Biology Reference
In-Depth Information
Version 7.10.0.499 (R2010a) 64-bit (win64) but should work on most of older ver-
sions of the MATLAB platform.
1.3.4.1
Description of histogram parameters
White noise background level: Corresponds to the measured pixel intensity
values for an image collected when the laser is off or to the mean pixel intensity
calculated for a region of the image where no cells or fluorophores are
present (e.g., area between cells). A histogram of an image is produced by
discretization of the intensity range into a number of bins.
Bin: We suggest that the bin size should be chosen in order to be larger than half
of the monomeric QB of the control, but not too large, so that the distribution
of the curve is not truncated. The smaller the intensity bin size, the longer
the computational time for SpIDA analysis.
Resolution (Res): Is defined in the GUI as being the number of pixels per beam
waist radius (i.e.,
o
xy
/pixel size). The resolution and beam waist radius
can be estimated by imaging a control sample of subdiffraction-size fluorescent
microspheres using the various zoom settings of the CLSM and analyzing
intensity lineout plots using ImageJ.
Threshold (Th): Sets the minimum and maximum values of intensities included in
the histogram analyzed.
1.3.4.2
SpIDA GUI procedures
This section is a step-by-step recipe explaining how to use the GUI. Note that all
details are also accessible on the website
http://www.neurophotonics.ca/en/tools/soft
ware
. An image showing the GUI is presented in
Fig. 1.8
. To analyze images using
the GUI, follow these steps:
1.
Set the Current Folder in MATLAB to the directory containing the GUI files and
launch GUI_SpIDA command.
2.
An explorer window (select an image) opens and offers you the option to load
image files. If the loaded file contains more than one image, a scroll bar will
appear to permit navigation through the image stack. To load another image, just
click on the “load image” textbox and select the image file.
3.
Set the correct parameters for the image file. The pixel size and the beam
waist radius in micrometer are needed as inputs. The GUI will attempt to
obtain the information from the image file itself. If the program cannot find the
spatial information in the header, a dialog box will appear and the correct
image information can be manually entered.
4.
To select an ROI to analyze, click on the button “Chose Region.” Using the
mouse, click, drag, and click again to delimitate a rectangular ROI. The two
chosen pixel positions are shown left of the two buttons “Chose Region”
and “Draw Region.” A zoom of the chosen region with enhanced contrast is also
presented in the GUI lower left panel. Set the check box “Normalize” to ON
