preparing samples
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what are the 4 steps to prepare samples
• fixation • embedding • sectioning • staining
what is the fixation step of preparing a sample
freezing the cells and tissues in their natural state to stabilise the cell structure and preserve the tissues shape. it prevents the sample from decaying
what are the advantages to fixation
preserves tissue structures, maintains cell components in their natural state, allows storage for longer periods of time
what are the disadvantages to fixation
over fixation can mask or change some cellular components
what is the embedding step of preparing a sample
after fixation the tissue if soft and fragile so needs to be strengthened so it can be sliced into thin sections. this is done by embedding the tissue in solid mediums, for light microscopy this is something like paraffin wax but for electron microscopy its something more rigid such as resin
what are the advantages to embedding
provides support, making thin slicing possible, enables consistent sectioning and better quality slides, relatively quick and inexpensive
what are the disadvantages to embedding
can sometimes extract or alter small molecules, resin is time consuming and requires special equipment and skills, improper embedding can cause deformation
what is the sectioning step of preparing a sample
thinly slicing to allow light or electrons to pass through
how thinly sliced do samples need to be for light microscopes
microtome cuts slices to 4 - 10 micrometers thick.
how thinly sliced do samples need to be for electron microscopes
ultramicrotome cuts into thinner slices of 50 - 100 nanometers thick. Electrons require thinner samples to generate detailed images
what are the advantages to sectioning
produced the necessary thin slices, enables visualisation of internal structures at different depths. Provide higher resolution
what are the disadvantages to sectioning
requires skill and precision, poor sectioning can tear / distort. Too thick or thin sections affect image quality, equip can be expensive and delicate
what is the staining step of preparing a sample
adding colour to create contrast by using dyes that bind to elements of the cell. it helps to distinguish between parts of the cell
what are the advantages to staining
adds contrast, makes structures visible, highlight cell components, simple and accessible
what are the advantages to staining
some stains not specific which causes background colouring or confusion. Overstaining can obscure details, takers time and complexity
what is Haematoxylin
dye that binds to acidic substances like DNA and RNA, found in the cell nucleus. stains the nuclei blue or purple, makes them stand out clearly.
what is eosin
stains basic substances, such as proteins in the cytoplasm and extracellular matrix, in pink or red.