Zeiss LSM 880 Operating Manual page 512

ZEISS Center Screen Area / Image Containers - Display and Image Analysis
N-FRET or Xia method:
Xia and Liu, Biophys J 81, 2395 (2001)
NFRET = FRET 1/ √(Dfd*Afa)
Displays an N-FRET image with intensities converted from the FRET index calculated for each pixel using
the Xia method. This method is similar to the Gordon method with the difference that for concentration
compensation the square root of donor and acceptor concentration is used. The resulting image is
properly corrected for variances in the fluorochrome concentration.
Fig. 701 FRET View Options Control Block,
Parameter tab, Last Analysis
The Donor coefficient include the Donor signal detected in the FRET channel/Donor signal detected in
the Donor channel (Fd/Dd) and the Donor Signal detected in the Acceptor channel/Donor signal detected
in the FRET channel (Ad/Fd). These values are saved for future use as a .fret file by choosing Donor in
the drop-down list and pressing afterwards the Save button.
To obtain the Acceptor coefficient values an image of a sample with only acceptor fluorescence is taken
and one or more regions are defined as objects and enabled in the FRET tab and the Analyze Acceptor
button is pressed.
The values for the Acceptor coefficient include the Acceptor signal detected in the FRET channel /
Acceptor signal detected in the Acceptor channel (Fa/Aa), the Acceptor signal detected in the Donor
channel / Acceptor signal detected in the Acceptor channel (Da/Aa), the Acceptor signal detected in the
Donor channel/Acceptor signal detected in the FRET channel (Da/Fa). These values are saved for future
use as a .fret file by choosing Acceptor in the drop-down list and pressing afterwards the Save button.
The Gordon factor G depends on the fluorphores used and is part of the Gordon and Xia analysis. Please
refer to the original publications for further details (Gordon et al., Biophys J 74, 2702 (1998),
Biophys J 81, 2395 (2001)).
The total Number of images is set using the slider or by editing the field next to the slider, to choose
how many images of a time series shall be taken into account. Their values will appear in the table below
the image.
To load the saved Acceptor and Donor coefficient values for FRET analysis Donor or Acceptor are chosen
in the drop-down list respectively. Afterwards the Load button is pressed and the previously saved .fret
file chosen.
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CHAPTER 1 - SYSTEM OPERATION
Parameter tab (Fig. 701):
Various Parameters of the image can be set in this
tab.
Prior to analysis the channels of the image need to
be assigned appropriately using the drop down
menu next to Donor ch.:, Acceptor ch.: and FRET
ch.:.
To determine the Donor coefficient values by
using a sample with only signal of the donor
fluorophore, one or more regions in the image are
defined and enabled as objects in the FRET tab. Of
this region the Donor coefficient values are taken
by clicking the Analyze Donor button.
000000-2071-464
LSM 880
Xia and Liu,
10/2014 V_01