Imaging of human meiotic chromosomes by scanning near-field optical microscopy (SNOM).
Centromeres and telomeres are key structures of mitotic and meiotic chromosomes. Especially telomeres develop particular structural properties at meiosis. Here, we investigated the feasibility of scanning near-field optical microscopy (SNOM) for light-microscopic imaging of meiotic telomeres in the sub-hundred nanometer resolution regime. SNOM was applied to visualise the synaptonemal complex (SC) and telomere proteins (TRF1, TRF2) after differential immuno-fluorescent labelling. We tested and compared two different preparation protocols for their applicability in a SNOM setting using micro-fabricated silicon nitride aperture tips. Protocol I consisted of differential labelling of meiotic chromosome cores (SC) by SCP3 immuno-fluorescence and telomeres by TRF1 or TRF2 immuno-fluorescence, while protocol II combined absorption labelling with alkaline phosphatase substrates of cores with fluorescent labelling of telomeres. The results obtained indicate that protocol I reveals a better visualisation of structural (topographic) details than protocol II. By means of SNOM, meiotic chromosome cores could be visualised at a resolution overtopping that of far-field light microscopy.
Hausmann
Michael
M
Department of Single Cell and Single Molecule Techniques, Institute of Molecular Biotechnology, P.O. Box 100813, D-07708 Jena, Germany. mihaus@ukl.uni-frieburg.de
Liebe
Bodo
B
Perner
Birgit
B
Jerratsch
Martin
M
Greulich
Karl-Otto
KO
Scherthan
Harry
H
D002877:Chromosomes, Human / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008540:Meiosis; D018625:Microscopy, Atomic Force / Q000379:methods*; D013090:Spermatocytes / Q000648:ultrastructure; D013573:Synaptonemal Complex / Q000648:ultrastructure*; D016615:Telomere / Q000648:ultrastructure*; D035321:Telomeric Repeat Binding Protein 1 / Q000378:metabolism; D035341:Telomeric Repeat Binding Protein 2 / Q000378:metabolism
D035321:Telomeric Repeat Binding Protein 1; D035341:Telomeric Repeat Binding Protein 2