General SpotMap Questions
Can I check my image quality?
When adding images to the project they are checked for quality and any issues/notes are highlighted for you.
Can I edit my images?
If your images require minor changes (flipping etc.) then you can edit them within the software without changing the original images.
Can I account for gel/blot distortions?
Alignment makes it possible to accurately compare images by removing the positional variation introduced during the gel running, blotting and imaging process.
Can I identify spots of interest?
Spots are categorised as present or absent during the analysis, this allows spots Common, Additional and Missing from an image comparison to be easily identified.
Can I measure percentage coverage of 2D gels and Western blots?Can I measure percentage coverage of 2D gels and Western blots?
Based upon the categorisation of spots, percentage coverage of one image vs another is calculated.
Can I compare spot measurements?
Can I calculate spot coverage?
A spot map is created of the fully resolved host cell protein. The presence of spots expressed on the blot is then used to calculate the relative coverage of the Western blot vs the 2D gel.
Can I export results?
All the data from the project can be easily exported from the program using the Data Access window or using the clipboard functions. .csv files of the numerical data are easily exported for all spots on all images, or for selected spots only. Images can be exported by copy and paste or exported as a .png file.
What is images mode?
What is Image QC?
When adding images to the project they are quality checked and any issues/notes are highlighted for you immediately before any further analysis is attempted.
Image QC flags any potential issues with:
• Image file size
• Manual editing of the images
Poor image quality can make analysis of your gels and blots difficult and may reduce the objectivity of your analysis.
What are the benefits of using high quality images?
Quantification of spot intensities may seem less important when only looking at spot numbers and coverage results. However, good quality images also improve:
• Spot Detection
• Filtering options when selecting small/low spots to remove
• Definition of spots on the blot (no saturation)
• Automatic alignment algorithm
Can I edit my images in the software?
Quick edits are available below each image to invert the colour intensities and flip or crop the images. More edits can be performed in the image editor window, accessed by right clicking on the image and selecting “Edit” from the menu. It is recommended that image edits are performed within the software as this will not affect the raw data of the images.
What problems does image editing solve?
What edit options are available?
• Flip images horizontally and vertically
• Rotate images 90 degrees clockwise and anti-clockwise
• Invert image
• Translate image
• Crop image
• Rotate image a variable angle
• Resize images to same size
What does the analysis mode do?
Why is a single spot map used for the analysis?
How does identifying spots as present or absent allow results to be calculated?
The present or absent categorizations from the spot map allow comparisons of images to be made. When any two images (the base and the secondary) are compared the spot presence/absence settings are used to determine which spots are Common, Missing or Additional and are used to calculate percentage coverage.
|Base image||Secondary image|
(Not included in calculation of spot coverage. Spots classified as absent should be present on another image in the analysis).
What is spot coverage and how is this calculated?
Spot coverage is the percentage of spots in the comparison being made which are present on the secondary image. Percentage coverage is calculated against the chosen base image using the calculation:
Percentage coverage = (Common + Additional)/(Common + Missing + Additional) x 100
What is a base image?
What does automatic spot detection do?
Why are spots detected on only one image?
Why are some spot edits required?
Why select spots using clauses?
What is a spot set?
Can I add molecular weight and pH markers?
Markers can be added to the map to identify the MW markers.
A linear or non-linear pI strip can be added to the map.
If you cannot find an answer to your question here
What is Alignment?
What problem does it solve?
Why is alignment better than spot matching techniques?
How is alignment different to warping?
Do I need to align DiGE images?
Can I “fix” alignment after spot detection?
The alignment window can be opened at any point in the project to align or correct the alignment of an image. Once the “Apply” button has been clicked the alignment will update. The images can be initially aligned prior to or following spot detection.
How does Alignment work?
What is the difference between automatic alignment vectors and manual vectors?
Results and Data Access
What does the results screen show?
Can I export data?
Using the Data Access feature, found from the main menu, all data from the analysis can be easily exported by dragging and dropping into reports and presentations or files.
What data can I export?
• Results of percent coverage
• Number of spots common, additional and missing between images.
• Colour coded spot maps overlaid on the images identifying the location of Common, Missing and Additional spots.
• CSV data file containing all measurements of all spots for all images or a spot set.
• All analysis parameters used in the analysis.
• Manually added images.
Images throughout the analysis can be added to the data access by right clicking and selecting “Add to Data Access”.
Can I annotate the image?
You can add text labels to the spot map in the analysis screen, these can point towards certain features of the map which are of interest.