Monitoring Receptor Oligomerization by Line-Scan Fluorescence Cross-Correlation Spectroscopy - Methods in Cell Biology - page 203

Oligomerization

0
@

1
A

0
@

1
A

F background ; a
F total ;a

F background ; b
F total ;b

G a;b ðÞ¼ 1 þ 1 1 G dynamics þ Offset

0
@

1
A

X
m F m

T e t=t T

ð Þ

1 T þ 1

where G dynamics ¼ t

G a;b ðÞ

1 þ t

0
@

1
A

ð 1 T Þ

2

s

1 þ t
t dif

o xy
o z

t dif

m Y m

with F m ¼ ð 11 : 5 Þ

2

X m m Y m

Here, the first two factors between brackets correct for the background fluores-
cence in the corresponding detection channel ( F background, x ). The value of this param-
eter is determined by averaging the fluorescence intensity in the plasma membrane in
mock-transfected cells. In most cases, this correction can be neglected since typically
F background <

F total .
Each molecular species, m , contributes to the autocorrelation curve function
according to its fraction ( Y m ) and molecular brightness (

0.01

m ). This information is
obtained from PCH or N&B analysis. The ratio o xy / o z is fixed to the value obtained
from the calibration measurement for each channel. An offset can be included in the
fit to correct for the effect of small and slow intensity drifts in the selected data trace.
The amplitude of the fitted correlation curves, G a,b (0), contains the information
of the number of labeled particles. G CY (0) scales to the number of doubly labeled
particles N CY , as observed in the cross-correlation observation volume, V CY . Equa-
tions (11.6)-(11.8) describe the auto- and cross-correlation amplitudes, when cor-
rected for the presence of monomers and dimers. In addition, one assumes the
probability of forming CC-, CY-, or YY-labeled dimers is equal. The absence of
higher-order complexes is validated analyzing the same data sets by PCH confirming
the presence of molecules having a similar and an almost double (1.92

,
respectively) molecular brightness compared to (monomeric) membrane-targeted
mTq2 or sYFP2.

and 1.89

1

G CC 0 ðÞ¼ 1 þ (11.6)

2 N CC

N C þ N CY þ ð 1 : 92 Þ

1

G YY 0 ðÞ¼ 1 þ (11.7)

2 N YY

N Y þ N CY þ ð 1 : 89 Þ

N CY

G CY 0 ðÞ¼ 1 þ (11.8)

ð N C þ N CY þ N CC Þ ð N Y þ N CY þ N YY Þ