You probably included images when creating a new project. The Quality Control step allows you to view these images and their properties, check their quality and consistency, edit them when necessary and define pI and MW markers for automatic pI/MW calibration. You can also add, remove or rename images. When finished, you can validate those gels you want to take forward for further analysis.
TIPS Most of the functionality in this step is specifically carried out on selected images. You can select images by clicking on their thumbnail. To select multiple images, hold the Ctrl or Shift keys. Note the difference between images and gels: gels (or blots) are the physical objects for which one or several images have been acquired. In the case of DIGE, DIBE and other multi-stain experiments, two or more images belong to the same gel (or blot).
NOTE If an image acquisition device supporting TWAIN is connected to the computer running Melanie, you can directly import images from that device. In such case (and only for non DIGE/DIBE projects), a drop down will appear next to the Add images icon with the option Import images from TWAIN scanner.
By default, the Quality control screen displays the thumbnails for all imported gel images, with below, the Quality control table. The table shows various properties of the gel images: Name, image Height and Width in pixels, Resolution, Bit depth, Estimated bit depth, number of Gray levels used, minimum and maximum gray levels before (Min/Max gray) and after (Min/Max value) calibration, the percentage of gray levels used (Gray levels %, calculated with respect to estimated bit depth), the percentage of the dynamic range in use (Dynamic range %, calculated with respect to estimated bit depth), the percentage saturated areas and background clipping detected (Saturated area % and Background clipping %), as well as the Calibration formula and Calibration unit. For Coverage and Coverage DIGE/DIBE projects, the Quality control table also contains the fields Image type and Primary reference. These are used to specify whether your image is:
- A Primary image – This is an image that represents a total population of host cell proteins. It contains both HCP that are recognized by anti-HCP antibody and HCP not recognized by anti-HCP antibody.
- A Secondary image – This is an image on which only immunoreactive host cell proteins were detected, that is, HCP recognized by a particular anti-HCP antibody reagent. When you assign an image as being Secondary, you must also indicate to which Primary image it must be compared, via the Primary reference field.
TIPS You want to compare replicate DIBE blots, different antibody reagents against the same antigen or the same antibody reagent against different antigens? Analyze all related images as part of the same project! You will not only be able to compare any image to another in terms of coverage or similarity, but also investigate differences at the protein level. Use the Ctrl or Shift keys to select several images and assign them the same Image type or Primary reference. NOTE When you create a Coverage DIGE/DIBE project and specify a particular dye for the primary images, the Image type and Primary reference are automatically assigned. When you choose the option Mixed as dye for the primary images, or when you create a Coverage project, you must enter the Image type and Primary reference information yourself before proceeding with the analysis.
You can change your preferred way of viewing images and image information in the Quality control screen. / Zoom in / zoom out Increase or decrease the size of the thumbnails. Display options Choose one of the options to view the images and image information:
- Images/Table – Display both the image thumbnails and the quality control table.
- Images – Only display the image thumbnails, and hide the quality control table.
- Table/Histogram – Display the quality control table with the gel image and intensity histogram columns included.
Potentially problematic items, issues or values are highlighted at various levels in the Quality control screen, through icons and color codes:
- Pass – green – All looks correct, you can confidently proceed with your analysis.
- Caution – yellow – Acceptable, but be careful, Melanie detected an issue that could weaken the quality of the analysis.
- Warning – red – Anomaly identified, Melanie detected an issue that will adversely affect the quality of the analysis.
When several images are concerned, you can click on the small Plus icon to get the list of images involved. Click the More icon to display detailed information about the detected issue, how it could affect the analysis, and what you can do to solve it.
The Edit images button will take you to a dedicated Image edition mode that provides the basic tools to edit images: (Only images that were selected in the Quality control step are opened in the Image edition mode.)
- Rotate left (90° CCW)
- Rotate right (90° CW)
- Rotate free
- Flip horizontally
- Flip vertically
- Invert gray levels
- Change resolution
Note that editing always occurs on a copy of the imported image file; never on the original file itself. In the Image edition mode, you will also be able to display a 3D view of your images, and to adjust their contrast. These two display options do not alter the underlying image data.
Choose Edit pI/MW from the Edit button drop down to create and edit pI and MW markers on known proteins. Melanie uses the pI and MW values of these proteins to compute approximate pI and MW values for all spots. The pI/MW information will automatically be available in all images and reports throughout the analysis. In the Edit pI/MW screen, first choose the image on which you want to define the markers. You can define markers on several images. But only markers on the image currently selected in the Image for pI/MW section will be used to calculate theoretical values for all other spots. Note that in the main Quality control screen, the image used for pI/MW calibration is marked with a small pI/MW icon.
In the Edit pI section, click the Edit pI icon and choose one of the three main options in the drop down:
Pi user markers
Choose Pi user markers from the list to define pI markers on individual protein spots or gel locations. Then double-click the position where you want to create a pI marker. In the Edit pI marker box, specify the pI value for the marker.
Predefined IPG strip
Alternatively, choose a predefined IPG strip (linear or nonlinear) from the list. A strip template will appear at the top of the image. Drag its extremities onto the effective strip ends of the gel so that all intermediary pI values are automatically positioned. This option especially improves pI estimates for nonlinear strips.
TIP Use the Measure Mode to check that the distance between the two strip extremities corresponds to the real length of the strip. If necessary, change the Coordinate units to Centimeters by going to the Display tab in the General options.
If your IPG strip is not in the predefined list, you can create a new one by selecting New strip. In the Edit pI strip window, you can edit the pI and % length values. You can add steps by clicking the Add step icon and store your strip for future use by clicking the Save icon. If you previously saved a strip definition, click the Load icon to reuse it. When done, enter a Strip name and click OK. Drag the extremities of the strip template to their appropriate positions, as indicated above for predefined IPG strips.
Click the Edit MW icon and then double-click the position where you want to create a MW marker. In the Edit MW marker box, specify the MW value for the marker (in Dalton).
Edit individual pI or MW markers
- To edit a marker’s value, double-click its label.
- To move a marker’s position, drag its base (disc).
- To delete a marker, right-click on its base (disc). Right-click and drag to delete all markers in an area.
Click the icons in the Display section to access helpful tools:
- Adjust contrast – Allows you to adjust the contrast of your image.
- 3D view – Shows the 3D view, to help more precise positioning of your markers.
- Grid – Displays the pI and/or MW grid on your image.
The pI/MW table lists the markers that have been defined, with their X and Y coordinates. If you created your own IPG strip, you can edit your strip by clicking on the Edit strip icon or remove your strip by clicking the Remove strip icon.
You can systematically review each gel to decide whether you want to take it forward for further analysis. Validate the gel for further analysis. Exclude the gel from further analysis. Your choice will be reflected in the Validation box at the top right of each gel image, and at the left of the gel or image name in the Quality control table. You can also click this box to choose between the three different states:
- The gel will be included in the analysis.
- The gel will be excluded from the analysis.
- the gel has not been validated, but will be included in the analysis by default.