Biology Reference
In-Depth Information
Oligomerization
lateral (
o
xy
)e
2
radii of the detection volume (
a
¼o
z
/
o
xy
) is obtained. Although the
shape of the detection volumes is close to a 3D Gaussian in “ideal conditions,” the
optical aberrations introduced by measuring inside the living cell allow to approx-
imate the detection volumes by a cylindrical shape. The amplitude of the autocorre-
lation function,
G
(0), equals
g
/
N
where
g
corresponds to the shape of the detection
volume. In all the equations used in this chapter, cylindrical detection volumes are
assumed with
g¼
1.
To validate the correct calibration of the microscope, the obtained fitting param-
eters are checked. For 3D diffusion in an open cylindrical volume, as measured here,
the diffusion times in
x
- and
y
-direction are equal and the diffusion time in the
z
-direction along the optical axis is given by
t
dif
;z
¼
o
z
t
dif
;x
(11.2)
o
2
xy
The diffusion times
t
dif,
x
for mTq2 or sYFP2 are in the order of 180
s and the shape
factors
a
range from 4 to 15. The observed brightness values vary between 8 and
12 kHz per molecule. Note that these values will be different for other microscope
systems. In general, the higher the brightness per particle, the better the quality of the
obtained data will be. The system has to be calibrated such that the highest possible
brightness is reached without photobleaching and/or saturation artifacts (
Gregor,
Patra, & Enderlein, 2005
).
By solving Eqs.
(11.3) and (11.4)
, using the translational diffusion coefficient (
D
)
of 90
m
m
2
s
1
for both mTurquoise as sYFP2 in buffer, the dimensions of the “cyan”
and “yellow” detection volumes,
V
, can be calculated by approximating these as
cylinders:
m
2
xy
4
D
o
t
dif
;x
¼
(11.3)
3
xy
V
¼
2
p
a
o
(11.4)
11.2.3
Fluorescence fluctuation measurements
A coverslip with the transfected HeLa cells is sealed in an Attofluor cell chamber
(Invitrogen) submerged in microscopy medium. Note that growth media should
not be used since this will acidify when incubated in the absence of 5% CO
2
during
the measurement.
The effective cross-correlation observation volume is estimated measuring cross-
correlation using a positive control, for example, cells producing a fusion protein of
mTq2-p63-sYFP2. To select transfected cells with FFS-compatible expression
levels, the sample is imaged by the confocal microscope using standard imaging set-
tings for the PMT detector sensitivity while the pinhole is completely open. The cells
