Biology Reference
In-Depth Information
time gates. Two synchronized LIMOs are used to capture the time-resolved anisot-
ropy decays of each polarization direction. An acquisition time of 3 ms per pixel was
chosen and a threshold of 300 counts was applied. At this dwell time, the maximum
number of counts per pixel amounted to
4500. All images covered an area of
50
160 pixels.
Determination of the time-resolved anisotropy using the two separate detection
channels requires careful synchronization in time and correction for their difference
in sensitivity. The former was achieved by using reference dye with fast decay time
(aqueous solution of Rose Bengal,
50
m
m at 160
70 ps). The correction factor for the difference
in transmission (
C
tr
) between both channels was determined by recording an anisot-
ropy image of an aqueous solution of fluorescein. In this case, fast rotation of the
fluorophore will result in emission that is completely depolarized before the second
gate opens. Consequently, the anisotropy in the last three gates will be zero, and
I
par
¼
t¼
I
per
. Finally, a correction was applied to correct for small difference in gate
width between the parallel and perpendicular channel. These differences were deter-
mined using a sample containing 10
C
tr
m
M GFP monomers in 50/50 glycerol/buffer. In
this solution, rotation of the fluorophore is much slower than the fluorescence life-
time. Consequently, the anisotropy will remain constant at the
r
0
level. For every
gate, a correction factor was determined that ensures that the anisotropy in this gate
is identical to the steady-state anisotropy. The absence of concentration-dependent
homo-FRET in the GFP solution was checked by lowering the concentration; no in-
crease in anisotropy was observed. Steady-state anisotropy images were obtained by
summation of the intensities in all four gates.
16.3
METHODS
16.3.1
Slide preparation
16.3.1.1
Reference measurements
NIH3T3 2.2 cells stably transfected with cDNA encoding the indicated reference
protein were seeded on glass coverslips and the day after treated with 1
M
AP20187 for 1-2 h at 37
C to allow controlled dimerization and oligomerization. Af-
ter treatment, cells were fixed with 4% paraformaldehyde (PFA) and autofluorescence
was quenched by 100 mMglycine/PBS. Finally, the coverslips were embedded in 10%
polyvinyl-alcohol (Mowiol), and the anisotropy values were determined and used as
reference for the determination of cluster size. Samples can be stored at
m
4
Cuntil
further use.
16.3.1.2
Predimerization measurements
NIH3T3 2.2 cells were transfectedwith cDNAencoding either mGFP or EGFR-mGFP.
The cells were seeded on 18 mmglass coverslips and allowed to adhere overnight. Cells
were fixed with 4% PFA followed by the quenching of autofluorescence with 100 mM
glycine/PBS for 10 min. The coverslips were embedded in Mowiol and stored at 4 or
20
C until further use. However, determination of anisotropy immediately after
