:
𝑛 =
(( 𝑘
)
−𝑜𝑜𝑜𝑠𝑠𝑠 )
2
𝜎
2
−𝜎
0
2
−𝑆 (( 𝑘 ) −𝑜𝑜𝑜𝑠𝑠𝑠 )
𝜀 =
𝜎
2
−𝜎
0
2
−𝑆 (( 𝑘
)
−𝑜𝑜𝑜𝑠𝑠𝑠 )
(( 𝑘 ) −𝑜𝑜𝑜𝑠𝑠𝑠 ) 𝑆
Legend for the fomulas:
N and n are apparent and corrected number of particles, respectively
B and
ε
are apparent and corrected brightness, respectively
〈
k
〉
is the mean signal expressed in digital levels (DLs)/pixel
σ
2 is the signal variance
σ
0
2
is the readout noise
offset is the dark signal (DLs/pixel)
S is the photon conversion factor (DLs/photon)
Min. Threshold sets a threshold for the intensity values to be taken into account for the calculation. This
is useful if the background in the image is interfering with the calculation. If the pixel value is below
[peak image value x Min. Threshold], the pixel is not processed. Intensity, number and brightness are set
to 0.
Detrend (Bleaching and Movement Correction) is a high pass recursive filter that corrects for
bleaching artefacts and the (much slower) movements for example of cells. When activated, the "trend"
(moving average – average over the whole time series) will be subtracted from the input time series prior
to N&B analysis.
Number of Frames is the number of images it takes for the recursive filter to decay to
1/e. Use a smaller number for detrending faster moving objects like vesicles and a higher number for
detrending bleaching effects or slower cell movements.
Photon Counting : If the input image has been acquired in Photon Counting mode, this button is
automatically set active.
Integration Mode : If the input image has been acquired in Integration mode, this button is
automatically set active. For images acquired in integration mode the following parameters are displayed
and can be adapted:
Offset : Is set to 0 per default and does typically not need to be changed. The value 0 is taken for
photomultiplying detectors. It is the value take for the calculation (see formulas above).
Readout Noise
: Is set to 1 per default. This is a parameter typical for PMT type detector. Other detectors
like cameras do have another value. It equals
σ
0
2
within the formulas above.
Photon Factor : Is the photon conversion factor taken from the meta data of the image (if available) and
describes the digital levels per photon (S in the formulas above).