Biology Reference
In-Depth Information
textbox will appear and set the value for the “Slope Variance.” Slope variance
determination is described in
Section 1.3.5
.
6.
When a mixture of monomers and dimers is expected in the image, the number of
populations “# Pop” can be set to 2. The monomeric QB can also be fixed in the
fit, using the previously determined value from control images using a single
population fit. A dimer will then be twice as bright as a monomer. If the
monomeric QB is set to 0, the QB will also be fit.
7.
The results of the fits are shown in the box “Results SpIDA.”
The following results are obtained as output:
2
“Area” is the area of the chosen region in beam areas corresponding to
pi
*
o
xy
(beam area is the fundamental unit of sampling where the pixel size is smaller
than the beam focus area).
“Amplitude” of the fit is the height of the histogram in pixels. The density of
each of the population, as units of particles per effective illumination
volume, is also given. This effective volume can be a surface if the region
chosen is on the cell membrane (
o
x
2
) or a volume if the ROI is completely
within a cell (e.g., cytoplasm or nucleus), and then the volume can be
approximated as the effective volume of a 3D Gaussian (
p
*
3/2
*
o
x
2
p
o
z
, where
o
z
is the axial radius of the PSF).
QB is the average brightness of a fluorescent entity in the effective volume
and has units of intensity.
8.
Save the results. To save the results of an analysis, just press the button “Save
all!” and the GUI will save a “.dat” file with the name of the image with all fit
values and set parameters. If “Save with Go!” button is activated, results will be
saved automatically in the chosen folder when the analysis is done, whereas when
the button “Save with Chosen region!” is set to true, the analysis will be launched
and the results will be saved automatically. These options allow for a faster
analysis by reducing the number of clicks for iterative analysis.
9.
Results are displayed in .txt format and the data may be extracted and opened in
other programs such as Excel. We strongly suggest programming a macro
command to extract the values from each file.
1.3.5
Determination of analog detector signal broadening
As SpIDA measures the fluorescence intensity fluctuations of the signal in the image
to return information on the number of particles and their QB values, it is important
to consider only the fluctuations that originate from true signal variations of the fluo-
rescently labeled proteins in the sample and exclude fluctuations inherent to the de-
tector. Analog PMT detector variance is empirically determined as described in the
succeeding text. Use either back reflection of the laser from a mirror placed at focus
or a bright fluorescent sample (e.g., a solution containing an extremely high concen-
tration of fluorophores or a commercial fluorescent slide). This determination needs
to be done with the same settings (PMT voltage, filter sets, and scan speed), which
will be used for later image acquisition for samples under investigation.
