Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS
10/2014 V_01 000000-2071-464 489
•
Switch to the FRAP View tab.
•
Mark the check boxes for background and reference region.
•
The intensity values of the ROI to be analyzed will be corrected for background intensity and changes
in intensity calculated for the reference region. The correction is done for each time point taking the
actual intensity difference in the reference region into account.
The remaining ROI or ROIs are used for analysis when checked active for either group 1, 2 or 3. ROIs
assigned to the same group are analyzed added for analysis.
•
Chose the
Kinetic Model
in the pull down list.
−
The result of the fit is displayed in the table. The result can be copied to the clipboard (Copy
Results ) or saved as a text file ( Save Results ) (right-mouse click). The following values are
calculated and shown:
−
The final signal intensity in the analyzed ROIs following recovery I0 (of the fitted curve).
−
The amplitude of the fitted curve (which equals the mobile fraction)
I1 mobile fraction
.
−
The fitted parameter T1 (seconds).
−
The rate constant for the exchange of molecules between the bleached region and the surrounding
area
K (per second).
−
The part of the immobile fraction of the protein I delta immobile fraction .
Checking the Table tick box opens a further table in the image display area. It shows all intensity values
for each channel and ROI analysis group over time. These values are corrected for background intensity
and intensity loss of the reference region.
The values can be saved as a text file (
Save Table
) or copied into Excel via right-mouse click (clipboard)
( Copy Table ).
The data can be normalized optionally when marking the check box Normalize in the FRAP View
Options control block.
The calculation of the parameters is based on the same ROIs unless other ROIs or moved ROIs
are selected again. The Kinetic Display is always available once the analysis has been performed.
The analysis is not stored with the image.
Note that this modeling is a very basic approach to your experiment. It offers a first hint on the
possible presence of only one or, in case of a bad fit, more than one mobile fractions of the
labeled protein within the cell or cell compartment examined. For a more advanced analysis refer
to the scientific literature.
The half time of the recovery can be calculated using the following formula: -t
half
= (ln 0.5)*T1