We have performed analysis of DNA ploidy with two different methods: Image cytometry and flow cytometry.
IMAGE CYTOMETRY
Equipment required for measuring DNA ploidy using image cytometry method.
Image cytometry is based on the feulgen tequnique, which is a frequently used staining procedure in biology. The technique is based on Schiffs or Schiffs-similar binding to released aldehyde groups from DNA molecules after hydrolysis by HCl, and thereby enables staining of DNA in situ. The intensity of staining is proportional with the DNA concentration and the amount of DNA in the nucleus is expressed as amount of light absorbed by the feulgen-stain over the entire nucleus. The feulgen reaction is today in use to quantify the DNA ploidy distribution in tumor nuclei.
(The image of each nucleus is transferred from a microscope to a computer through a digital camera. The computer measures the amount of absorbed light as an expression of the amount of DNA.)
FLOW CYTOMETRY
Principles for the method in brief: A DNA specific fluorocrome binds to DNA. Nuclei is going by a liquid stream, through a optical measuring point. A laser beam splits of photons and the emmited light is measured and thereby expresses the amount of DNA in the tissue sample.
Preparation of samples (in brief) from paraffin embedded tissue :
Flow cytometry
Image cytometry
Sectioning (100mm)
Sectioning (50mm)
Deparaffination
Deparaffination
Enzyme treatment (pepsine, RNase)
Enzyme treatment (protease)
Cytospin centrifugation (monolayer)
Staining with ethidium bromide
Staining with feulgen-schiff
Measurements
Measurements
Advantages and disadvantages of flow cytometry and image cytometry:
Flow cytometry
Image cytometry
Advantages
Fast Abuntant nuclei Nuclei may be sorted
Morphological control No background noise Unlimited number of features may be evaluated
