LSM 710 and LSM 780
CENTER SCREEN AREA / IMAGE CONTAINERS - …
Systems
FRET View
Carl Zeiss
02/2010 M60-1-0025
e
237
Fc
or
Youvan
method:
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 cells that have higher FRET molecular concentrations.
FRETN
or
Gordon
method:
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
by overdoes it. As a result cells with higher molecular concentrations report lower FRET values.
N-FRET
or
Xi
method:
Displays an N-FRET image with intensities converted from the FRET index calculated for each pixel using
the Xi 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.
Parameter tab (Fig. 267):
In case the image series for
Sensitized Emission
has not been acquired with the FRETplus Macro,
various Parameters of the image series can be set
in this tab for the
Last analysis
, the
Acquisition
and the
Image analysis
.
The values for the different Analysis methods to be
set (from the images) for the
Donor coefficient
include the FRET signal detected in the donor
channel/Donor signal detected in the Donor
channel (
Fd/Dd
)
and the Acceptor Signal detected
in the Donor channel/FRET signal detected in the
Donor channel (
Ad/Fd
).
The values for the
Acceptor coefficient
include the FRET signal detected in the Acceptor channel /
Acceptor signal detected in the Acceptor channel (
Fa/Aa
), the Donor signal detected in the Acceptor
channel / Acceptor signal detected in the Acceptor channel (
Da/Aa
), the Donor signal detected in the
Acceptor channel/FRET signal detected in the Acceptor channel (
Da/Fa
), and the Gordon factor
G
.
The image of the Donor the Acceptor and the FRET are assigned to the appropriate channel using the
drop down menu next to
Donor ch.:
,
Acceptor ch.:
and
FRET ch.:
.
The total
Number of images
is set using the slider or by editing the field next to the slider if necessary.
By clicking the button
Donor
or
Acceptor
the analysis is performed with the new/edited parameters and
a new image view is added to the image container (if not already present).
Use the buttons for Donor, Acceptor and FRET in the lower left corner of the image view to alternatively
display the three different images.
Fig.
267
FRET View Options Control Block,
Parameter tab, Last Analysis
