The image correction program is used to correct systematic errors that occur during data collection. The three basic errors are caused by photobleaching, inconsistent illumination intensity, and CCD defects. Under most circumstances, image correction should be performed before all other image processing such as deconvolution.
Options for selecting the region to process and aspects of the graphical front end that CCDCor shares with other applications are described in BatchRegion.html.
Overview | Region for analysis | Special Parameters
BatchRegion.html | Deconvolution | Priism
In certain cases, you may wish to have a correction calculation based on a subset of the data processed. To do so, specify the first and last pixel coordinates along the x axis in the X Region for analysis field and the first and last pixel coordinates along the y axis in the Y Region for analysis field. These ranges should fit within the region processed as shown in the X range and the Y range fields. By default, the region for analysis has the same size as the region processed.
On the command line, the region for analysis is specified by the area option: -area=x_first:x_last:y_first:y_last.
Overview | Region for analysis | Special Parameters
Press the Special Parameters button in the main menu to access advanced correction settings. Most of the time, you will not need to change any of the advanced settings.
Overview | Region for analysis | Special Parameters
Polynomial orders | Polynomial orders to plot | Intensity Normalization | Use Photosensor Data | Bleach Correction | Z-Line Correction | Intensity Reference Time
When correcting for changing illumination levels, a polynomial fit is used to smooth the estimates of the illumination level (either the photosensor readings or averages from the image data). The order of the polynomial fit can be specified for each wave in the Polynomial order for fit field. You should not need to change these values; the default, using a fourth order polynomial, is adequate for most images.
On the command line, specify the order of the fitting polynomials
with -norder=order_1:order_2:order_3:order_4:order5 where
order_x is the order of the fitting polynomial for wave x.
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Results for the polynomial fits (one per wave) specified by Polynomial order for fit are written to the file shown in PlotFile. If you want to see the results for other lower-order fits, use non-zero values in # of orders to plot. In detail, for a wave, w, polynomial fits with orders from o(w) - p(w) to o(w) are recorded where o(w) is the wth entry in Polynomial order for fit and p(w) is the wth entry in # of orders to plot.
2d_plot is useful for displaying the file with the polynomial fits.
From the command line, specify the number of lower-order fits to plot
with -nout=n_1:n_2:n_3:n_4:n_5
where n_x is the number lower-order fits to plot for wave x.
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The toggles next to Normalize intensities set whether or not to correct for illumination levels that vary from section to section. The illumination levels can be estimated from the photosensor readings (the Use photosensor data toggles control this); these estimates are smoothed using a polynomial fit (see Polynomial Orders and Polynomial Orders to Plot.
On the command line, use the option
-polyfit=do_1:do_2:do_3:do_4:do_5
where do_x is one if intensity normalization should be applied to
wave x and zero if it should not be applied.
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Use the Use photosensor data toggles to set whether the photosensor readings recorded during data collection are to be used to calculate the intensity normalization correction. When the photosensor readings are not used, intensity normalization is based on a polynomial fit to average intensities from the input images. By default, the photosensor values are used for all waves.
On the command line, use the option
-photon=do_1:do_2:do_3:do_4:do_5
where do_x is one if the photosensor readings should be used when
normalizing the intensities in wave x and zero if they should not be used.
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For situations where image intensity changes in a wave are due to the specimen and not photobleaching, turn this option off. The default is to apply a bleach correction to all waves.
On the command line, use the option
-bleach=do_1:do_2:do_3:do_4:do_5
where do_x is one if bleach correction should be applied to wave
x and zero if it should not be applied.
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The Z-Line correction is designed to detect CCD defects by looking for unusual intensity profiles along the Z-axis. For example, a dead CCD element will appear as a straight, dark line along the Z-axis, and a hot CCD element will appear as straight, bright line along the Z-axis. Voxels identified as Z-line errors are replaced with the average of their neighbors. The default is to apply this correction to all waves.
On the command line, use the option
-zline=do_1:do_2:do_3:do_4:do_5 where
do_x is one if Z-line correction should be applied to wave x and
zero if it should not be applied.
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When correcting for varying illumination levels (see Intensity Normalization) and the photosensor data is used as a measure of the illumination level (see Use Photosensor Data), CCDCor uses the photosensor reading from the first z section at the reference time as the reference photosensor value. The default is to use the first time point (zero) in the input file as the reference time.
On the command line, use -itref=t to specify
the reference time point, t.
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