I am hoping I can get some advice on the following problem. I compiled and put all my ideas/methods/approach in detail in order to verify what is the best method to analyse shape profiles (angle or orientation, amount of migration 0-180 degrees) from an internal circular body outwards.
I have spheroids migrating on different surfaces, such as standard tissue culture polystyrene, and fibres. I tried multiple methods to measure the amount and angle of cell migration. The methods I have tried gave different results. So I am here asking for clarification in terms of what is actually being measured and whether I am implementing the right methods/tools to extract the features I am looking for.
I attached a PPT showing the workflow, and please correct me if i’m wrong with the steps implemented towards quantifying the migration profile: area, and the amount/angle of migration from spherical body. The methods used include: Directionality plugin, Moment analysis, Analyze particles, Measure.
In my analysis I tried complex and simplified shape versions of the same migration profile.
Method 1a: simple shape, Directionality [slide page # 3, 5 ]
Load image- Make 8-bit - adjust brightness- threshold- Make binary - Plugin shape analysis - Hull and circle (unclick circle)- click on hull - clear outside - invert
Method 1b: complex shape, Directionality [slide page # 4, 6]
Load image - Make 8-bit - adjust brightness - threshold - Make binary - Fill holes - Noise despeckle - select object with largest area - clear outside
Method 2: Analyze - measure
2a: complex shape [slide page # 6]
2b: complex shape [slide page # 7]
Method 3: Moment analysis A and B [slide page # 8]
3a: Complex shape
3b: Circle cleared in centre of 3a (above) to show migration from body.
Method 4 [slide page # 9 ] Amount and angle from internal circle boundary
Merge channels (Bright-field and processed imaged such as the one in Method 1b
Draw circle around bright red area in centre - clear circle - split channels
1a: direction 65.97 degrees (simple shape)
1b: direction -7.49 degrees (complex shape)
2a: Angle 167.413 degrees (complex shape)
2b: Angle 17.932 degrees (simple shape)
3a: Angle = 167.400 degrees, orientation = 12.857 degrees (complex shape)
3b: Angle = 167.400 degrees, orientation = 9.398 degrees (complex shape with cleared circle in the middle)
4a: still in progress but an attempt is shown in 3b.
As you can see from the results above, the angle extracted is different depending on the method. I would like some clarification on how each method is defining the measurement.
What i would like to do? I would appreciate if someone could explain and/or guide me through this
Method 4: [slide page # 9 ] Is it possible to measure from image attached as example (picture1)
- Distance of cell fronts from Centroid of cleared middle circle, or circle boundary?
- The amount and angle/direction/orientation of cell fronts wrt. centroid of cleared middle circle, or circle boundary?
- The furthest distance from centroid of cleared middle circle, or circle boundary
Thank you all in advance