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A
B
Fluorescence
Measured fluorescence
Field illumination
Inhomogeneous field
illumination on a dual view
l FRET / l donor
X
=
=
X
=
Differences between
l FRET and
l donor fields illumination
C
D
Cell movement between l FRET and l donor acquisitions
Figure 5.24 Field illumination and cell movement between
I donor acquisition
channels effects on ratio imaging. For each panel, the cell exhibiting an equal
I FRET and
I FRET and
I donor is imaged through a nonhomogeneous field illumination and ratio image is cal-
culated. Procedure is detailed in (A). The expected ratio value is thus equal to one for the
whole image. In (B)-(D), we can see erroneous ratio images due to different sources of
artifacts. See text for more details.
and will strongly depend upon the acquisition technique used for measuring
the ratios (sequential ratio, SR; dual view ratio, DVR; dual channel ratio,
DCR).
7.1.1 Field illumination
Nonhomogeneous field illumination and light collection is a classical prob-
lem encountered both in wide-field and in confocal microscopy and can
yield to significant differences in intensity measurements from the center
to the border of an image. We encourage users to verify the field homoge-
neity of the employed system using a reference sample. In ratio imaging, this
may not be a major issue if the field nonhomogeneity is the same for the
sequential ratio (this is most of the time the case for SR, Fig. 5.24A ). Indeed,
if each pixel of the emitted I FRET and I donor images is multiplied by a same
factor, the ratio measurements will stay accurate despite nonhomogeneities
in the field. On the contrary, for DVR or DCR, this may be a major issue
because both images are measured on either two distinct detector parts or
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