Biology Reference
In-Depth Information
wavelength and NA is the numerical aperture of the objective. The number of
molecules in the observation volume (
N
) is calculated as in Eq.
(10.2)
:
1
G
0
N
¼
ðÞ
g
(10.2)
where
G
(0) is the amplitude of the autocorrelation curve (
y
-intercept) and g is
the point spread function describing the shape of the observation volume (for
calibration dye, use g
0.35 for a 3D Gaussian confocal observation volume).
Commercially available software designed for FCS analysis will automatically display
G
(0), the number of molecules, the count rate, and the diffusion time in a table. Use the
number of molecules (
N
) to calculate the confocal volume (
V
)asinEq.
(10.3)
:
¼
N
V
¼
(10.3)
C
Na
where
C
is the concentration of calibration dye (20 nM) and Na is Avogadro's num-
ber. Finally, calculate the structural parameter (
s
, the ratio of the axial length to radial
width of the observation volume) as in Eq.
(10.4)
:
V
po
0
s
¼
(10.4)
Successful instrument alignment and calibration will produce a structural parameter
between 3 and 8.
10.2.3
Data acquisition
10.2.3.1
FCS recording
Always perform a pinhole adjustment using a sample of the fluorescent probe chosen
to label the receptor of interest before beginning an FCS recording session. Begin and
end each session with control samples and perform FCS recordings on the isotonic
viewing solution and in the middle of untransfected cells to establish autofluores-
cence levels.
a.
Prepare a coverslip of control cells transfected with plasmid containing the
chosen fluorescent probe (e.g., monomeric GFP or YFP expressed in the cytosol)
by washing three times using an isotonic solution such as PBS, Krebs' ringer, or
phenol red-free MEM. Place the coverslip in a viewing chamber and add 1 ml of
room temperature isotonic solution buffered with 10 mMHEPES. Place a drop of
HPLC-grade water on the 40
objective, place sample on the microscope stage,
and raise the objective until the waterdrop touches the coverslip. Working
quickly, illuminate the sample and focus upward until the cells are visible, and
then turn off. It is important to minimize illumination and viewing of the cells to
minimize photobleaching.
b.
Collect an image of the cells and display on the computer screen. Choose a cell
of low to medium brightness (avoid bright cells) and adjust the zoom setting to
3. Working quickly, use fast continuous scanning to position the cell in the
