So basically you have a coverslip on a microscope slide and you want to make an unbiased observation of certain representative areas of the coverslip, so that you can later generalise the result to the whole coverslip (similar to random sampling from a population). I know one way to do it is to image 5 photos in an X manner, so 4 images at 4 corners and 1 at the the centre. Data from each image can later be used to generalise to the whole coverslip.
But I'm wondering is there any other method to do this?
Also, sometimes the cells just clump together or distributed unevenly which make this X method a bit inaccurate. Is there any way to troubleshoot this apart from evenly seeding your cells next time?
Thank you very much
I always randomly select regions on my coverslip for imaging - to avoid biasing, you can refrain from looking down onto the coverslip while you move in the XY plane. You can do the corners and centre but as your cells clump in middle it's not a good representation.
Just as a matter of interest, when you seed your coverslips with cells, do you swirl the cell mix or do you rock the cell mix once it's in the tissue culture dish/plate? Normally one should not swirl the mix as it causes a vortex in the medium thereby pulling all the cells to the centre of the plate - resulting in uneven distribution and clumping. If you gently rock from left to right, up and down it should give a better distribution. Also preparing the cell mix prior to addition to coverslips is better than having coverslips in fresh medium and adding a small volume of concentrated cells directly to it.
Hi John,
Thank you for the reply.
About the random selection, I still feel like even if you move the coverslip randomly, it still feels like it's not random enough. Maybe it's just because my cells sometimes they group together and not very spreading evenly. If the cells spread evenly, then random movement is good. But if they clump, you might just move your coverslip to an empty area tho. Maybe the problem is how well the cells spread on the coverslip, right?
And no, I don't swir the dish. I always make sure that I shake the plate sideway and then far-near direction several times. But the cells still don't distribute very nice.
Can you explain this clearer to me "Also preparing the cell mix prior to addition to coverslips is better than having coverslips in fresh medium and adding a small volume of concentrated cells directly to it"? The two ways seem similar to each other? What I usually do is I have my coverslip in media in a 12-well plate let say ready. And then I trypsonise the cells and resuspend it in a certain amount of media, then take aliquot of this cell suspension and add to each well dropwise and spreadingly. But maybe i suspend the cells in too few media, 6ml sounds fine to me though. But i can try to suspend it in larger volume.
One approach is to image the whole coverslip in a tile-like manner if you have an automated microscope capable of doing so. Then, have it stitch the images into one image and analyze the whole coverslip image using a software program like Image J. Depending on what you're actually analyzing, this may or may not be practical, but it sure does remove the bias from the analysis because as you say, choosing random representative fields is very subjective and often not representative at all.
Erick Martin: Thank you. I think if we have that, I may just use that method. But I will need to double check with the service.
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