Zeiss LSM 880, Руководство по эксплуатации

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS
10/2014 V_01 000000-2071-464 505
6.18.2 Tools in the FRET View Options
Control Block for Sensitized
Emission
FRET tab (Fig. 700):
Within this tab overlay regions can be defined.
(The drawing tools correspond to the drawing
tools described in section
Graphics ).
The checkboxes Numbers and Measure refer to
the overlay regions and annotate the number of
the region and its area to the overlay in the image.
Export
allows the resulting FRET image to be
saved as a separate image file.
The regions defined can be set as
Object (Region where FRET should be calculated or the reference
coefficient values are determined from) or Background (from which the threshold for the image analysis
is deduced and automatically used for analysis and displayed in the Threshold tab ).
Regions and the assigned status as Object or Background can be selected or deselected for the individual
analysis using the corresponding check box under
Enabled .
To start analyzing the
Analyze
button must be clicked once. If all further changes shall be effective
immediately the On the fly checkbox can be checked.
Three different methods for FRET analysis are available in the drop down list:
−
Fc (Youvan) ,
−
FRETN (Gordon)
and
−
N-FRET (Xia) .
Fc or Youvan method:
Youvan et al., Biotechnology et alia 3, 1 (1997)
Fc = Ff-Df(Fd/Dd)-Af(Fa/Aa)
Displays the Fc image with intensities converted from the FRET index calculated for each pixel using the
Youvan method. This method assumes that the signal recorded in the FRET channel is the sum of real
FRET signal overlaid by donor crosstalk and acceptor signal induced by direct (donor) excitation. There is
no correction for donor and acceptor concentration levels and as a result the FRET values tend to be
higher for areas with higher intensities.
FRETN
or
Gordon
method:
Gordon et al., Biophys J 74, 2702 (1998)
FRETN = FRET1/ Dfd * Afa
Displays the FRET image with intensities converted from the FRET index calculated for each pixel using the
Gordon method. This method calculates a corrected FRET value and divides by concentration values for
donor and acceptor. This method attempts to compensate for variances in fluorochrome concentrations
but overdoes it. As a result cells with higher molecular concentrations report lower FRET values.
Fig. 700 FRET View Options Control Block,
FRET tab