How to Look at Cells

My research involves slicing up pieces of tissue and then looking at it under a microscope.  I do this after I have already stained special cells within the tissue, making them easier to see; this staining, however, can only be accomplished by getting tissue slices to be really thin.  And I mean REALLY thin!  In order to see the parts of a mouse skull that I am looking for, the slices of tissue cannot be any more than 10 micrometers thick; that is 10 MILLIONTHS of a meter, or about 1/100th of a millimeter!  For a better idea of just small that is, here is a website that will help you visualize such small distances.  Animal tissues, even bones, are too squishy and fragile to cut using a normal knife at normal temperatures, so I have to use an extremely sharp razor blade (much sharper than normal razor blades) and I need to freeze the tissue in order to make it very hard.

This is a cryosectioning machine with a frozen sample embedded at the center, ready to be cut.  (image by Thermo Scientific, used with permission)

The cutting process is actually a lot of fun!  First, I embed the tissue sample (which in my research is always a part of the mouse’s skull) into a special gel called OCT which protects the tissue and freezes rock solid to allow for easier cutting.  I then freeze the sample to -80 degrees Celsius (about -112 degrees Fahrenheit) so that the slices do not bend or flex in any way while I cut them.  Then, using more embedding gel, I freeze the sample in place inside a machine called a cryosectioning machine that allows me to adjust the temperature of the sample along with the thickness of the slice, down to just a micrometer or two!  This cutting process is easily the most difficult procedure in my experiments, as no matter how careful I am, many of the slices end up folding over or getting stuck to surfaces inside the machine, leaving them unusable.  After creating a good tissue section, I place it on a microscope slide (I usually place between 4 and 6 sections on each slide) and prepare the slices for staining.

Specific cells are easily differentiated after staining because the dark blue color which they turn is easily contrasted by the surrounding pink tissues. (Images courtesy of the Liu Lab at the University of Michigan)

Staining the tissues is the next step of the cell-viewing process; while unstained cells can be easily seen under a microscope, it is often almost impossible ti distinguish one cell type from another.  To make these cells easier to view, I use a differential staining process called lacZ staining, which turns the cells that I am interested in counting a deep blue color and cells that I care less about a light pink, making it extremely easy to identify and count the stem cells located in the tissue sample. (University of Michigan lacZ staining procedure)


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