2.31 MB

Create Direct Warpings

SmartVectors^TM HQ: Find Match Vectors Automatically

The goal of this step is to produce a warping that aligns spots on one image with corresponding spots on the other image. In the 'Warping Setup' window, each yellow or green line with a yellow or green icon connecting two images represents one direct warping using so-called match vectors to align the images. Delta2D's SmartVectors^TM HQ Technology automatically produces match vectors. You can batch all the warpings so that a first iteration using the SmartVectors^TM Technology is done in the background:
From the third step of the Workflow 'Create Direct Warpings' you can invoke the 'Job Manager' by clicking on the link for it. In the Job Manager click the 'Run' button to let it monitor and perform all automatic warping jobs.

In order to open the completed warpings you can use the table below 'Review Direct Warpings' in the third step of the Workflow: Double-click on a row where the symbol in the yellow icon changed from a toothed wheel to two arrows:
A Dual View window (the dual view of these images) opens and shows the combination of the two images of the respective table row. Thus, you can see the differences in spot positions at a glance: black or gray spots have about equal volume in both images. Orange spots are stronger on the first image while blue spots are weaker. The more their color tends to one extreme, the bigger the difference is in expression.

Dual Channel image of two unwarped gel images

Of course you can navigate any time through the dual channel image, zoom in and out or view the single images only. You can also correct the presentation of the images by choosing 'Histograms...' () from the menu 'Images' and by using the sliders to adjust the settings and clicking on the 'Equalize Image Button' () in the tool bar to make sure that both images are balanced. Changes will affect the visualisation only, while quantification will always be done on the raw images. Please also check if the gel image background has been hidden by using the 'Show/Hide Background' button () in the tool bar. This adjustments help to better see the differences in spot positions. Confirm with OK.

Histogram settings dialog

Now press the warp button () and Delta2D will warp the orange image in accordance to the match map using the exact warp mode. In the resulting warped view, it is usually easy to see if all corresponding spots are aligned. Regions that are not sufficiently aligned show up as similar spot patterns in the blue and in the orange part of the image. Delta2D will only create match vectors in regions where it is reasonably certain that they correspond. You can unwarp the images (press unwarp ) whenever you want to review the match vectors with respect to the original images.

Delta2D distinguishes between match vectors that were set by hand (shown as solid lines), and those that were created automatically (shown as dashed lines). By 'approving' a match vector you declare that it was checked by you, so it can be used to guide the finding of more match vectors.

If there are regions that are not properly warped yet, you can use the 'Find Match Vectors' button. Delta2D's SmartVectors^TM HQ technology will use the vectors that are already present to guide the process of finding new ones:
Press the 'Find Match Vectors' button. Delta2D will ask you what to do with the vectors that were automatically created in the previous step: You can decide to use them as basis of another iteration with SmartVectors^TM technology or to reject them.

Alternatively, you can create, delete or change match vectors manually as described in the following section.

As soon as you have approved any match vector in the gel image pair, the symbol in the project manager will change from yellow to green, meaning that you have already worked with the match vectors. It is good practice to approve all match vectors when you are finished with a gel pair (use Matches -> Select Non-Approved, and then Matches -> Approve Selected).

Creating, deleting, and changing match vectors by hand

In order to work with match vectors, you have to select the Match Vector Tool first. Click on the top-most button in the vertical tool panel at the left of the dual view window.

Sometimes you will want to correct or improve the match vectors that were found auto-matically by Delta2D. The general approach is to work with whole image regions (as op-posed to single spots). If one or more match vectors in a region are not correct, you just delete all match vectors in that region and press 'Find Match Vectors' again to find better ones; vectors from surrounding regions will guide the process.

The Dual View after Warping

Of course, you can set and change all match vectors by hand if you want:

• Selecting a single match vector
  You can select a single match vector by clicking on it. Selected match vectors are highlighted.
• Selecting match vectors in a region
  Drag a rectangle with the mouse (keep the left mouse button pressed). All vectors in that rectangle will be selected.
• Deleting match vectors
  Right click on a match vector and choose 'Delete' or 'Delete Selected' from the menu. This will delete the selected or all selected match vectors, respectively.
• Setting a new match vector
  Click first on the spot in the orange image, then on the corresponding spot on the blue image. Hold down the CTRL key while you click in order to let automatically snap the ends of match vectors to spot centers.
• Changing a match vector
  Drag one end of the match vector in order to change it. Hold down the CTRL key while you drag in order to switch off spot snapping.

You can undo match vector operations by selecting 'Undo' () from the Edit menu.

Click on the Warp button to apply your new match vectors. You can iteratively add, correct or delete match vectors until the result is satisfying.

Verifying the results

Since the quality of the whole analysis relies on the exactness of warping, it is worth working carefully in this step of analysis. In order to control the quality of the warping step, please check this list:

• In the Warping Setup window you should see green lines with green icons only.
• For each gel pair with a green circle, open the Dual View and check the warped dual channel image.

Even if you set the warp mode to automatic you should still review the resulting warping in the Dual View to check if you are satisfied or if a correction is necessary.

The demo data set includes prepared match maps. To save time, just import the respective match map for a pair of gel images in the Dual View. In the menu choose Matches -> Import. If the blue match vector ends point into orange spots and vice versa the match vectors have the wrong direction and you need to convert them by choosing 'Matches > Invert'.