Dear all,
I am imaging HEK cells using TEM for the first time. I have currently seeded the cells into a 6-well plate and I am using a single well as n=1. So cells are a monolayer and the facility I am working with asked me how many images they have to acquire per cell? and how many cells into a single well do I need to images for a n=1? Also a total n=3 is enough for this type of analysis?
thank you very much
Are you sure you'll be using TEM, and not, say, SEM? For TEM your cultures should be embedded in plastic and cut in thin slices. Anyway, the number of pictures depends only on your task (which is so far unknown).
Hello Federica Albanese. To prepare your ultrathin sections, you should have a suitable pellet volume (which depends on cell density). For us, we culture the cells in flask 25T and collect the cell pellets to have a suitable pellets volume. For TEM micrographs number, is dependent on you. We generally take ten micrographs to demonstrate the change in the nucleus (marginated chromatin, fragmentation, etc.), the existence of lysosome, endsome, and mitochondria, ER, Golgi, as well as images demonstrate more than one cell in the micrograph to check the general morphological appearance, make length and width measurements, etc. You can handle the number based on your search purpose.
The number of images will depend on your objective. But in general, ten images of different cells is enough to see morphological differences in cells.
Well, number of images depends upon on your objective and quality of the sample. I generally take 1-2 images at lower magnification to cover the overall cell to comment about physiological state of the cell, 2-5 images of interested cell organelles at medium magnification to verify its respective location inside the cells and its connection with other cell organelles; and 2-5 higher magnification images to identify shape, size and consents (if inclusions) of the cell organelles.
Regarding preparation, I am assuming cells will be detached from well and processed. Or if you have grown cells on coverslips then it should be process as monolayer which is little tedious work.
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