Application of an optical immersion-gel in a flow cytometer with horizontally oriented objective.
In certain flow cytometry systems, it is desirable to use immersion optics to obtain optimum fluorescence yield. This is important when propidium iodide and other DNA fluorochromes are used that have weaker fluorescence emission compared to DAPI, when a lamp is used instead of a laser and when the DNA concentrations are low. Our Partec PA II with a horizontally oriented objective and a vertically oriented flow chamber precludes using a liquid immersion medium. The problem was solved using an optical gel with appropriate characteristics. This gel is commercially available and commonly used for connecting glass fiber cables, but has never been used for microscopy before. Compared to the manufacturer's objective (40 x, aperture 0.8), the fluorescence yield was improved approximately four-fold using the optical gel and a 40 x glycerol objective (aperture 1.25). This innovation widens the applicability of flow cytometers with horizontally oriented objectives and vertical flow chambers. We expect it to facilitate the use of propidium iodide as a DNA stain, especially when interspecific genome size comparisons are to be done and base ratio dependent bias must be avoided.
Temsch
E M
EM
Institute of Botany, University of Vienna, Austria. temsch@s1.botanik.univie.ac.at
Obermayer
R
R
Dolezel
J
J
Greilhuber
J
J
D002467:Cell Nucleus / Q000648:ultrastructure; D018744:DNA, Plant / Q000737:chemistry / Q000302:isolation & purification; D005434:Flow Cytometry / Q000379:methods*; D005782:Gels; D018745:Genome, Plant; D059828:Plant Cells; D010944:Plants / Q000235:genetics*
D018744:DNA, Plant; D005782:Gels