Zeiss LSM 880 Operating Manual page 510

ZEISS Center Screen Area / Image Containers - Display and Image Analysis
Fig. 697 FRET View Options Control Block,
Thresholds tab
Fig. 698 FRET View Options Control Block,
Settings tab, General
Exclude saturated (max intensity) pixels: Pixels that are saturated in at least one channel will not be
considered for analysis.
Exclude zero intensity pixels: Pixels with zero intensity in any channel are not part of analysis. This
checkbox is always checked.
Include thresholded pixels: Pixel with the same grey value as the defined thresholds will be part of
analysis when checked. This is usually not activated.
Generate palette: The palette of the FRET image is displayed color coded when checked.
Show palette: This checkbox allows to choose if the palette of the FRET values is displayed within the
FRET image or not.
Fig. 699 FRET View Options Control Block,
Settings tab, Acceptor Photobleach
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CHAPTER 1 - SYSTEM OPERATION
Thresholds tab (Fig. 697):
The threshold for the image analysis can be set
manually using the slider or editing text box next
to Donor, Acceptor or All, where All moves both
sliders to the same value.
Alternatively, the threshold can be set by defining
and enabling a background region in the FRET tab.
The values for the thresholds are either displayed
as grey value levels (Raw data) or Normalized to
the value 1.
Settings tab (Fig. 698):
This tab provides access to the parameters used for
the image analysis.
The General tab applies to both methods,
Acceptor Photobleaching and Sensitized Emission.
If checked the listed options will be taken into
account for the analysis of FRET.
Subtract background using thresholds: The
previously defined grey values of the thresholds
(FRET tab, Thresholds tab) are subtracted from
each pixel prior to analysis. If not checked, pixel
below the defined thresholds will not be displayed
in the FRET Image and are not part of the analysis
(value and area).
The Acceptor Photobleach tab (Fig. 699) sets
additional parameters to analyze the Acceptor
Photobleaching experiment such as the choice for
Truncation (as Remove Max or No Truncation)
and the option not to display negative values in the
image (Don't show negative values in the FRET
image).
000000-2071-464
LSM 880
10/2014 V_01