I did DIC live imaging of my cells for 2 minutes using metamorph acquisition software. Live Images were collected at every 5 seconds for 2 minutes. The membrane ruffling of the images of one group shows highly active and moving fast, whereas second group is moving slowly. I wanted to quantify there movement in terms of kymograph as well as velocity. Can please provide me the step by step guidelines to perform these analyses using ImageJ or Metamorph or Velosity software.
Hi Ethesham,
I have done similar work to this with Primary Drosophila macrophages. The description I provide in the paper is quite informative: Sampson, et al., 2013, Real-time analysis of Drosophila post-embryonic haemocyte behaviour. Plos One (7)1: e28783.
I used imageJ to produce kymographs that were perpendicular to the cell centroid region, in the direction of the cell membrane protrusion. The kymograph produced was of 1 pixel width (or subsequent conversion into microns) and over a time frame of >10 seconds I believe. From there I used the kymographs to calculate the retrograde flow, lamella flow, and also made an attempt at calculating the 'protrusion frequency' (open to further optimisation). These are noted in the paper, and how to calculate these variables.
In terms of plugins to analyse the kymographs, you can google search 'tsp050706', which is the name of the plugin I found and downloaded for use in imageJ to calculate retrograde flow velocities. But you may want to look further into Metamorph as I remember that I could analyse retrograde flow from kymographs with pre-designed software tools in the Metamorph package. I used Metamorph in Germany and it was amazing, easily the best microscopy software out there in my opinion. Plus the numbers produced from metamorph are more reliable than that of imageJ for the same analyses. I only used imageJ as my home institution did not have metamorph.
Lastly, just as another detail regarding time-lapse microscopy. As far as I am aware, the gold standard for cell behaviour/ membrane behaviour analysis comes from a time-lapse of at least 20 mins with an image taken every 15 seconds (~80-81 frames total). Also you want to time-lapse multiple foci per experimental set-up, at least 3 (with around > 5-10 cells per foci). Outside of that you just need to collect enough to run stats on, I usually stick to the minimum of 32 randomly selected data points from a pool created from at least 3 experimental repeats.
I hope this all helps you!
Best of luck,
Chris
Hi Chris,
Thanks for details explanation of using Image J for making and analyzing Kymograph. I am going to follow the instructions as given and if there is any problem I will get back to you. Thanks
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