When you created a new project, you likely included images. The Quality Control step allows you to view these images along with their properties, assess their quality and consistency, make necessary edits, and set pI and MW markers for automatic pI/MW calibration. For each image, you can assign or import an alias – a more descriptive name used throughout the analysis – and import image metadata from Excel. You also have the option to add or remove images. Once you’re satisfied, you can validate the gels you wish to proceed with for further analysis.

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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).

How to

Add or remove images

Add images
To add one or several images to the project, click the Add images icon. For recommendations on image capture and information on supported file formats, refer to our Imaging guide.

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To compare replicate DIBE blots or different antibody reagents, analyze all related images within the same project. This facilitates comparisons in coverage or similarity and allows detailed protein level investigations.

NOTE

Images originating from the same gel or blot should be added at the same time and stored in the same folder.

If a TWAIN-compatible image acquisition device is connected to your computer, you can directly import images from it. This option is available only for non-DIGE/DIBE projects and can be accessed via a dropdown next to the Add images icon labeled Import images from TWAIN scanner.

Remove images
To remove images, select images and then click the Remove images icon.

View images and their properties

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, Alias, 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, additional fields in the Quality control table include Image type and Primary reference. The former identifies images as:

• Primary image – 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.
• Secondary image – Only shows immunoreactive HCP detected by a specific anti-HCP antibody. It must be linked to a Primary image through the Primary reference field for comparison.

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Use the Ctrl or Shift keys to select multiple images for assigning the same Image type or Primary reference.

NOTES

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.

If additional image metadata has been imported – either directly from the image file or through an Excel import, you can opt to display the imported fields. Access this feature by clicking the Options icon in the upper right corner of the Quality control table, selecting Column settings, and checking the fields you wish to display.

Some imported metadata fields are editable, except for default fields and those prefixed by ‘I-‘. To edit, click the Options icon in the upper right corner of the Quality control table, select Edit image description, and make adjustments in the Gel table on the Image view screen.

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.

Define or import image aliases

As discussed in this article, effective naming of gel and blot image files is essential for streamlined analysis and research. We advocate for a robust file naming policy that ensures names are consistent across various platforms – including lab notebooks, image acquisition devices, storage systems, and analysis software. Names should also be unique and enable tracking of all images from a given IPG strip.

While such names are functional, they might not provide enough detail for in-depth image analysis. Descriptive names like ‘CHO-HCP LMW’ or ‘anti-CHO HCP Ab’ can offer insights into antigens, antibodies, or other specific parameters. You can therefore assign or import aliases – meaningful names used throughout the analysis, while preserving the original file Names. Once established, aliases replace the original Names in all tables, graphs, plots, and image labels. Whether you need to customize image names for an internal presentation or anonymize your data for publication, simply modify the aliases.

To assign or edit aliases:

• Manually edit an alias by double-clicking a cell in the Alias column of the Quality control table and typing the new name. You can also edit the names above or below the image thumbnails (depending on the project type) in the Quality control screen.
• Import aliases along with other image metadata from an Excel file. This method allows for a batch update, streamlining the process when handling multiple images.

Import image metadata from Excel

Due to third-party component restrictions on file path lengths, file names incorporating a lot of metadata such as timestamps, sample information and experimental conditions can become too long for Melanie. Instead, we recommend keeping file names short (read our file naming recommendations) and storing detailed metadata in an Excel file located in the same directory as the image files, ensuring that it always accompanies the image data. You can then import selected data fields from that file for use during analysis.

Refer to our Excel Metadata File template for a structured format to log detailed experiment information, including columns for file name, alias, acquisition date and time, detailed acquisition parameters, sample information, and experimental notes.

To import metadata, click the Import metadata button at the top of the Quality control screen and browse for your Excel file. In the Import metadata window, the fields eligible for import will be displayed in one of three sections:

• Import as Property: Fields here are imported into predefined and editable Melanie fields, such as Image type, Primary reference, Staining, Comment. The name of the field in the Excel metadata file (first row) must be identical to the name of the field in Melanie.
• Import as Description: Fields here are imported as Descriptions, accessible in the Quality control table and Gel table. Ensure these field names do not coincide with any predefined Melanie field names, such as Resolution.
• Available: Move fields to this section if you prefer not to import them.

Ensure the Import alias box is ticked to import alternative image names into Melanie’s Alias field. The field in the Excel file must be labeled as Alias.

Click OK to complete the import of the selected fields.

Check image quality and consistency

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.

Edit images

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.)

• Crop
• 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.

Define pI and MW markers

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.

Create pI markers

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, select a predefined IPG strip (linear or nonlinear) from the list. A strip template will appear at the top of your image. To align the template with the actual strip on your gel, drag the red ends of the template to the strip’s ends. This placement ensures that all intermediate pI values adjust automatically. This option especially improves pI estimates for nonlinear strips.

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In the 2D view, the name and calculated length of the selected IPG strip appear above the template. Use this information to adjust the template’s length with its actual physical dimension. You can then reposition the strip template using a simple drag-and-drop action. You can also adjust the extremities or reposition the IPG strip in the 3D view.

New strip

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.

Create MW markers

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.

Display tools

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.

pI/MW table

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.

Validate gels for further analysis

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.