Biology Reference
In-Depth Information
Eq.
(10.1)
as described in the preceding text. Diffusion rates for different types of
plasma membrane receptors are summarized in
Briddon & Hill, 2008
.
10.2.4.2
Molecular brightness
The average photon count rate (
k
) of a given sample is determined by the number of
fluorescent molecules (
N
) and their molecular brightness (e) as in Eq.
(10.6)
:
k
¼
N
e (10.6)
Thus, dividing the photon count rate (in kHz) by the number of molecules (
N
) yields
the molecular brightness (e) of the sample expressed as counts per second per mol-
ecule (CPSM). Note that the absolute numerical values of molecular brightness will
vary depending on whether a 2D or 3D model is used to calculate
N
in Eq.
(10.2)
.
This is due to the different numerical values of g used for 2D samples (where g
¼
0.5)
versus 3D samples (where g
0.35). Even though the surface of a cell may be 3D, the
plasma membrane is only 5 nm thick and doesn't fill the observation volume in the
axial direction (
¼
m). Thus, a plasma membrane is 2D with respect to the FCS
observation volume. However, as long as the same model is used for control and un-
known samples, the choice of 2D versus 3D for molecular brightness analysis of
plasma membrane receptors becomes less critical. This is illustrated in the example
provided in the succeeding text:
1
m
a.
FCS: FCS software packages automatically calculate the counts per molecule
(molecular brightness) by dividing the photon count rate by the number of
molecules. A 2D analysis of the FCS data presented in
Fig. 10.3
is performed by
dividing the average count rate obtained from the fluorescence intensity trace in
Fig. 10.3
A (130 kHz) by the number of molecules calculated as in Eq.
(10.2)
where
G
(0) is the amplitude (
y
-intercept) of the autocorrelation curve shown in
Fig. 10.3
B(
N
10). This yields a molecular brightness of 13,000 CPSM. In this
example, the beta2-adrenergic receptor and the dimeric CD-28 control have
similar molecular brightness, indicating that beta2-adrenergic receptors form
homodimers. Membrane receptor expression level can be determined by dividing
the number of fluorescence-tagged receptors in the observation volume (
N
,
calculated using Eq.
(10.2)
where g
¼
¼
0.5) by the area of the observation volume
calculated as po
2
(where o
0
is the radius of the observation volume determined
experimentally using Eq.
(10.1)
).
b.
PCH: Software packages with PCH modules are available commercially. PCH
analysis uses a 3D Gaussian approximation of the laser beam profile and Poisson
statistics to predict what the molecular brightness of the fluorescent particle
would be when it is at the center of the observation volume (
Chen et al., 1999
).
For PCH analysis, choose cells with a count rate of 50-250 kHz and use a bin time
of 10
s yielding 0.5-2.5 counts per bin (a good range for molecular brightness
analysis). If studying membrane receptors, photobleaching of the immobile
fraction will occur during the first two 10 s runs of the 100 s observation
period. Therefore, molecular brightness values from runs 3-10 are averaged.
m
