Novel approaches for scanning near-field optical microscopy imaging of oligodendrocytes in culture.
Newborn rat oligodendrocyte cultures were investigated by scanning near-field optical microscope (SNOM), a versatile new tool able to map cell membranes in 3D and simultaneously obtain images of the cytoplasm. Topography, error, transmission and reflection signals were acquired to describe cell morphology with nanometer-scale resolution. Oligodendrocytes were studied as a model because their extensive membrane processes (typical of their physiological role in myelination) made them particularly suitable to test the sensitivity of the new method. Furthermore, we combined a classical histochemical method with SNOM, to identify specific intracellular proteins at high definition. In particular, with this technique, cytoskeleton elements of oligodendrocytes, such as microtubules, were observed with tubulin antibodies. Images obtained with SNOM were also compared with those from conventional scanning electron microscopy (SEM) and optical microscopy. Our results showed that SNOM allowed to observe cell nanostructures otherwise undetectable all together with other microscopies. In conclusion, SNOM, combined with rapid and non-invasive methods of specimen preparation, appears to be a powerful tool that can offer new possibilities in the field of neuroscience imaging at nano-scale level.
Trevisan
E
E
University of Trieste, Clinical Department of Biomedicine, Via Manzoni 16, Trieste, Italy.
Fabbretti
E
E
Medic
N
N
Troian
B
B
Prato
S
S
Vita
F
F
Zabucchi
G
G
Zweyer
M
M
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D008870:Microtubules / Q000648:ultrastructure; D009836:Oligodendroglia / Q000648:ultrastructure*; D051381:Rats; D016707:Tissue Fixation; D014404:Tubulin / Q000648:ultrastructure
D014404:Tubulin