. Please refer to section 6.3 for a detailed description of each of the
parameters.
Spot detection is done automatically, controlled by a few parameters that are set by the user. In Delta2D, any "spot painting" or "trimming" by hand is obsolete.
Once spots have been detected, quantitation is also done automatically – with only one parameter that is necessary for background subtraction.
Since Delta2D includes image warping (introduced into 2DE gel image analysis by DECODON in the year 2000), spot matching is very reliable, even with individual spot detection for each gel image. In traditional packages for the analysis of 2D gel images, where spot matching is based on spot patterns rather then spot positions, these steps are error-prone and require extensive manual corrections. In Delta2D spot matching is done automatically since after warping corresponding spots already have the same position throughout the whole experiment.
With its intuitive and modern approach, Delta2D tries to automate the analysis as far as possible, leading faster to results you can rely on.
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Read more about the benefits of 100% Spot Matching at www.decodon.com/Solutions/Delta2D/100_Percent_Spot_Matching.html.
The Quantitation dialog comes with a proposal for three numerical parameters. The numbers are
derived directly from the images and should lead to reasonable results. However, you can change
the parameters according to your individual preferences. Having changed the parameters
proposed by Delta2D, you can restore the proposal again by using the feature Parameter
estimation. Simply click on . Please refer to section 6.3 for a detailed description of each of the
parameters.
The appearance of the Quantitation dialog is shown in figure 6.1. Note that when you have chosen to quantify only one gel, only the parameters corresponding to that gel will be editable.
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Besides from the Project Manager, you can invoke this dialog from the Gel Image View from the
Spots menu, by selecting the Spots 
Quantify Gel name of first gel. . . or Spots Quantify Gel
name of second gel. . . menu item , as appropriate.
The meaning of a parameter is explained in the panel on the right-hand side. Please refer to section 6.3 for a detailed description of each parameter.
Parameter sets can be saved and loaded using the buttons in the top right panel.
The set of Parameters, i.e. the list of parameters that were used for quantitation, normally is saved within the *.qnt files which contain the quantitation information of a 2D gel image. You can load a parameter set from a previously exported quantitation file by loading the corresponding *.qnt file in this dialog.
Clicking on the OK button will start the spot quantitation process, Cancel will discard your settings.
Quantitation is always done using the original images while warping and histogram adjustment have no effect on the results. The background for a spot is computed and subtracted automatically, it is the very same background that is switched on and off using the layer panel.
When the quantitation process is finished, spot boundaries will be shown in the main window of the
Gel Image View. The spot boundaries for the respective gel image are overlaid on the image, placed
on a separate layer (one spot layer per gel image). These layers can be switched on and off, just
like the image layers, using the overlays rollup. Select Rollups Overlays to open the
rollup.
Spot centers are marked by points. The center is located where a spot cutter would obtain the maximal protein amount.
Quantitation data can be saved as a complete set of data (spot boundaries and quantities for the whole
gel image) using the Spots Export menu.
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You can invoke this dialog only from the Gel Image View, from the Spots menu, by selecting the
Spots Quantify Both Gel Images. . . menu item, by using the keyboard shortcut sequence
Ctrl+Alt-Q, or, if there are no quantitations available, by clicking on the Segments tool icon 
in the tool
panel.
Clicking on the OK button will start the spot quantitation process, resulting in the display of the quantitation table. It will take a few moments for Delta2D to find all the spots in your gel images and prepare the table: if you realize you have made a mistake with your parameters or match vectors, you can stop the computation at any time by clicking on the Cancel button in the progress window that appears. You can then go back and make corrections before starting the quantitation process again.
Spot matching is integrated into the quantitation process and happens mostly unnoticed by the user. Delta2D just checks if two spots overlap sufficiently, taking overlap quantities into account. It may happen that a spot has more than one corresponding spot on the other gel, or no corresponding spot at all.
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Reasonable values should be 1.5 to 2.0 times the diameter of the largest spot in the image.
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Higher values will decrease noise sensitivity. Use lower values to separate spot clusters better and to detect very small spots.
Note that you can get an idea of the size of the spots in your image by looking at Delta2D's status bar, at the bottom of the main frame. The status bar displays information about the current pixel position of the mouse pointer within a gel image, so you can see how large your spots are by moving the mouse over the spots and observing the number of pixels covered directly.
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Just type a percentage value directly into the text entry field corresponding to either the master or sample gel image.
Specifying a higher value will result in Delta2D detecting spots with weak intensity more reliably. Specifying a lower value will mean that more noisy background artifacts in the images will be suppressed successfully.
Generally, a value between 5 and 20 % is suitable.
To save the current set of options to a file, click on the Save icon 
. A dialog will appear, enabling you to
specify a file to save your options to.
To load previously saved options, click on the Load icon 
. A dialog will appear, enabling you to
choose a file from which to load a set of options.
Pixel based spot boundaries directly reflect the raw gray value distribution within the scanned gel image. Since the gel images usually include noise and spots divided into pixels, this kind of spot boundaries regularly look erratic.
For different reasons, be it that, due to a low resolution of your image, the spot outlines look to rough, be it for purposes of printing or presenting results, or simply for a better overview, a smoother appearance of the spots its often preferred. Delta2D includes the option to model spot boundaries within the process of the spot detection. Simply check the box Create Modeled Spots when defining the parameters for spot detection and quantitation. (Fig.6.4).
 
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You have access to the option in the dialog for the spot detection parameters only. The option will be used for the next spot detection, existing spots on other gel images will not be affected. For changing the spot shape a re detection by using the altered parameter is necessary. "Keep atttributes" preserves already done classifications like hidden, canceled, exclude from normalization, etc..
You can correct the results of Delta2D's automatic spot detection by setting "markers". Using markers you can control where a spot should be detected; Delta2D will then compute the new boundary accordingly. There are two basic operations for spot editing: creating a new spot, and joining two or more spots. In any case, Delta2D will compute spot boundaries automatically, using your input. Delta2D's approach to spot editing maximizes reproducibility while giving you a lot of control over which spots are detected.
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To add a spot, click on the Spot Editing Tool 
in the tool bar of the Gel Image Pair View. Now click
on the position in your gel image where the spot should be detected, trying to hit the darkest point of the
aspired spot. Where you clicked, a "+" will appear and according to this manually added marker a new
spot will be detected instantly. If the result is not satisfying, you can either move the marker by
dragging it, or you can remove the marker by right-clicking on it and set a new marker somewhere else.
Your manually added spots will look and behave exactly like the automatically detected spots
but still keep their marker as manually added, visible when switched to the Spot editing
tool. Thus reproducibility is granted and at any time you have the possibility to edit them
again.
In some cases it can be necessary that you drag a spot marker instead of setting it by a click. The dragged line markers have some influence on the spot shape and help for the correct detection of by gel breaks separated by spots.
To split a detected spot in two parts, simply override the detected spot with two manually set detection markers: select the edit spots tool as described above. Now click on the two sections of the spot you want to divide, trying to hit the centers of the aspired spots.
If a spot mistakingly was detected as two spots, you can easily join the two spots: switch to the edit spots tool, and drag a line from one to the other half of the aspired spot. Delta2D will join the two spots touched by the line.
To remove an edited spot, choose the edit spots tool as described above. Now right click on the marker you want to remove.