Month: August 2015

Using Thresholds to Measure and Quantify Cells in Image J

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I often get asked how to uses Thresholds to measure things in Image J.

There are some great guides on the web explaining how to use Thresholds in Image J, and here are a few that are well worth checking out [Link1][Link2].

Below are some of the Basic Steps for using Thresholds:

  1. Open your image and duplicate it (Image>Duplicate)
  2. On the duplicate go to Image>Adjust>Threshold
  3. Play with the sliders until all of your cells are red.
  4. Click ‘Apply’
  5. You should now have a ‘binary’ black and white image
  6. Now go to menu Process>Binary and select ‘fill holes’
  7. You may also want to select erode, dilate, open or close to optimise the binary image so that you have nice solid filling of your cells.
  8. Now go to menu Analyse>Set Measurements. Select all the things you want to measure.
  9. Critical steps: make sure that you select your original image (not the binary) in the ‘Redirect to:’ pull down Menu
  10. Also make sure the ‘Limit to threshold’ checkbox is ticked and also tick the ‘Add to overlay’ and ‘Display label’.
  11. Click ok to close the ‘Set Measurements’ box.
  12. Now go to Analyse>Analyse Particles
  13. Here you will need to play around with the size and circularity settings (bit of trial and error) in order to get accurate identification of your cells or ROIs. I suggest making duplicates before you start so that you can quickly try different things to see which one works best.
  14. Make sure you have the Display results tick box selected.
  15. Once you click ok you should have a the measurements box appear with all your measurements for each cell.
  16. You can copy and paste these into Excel or what ever program you like to use.
  17. Go get a coffee and cake you deserve it!

Good luck!

 

Using ImageJ to Measure Cell Fluorescence

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Image J can be downloaded for free from here .
This guide can also be downloaded as a complete PDF here: Measuring Cell Fluorescence using ImageJ

Here is a very simple guide for determining the level of  fluorescence in a given region (e.g nucleus)

  1. Select the cell of interest using any of the drawing/selection tools (i.e. rectangle, circle, polygon or freeform)
  2. From the Analyze menu select “set measurements”. Make sure you have AREA, INTEGRATED DENSITY and MEAN GRAY VALUE selected (the rest can be ignored).
  3. Now select “Measure” from the analyze menu or hit cmd+m (apple). You should now see a popup box with a stack of values for that first cell.
  4. Now go and select a region next to your cell that has no fluroence, this will be your background.
    NB: the size is not important. If you want to be super accurate here take 3+ selections from around the cell.
  5. Repeat this step for the other cells in the field of view that you want to measure.
  6. Once you have finished, select all the data in the Results window, and copy (cmd+c) and paste (cmd+v) into a new excel worksheet (or similar program)
  7. Use this formula to calculate the corrected total cell fluorescence (CTCF).
    NB: You can use excel to perform this calculation for you.
    CTCF = Integrated Density – (Area of selected cell  X Mean fluorescence of background readings)

     
  8. Make a graph and your done. Notice that in this example that the rounded up mitotic cell appears to have a much higher level of staining, but this is actually due to its smaller size, which concentrates the staining in a smaller space. So if you just used the raw integrated density you would have data suggesting that the flattened cell has less staining then the rounded up one, when in reality they have a similar level of fluorescence.

How to Cite this if you wold like to:

We have used this method in these papers:

McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., and Burgess, A. (2014) Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle 13, 1400–1412 [Link]

Burgess A, Vigneron S, Brioudes E, Labbé J-C, Lorca T & Castro A (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA 107: 12564–12569

But you can also find a similar method published here:

Gavet O & Pines J (2010) Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev Cell 18: 533-543

And here:

Potapova TA, Sivakumar S, Flynn JN, Li R & Gorbsky GJ (2011) Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited. Mol Biol Cell 22: 1191–1206

And my apologies to any others that I have not mentioned.