Biology Reference
In-Depth Information
samples. It is also important to note that N&B analysis accounts for limitations such
as autofluorescence, scattering, bright immobile particles, or fast-moving particles
that are dim by calculating the total variance. Variance is proportional to the particle
brightness for particles fluctuating in the focal volume; however, the variance of the
immobile particles, scattering, autofluorescence, and detector noise is proportional
to the intensity of these components. Thus, only fluorescent fluctuations that are de-
pendent upon the mobile particles have a ratio of the variance to intensity
1. Bright-
ness maps then allow for pixel resolution of the clustering of fluorescently labeled
proteins (see
Figs. 19.1 and 19.2
).
It is also important to consider that a fluorescent molecule can move faster than
the scanner during line scanning (
Digman et al., 2013
). This would mean that
fast-moving particles would not be counted accurately and the average diffusion co-
efficient would be lower than the true diffusion coefficient. Digman and colleagues
recommend keeping the pixel time faster and pixel size larger to detect the fast-
moving particles before the next line scan (
Digman et al., 2013
). Typical settings
of a pixel size of 0.05
>
m
m
m and a slow pixel dwell time (25
s) can detect particles
m
2
/s (
Digman et al., 2013
).
The spatial resolution of RICS is usually larger than the size of a diffraction limited
area but smaller than the entire cellular cytoplasm meaning multiple boxes around a
cell can be examined independently. This is advantageous for collecting large data
sets from single or multiple cell measurements, which is especially important when
determining N&B of particles or the molecular stoichiometry of a protein complex
(
Digman et al., 2009
).
m
diffusing in cells with diffusion coefficients of
20
SUMMARY AND CONCLUSION
RICS and N&B analysis are used to monitor the diffusion of fluorescently labeled
molecules in live cells or in solution. Together, they can measure the formation
of protein oligomers and their stoichiometry while determining the number of mol-
ecules that are both free and in complexes. These techniques are advantageous for
studying oligomerization in live cells as harsh conditions do not have to be used
to lyse the cells and assess oligomerization. Imaging fluorescent fluctuations also
does not require the large sample size that would be required for performing size
exclusion chromatography with multiangle light scattering, which can accurately
determine oligomerization state (
Wyatt, 1998
). Of course, creating fusion proteins
with fluorescent tags can influence cellular membrane binding or oligomerization
so independent methods should be employed to verify that tagged and untagged
proteins behave similarly. This approach has been useful and informative for
investigating the basis of assembly and oligomerization of the Ebola virus matrix
protein VP40, which regulates egress of the virus from human cells (
Adu-Gyamfi
et al., 2012, 2013
).
Oligomerization of VP40 is a critical step in the assembly and replication of the
Ebola virus. Inhibition of this step of the viral life cycle has been found to
