Interference microscopy delineates cellular proliferations on flat mounted internal limiting membrane specimens.

Aim: To demonstrate that interference microscopy of flat mounted
internal limiting membrane specimens clearly delineates cellular
proliferations at the vitreomacular interface. Methods: ILM
specimens harvested during vitrectomy were fixed in glutaraldehyde
0.05% and paraformaldehyde 2% for 24 h (pH 7.4). In addition to
interference microscopy, immunocytochemistry using antibodies
against glial fibrillar acidic protein (GFAP) and neurofilament
(NF) was performed. After washing in phosphatebuffered saline 0.1
M, the specimens were flat-mounted on glass slides without
sectioning, embedding or any other technique of conventional light
microscopy. A cover slide and 49,6-diamidino-2-phenylindole (DAPI)
medium were added to stain the cell nuclei. Results: Interference
microscopy clearly delineates cellular proliferations at the ILM.
DAPI stained the cell nuclei. Areas of cellular proliferation can
be easily distinguished from ILM areas without cells.
Immunocytochemistry can be performed without changing the protocols
used in conventional microscopy. Conclusion: Interference
microscopy of flat mounted ILM specimens gives new insights into
the distribution of cellular proliferations at the vitreomacular
interface and allows for determination of the cell density at the
ILM. Given that the entire ILM peeled is seen en face, the
techniques described offer a more reliable method to investigate
the vitreoretinal interface in terms of cellular distribution
compared with conventional microscopy.