Biology Reference
In-Depth Information
11.2.2
Microscope calibration
The microscope is placed in an air-conditioned room to ensure temperature stability,
and the calibration samples are stored in glass-bottomed 96-well plate. In order to
reduce the amount of detected background light, a protective blackened aluminum
cover is placed on top of the sample during the measurements. The microscope and
lasers are turned on roughly 30 min before starting the measurements in order to sta-
bilize. The mTurquoise2-labeled samples will be excited with the 440 nm laser line
pulsing at 20 MHz and the sYFP2 samples are excited using the 514 nm line of an
Ar
þ
laser.
To optimize the microscope settings, the fluorescence of a
100 nM solution of
12 kW cm
2
in the
focal plane. The correction collar of the objective lens is adjusted to the position such
that the highest fluorescence count rate is observed in one of the two detection chan-
nels. In order to prevent that scattered excitation light or fluorescence from adsorbed
probe at the bottom of the sample holder is optimized, instead of the fluorescence
signal from the sample of interest, the focus should be set at least 10
purified mTurquoise2 in PBS is measured at a laser power of
m into
the solution. Furthermore, the position of the pinhole is optimized by moving the
X-, Y-, and, if available, the Z-position of the pinhole that yields the highest detected
fluorescence of mTq2. The lens position in front of the APD is optimized as well in a
similar way.
The purified mTq2 or sYFP2 samples are measured, using their corresponding
laser lines at 12 (440 nm) and 9 kW cm
2
(514 nm). The acquisition time, typically
2 min, was adjusted such that the resulting correlation curves are smooth in the
decaying part of the curve. The intensity traces are imported into the FFS data pro-
cessor 2.3 software, autocorrelated, and fitted to a model (Eq.
11.1
) including terms
for triplet state kinetics and three-dimensional Brownian diffusion (
Skakun et al.,
2005
). Because of the relatively slow scanning speed and the oversampling (pixel
size of
m
100 nm) in the line-FCCS experiments, the
y
- and
z
-dimensions of the de-
tection volumes can be estimated from point FCS measurements. In order to estimate
the size of the cross-correlation detection volume, the mTq2 signal, as acquired in
both detectors, is cross-correlated, using the relative large number of mTq2 cross-
talk photons that are detected in the YFP channel.
0
@
1
A
T
e
t=t
TRIP
ð
Þ
N
1
T
þ
1
q
1
q
1
q
1
G ðÞ¼
1
þ
(11.1)
ð
T
Þ
1
þ
t
t
dif
;x
þ
t
t
dif
;y
þ
t
t
dif
;z
The autocorrelation function,
G
(
t
), contains a parameter
N
, which corresponds to the
average number of fluorescent particles in the detection volume.
t
dif
denotes the av-
erage diffusion time of the molecules,
T
denotes the fraction of molecules present in
the dark state, and
t
TRIP
is the average time a molecule resides in the dark state. Pa-
rameter
g
represents the shape factor of the observation volume and equals 0.3535 for
a 3D Gaussian or 1.0 for a cylindrical-shaped observation volume. From the auto-
correlation fits a shape factor,
a,
describing the ratio between the axial (
o
z
) and
