Biology Reference
In-Depth Information
analysis is based upon moment analysis and allows for measurement of the average
number of molecules as well as brightness in each pixel of a fluorescent microscopy
image (
Digman, Brown, et al., 2008, Digman, Dalal, Horwitz, & Gratton, 2008
).
N&B utilizes the first and second moment of amplitude fluctuations from the histo-
gram of amplitude oscillations, which is originally derived from the photon-counting
histogram method (
Chen, Muller, So, & Gratton, 1999
). The average brightness of a
particle is determined from the ratio of variance to intensity at each pixel. Fluctuating
particles can be determined by dividing the average intensity by the brightness at
each pixel. For particles fluctuating in the focal volume, the variance is proportional
to the square of the particle brightness; however, the variance of the immobile
particles and detector noise is proportional to the intensity of these components.
Thus, only fluorescent fluctuations that are dependent upon the mobile particles have
a ratio of the variance to intensity
1. Aggregation of mobile fluorescent particles
can then be determined from a brightness map with pixel resolution.
For an electron-multiplying CCD camera, the following equations are used to
compute the N&B:
>
2
ð
hi
>
I
Þ
offset
N
¼
(19.1)
2
s
0
s
2
0
s
s
B
¼
(19.2)
hi
I
offset
In these equations,
N
and
B
are the apparent number and the brightness of the mol-
ecule,
h
I
i
is the average intensity of a pixel throughout a stack of frames (time av-
s
2
are properties that depend on the
camera hardware. With these parameters properly calibrated, the distribution of
the brightness of each individual pixel in the image of a section of a cell can be in-
vestigated. This approach is therefore useful to study interactions of fluorescently
labeled proteins that occur on cellular membranes.
Cellular membranes contain hundreds if not thousands of different lipids, which
are highly dynamic. Specific lipids in the membrane bilayer (
Lemmon, 2008;
Stahelin, 2009
) or lipid anionic charge (
Yeung et al., 2006
) can regulate recruitment
of peripheral proteins to the membrane interface. These signaling cues are vital to
membrane trafficking and signal transduction as it is estimated that nearly half of
all proteins in the human genome are localized in or on membranes. A large number
of peripheral proteins are recruited to cellular membranes through modular lipid-
binding domains that are often found in signal transduction and membrane-
trafficking proteins (
Cho & Stahelin, 2005
). These modular lipid-binding domains
can bind, often with nanomolar affinity, to membranes containing a specific lipid
ligand or recognize membrane physical properties such as charge or curvature
(
Lemmon, 2008
). Binding to the membrane interface and restricting the protein to
two dimensions reduce dimensionality and provide a platform for signal transduction
to occur. In some cases, peripheral proteins may interact or oligomerize on the mem-
brane interface, an event that is important in biological processes such as generation
2
erage),
is the variance, while offset and
s
