Biomedical Engineering Reference
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Fig. 3.4
Tracking the internalization of transferrin receptor-ligand complexes in live polarized
MDCK cells using confocal FRET microscopy. (
a
) The organization and distribution of pIgA-R
and TFR complexes in endosomes that are localized at the basolateral (
blue rectangle
)and
perinuclear (
green rectangle
) regions was assayed using confocal FRET microscopy after coin-
ternalization of Cy3-Tfn (FRET acceptor) and Alexa488-pIgA-R ligand (FRET donor) from the
basolateral PM of live polarized MDCK-PTR cells. (
b
) Pseudocolor images, processed using
the PFRET algorithm in combination of single-label images (see Sect.
3.3.4
), depict the pixel-
by-pixel distribution of the “donor to acceptor” ratio (D/A - a, d), acceptor (b, e), and apparent
FRET efficiency (E% - c, f) levels at the perinuclear (a-c) and the basolateral (d-f) regions.
Examples of selected ROIs that were selected according to the definitions of basolateral and
perinuclear endocytic regions are shown as
white squares
.(
c
and
d
) The E%, D/A, and acceptor
levels were calculated for a wide range of ROIs (
white squares
). E% values were plotted as
a function of the acceptor levels for D=A 1 () and for D=A 2./, with
trend lines
(D=A 1,
dotted line
;D=A 2,
dashed line
). (
c
) E% is largely independent of the acceptor
levels and increases with decreasing D/A ratios, suggesting a clustered organization of TFR and
pIgA-R complexes in perinuclear endosomes. (
d
) E% shows a variable dependency on the acceptor
levels depending on D/A, indicating a mixed/random organization of TFR and pIgA-R complexes
in basolateral endosomes. More details about this assay are described in the literature [
52
-
54
]
(Adapted from [
54
])
approximately half of the energy to excite a molecule in a single-photon excitation
event, are simultaneously absorbed by the molecule, resulting in the emission of a
fluorescence photon. The probability for the two-photon absorption depends on the
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