Biology Reference
In-Depth Information
1.1
THEORY OF SPATIAL INTENSITY DISTRIBUTION ANALYSIS
Spatial intensity distribution analysis (SpIDA) is based on fitting super-Poissonian
distributions to pixel intensity histograms calculated from CLSM images and returns
information on the densities of the underlying fluorescent molecules and their quan-
tal brightness (QB). As described previously (
Barbeau et al., 2013; Godin et al.,
2011; Swift et al., 2011
), SpIDA is inspired by the temporal photon counting
histogram (PCH) approach (
Chen, Muller, So, & Gratton, 1999
) but is applied to
the spatial domain rather than the time domain, enabling measurements on subre-
gions within single images collected on conventional fluorescence microscopes
equipped with analog photomultiplier tube (PMT) detectors.
1.1.1
Theoretical basis of SpIDA
The intensity histogram of an imaged region of interest (ROI) is simply calculated by
counting the number of pixels for each intensity value or intensity bin of values. Each
pixel intensity in a CLSM image is the integrated fluorescence intensities collected
from detected fluorescence photons originating within the region excited by the
laser beam focal volume, the point spread function (PSF), at a given position within
the sample.
The theoretical intensity distribution for an infinite region containing N particles on
average per PSF, randomly distributed in space, with each particle of QB
e
, gives a unique
intensity histogram. Recovering the set of parameters (N and
) that best fit the experi-
mental intensity histogramprovides information on the distribution of particles in the im-
age and their QB (which can be used as a measure of oligomerization states).
A more detailed description of the underlying basis of SpIDA is provided in
Godin et al. (2011)
and
Barbeau et al. (2013)
.
e
1.2
SpIDA: EXAMPLES OF APPLICATION TO RTK
We provide two examples of the applicability of SpIDA to quantify RTK transacti-
vation. The first example involves the use of stable cell line expressing a green
fluorescent protein (eGFP)-tagged epidermal growth factor receptor (EGFR) trans-
activated by several different GPCRs. In a second example, we demonstrate
the transactivation of endogenous neuronal tropomyosin receptor kinase B (TrkB)
by dopamine receptors. It should be noted that, in both examples, SpIDA was also
used to quantify direct RTK activation by their cognate ligands, validating the use of
SpIDA for quantitatively describing dimerization (
Sergeev, Swift, Godin, &
Wiseman, 2012; Swift et al., 2011
).
1.2.1
Quantification of EGFR-eGFP
Using SpIDA, we were able to focus on direct measurement of early transactivation
events, specifically EGFR dimerization, following stimulation of a transfected
GPCR (
Swift et al., 2011
).
Figure 1.1
illustrates a typical EGFR dimerization curve,
