Biology Reference
In-Depth Information
sensitivity of the instrument (on the Zeiss ConfoCor 3, we use 1%, and on the LSM-
780, we use 0.1%) and must be determined experimentally. Select a correlation bin
time of 0.2
s, and data acquisition time as 10 s for 10 con-
secutive repetitions (runs). For proper signal statistics, the acquisition time should be
s, PCH bin time of 10
m
m
1000-fold over the diffusion time of the protein being studied. For membrane re-
ceptors, with diffusion times on the order of 10-100 ms, a total acquisition time of
100 s is sufficient. It is essential to use the same settings for all FCS recordings.
10.2.2.4
Instrument alignment and calibration
Align the light beam path and pinhole, and determine the dimensions of the obser-
vation volume.
a.
Clean the lens of the 40
objective and adjust the collar to match coverslip
thickness (0.17 mm for no.1.5 and 0.15 mm for no.1.0 coverslip thickness). Place
a drop of HPLC-grade (ultrapure, low fluorescence) water on the objective lens.
b.
Prepare a 20 nM dilution of calibration dye, such as rhodamine 6G, fluorescein
(pH
l drop on a coverslip or MatTek dish. Be
aware that sample evaporation will concentrate the sample and introduce error
into the calibration process.
c.
Place the holder with dye solution on the microscope stage and raise the objective
until the water just contacts the surface of the coverslip. Adjust the focus upward
until the focal plane of the laser is in the middle of the dye solution.
d.
Using the imaging and FCS settings established in the preceding text (for the
fluorescent probe used to label your receptor of interest, not the calibration
dye), capture an image of the dye solution. In the imaging setup menu, set the
zoom to 3 and capture another image. Locate the “region of interest” marker
and position the marker in the center of the screen. Perform pinhole adjustments
for the
X
and
Y
planes.
e.
Begin an FCS recording and adjust the laser setting to give a count rate of
approximately 50 kHz. Begin an FCS recording for 10 consecutive 10 s intervals.
Repeat. Fit the data to a 3D model for Brownian diffusion and perform an
autocorrelation analysis. The autocorrelation curve represents the time-dependent
decay in the fluorescence signal. The midpoint of the curve provides a measure of
t
D
(the average dwell time of the fluorescent particles in the observation volume).
Use t
D
to calculate the radius of the observation volume (o
0
)asinEq.
(10.1)
:
7.5), or GFP. Place a 100
>
m
p
4
D
t
D
o
0
¼
(10.1)
where
D
is the known diffusion coefficient of the calibration dye (rhodamine
6G, or fluorescein, 400
m
2
/s; GFP in solution, 87
m
2
/s). The theoretical value
m
m
of o
0
is calculated as 0.61
l/NA where l is the laser
