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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.
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By Ludwig-Maximilians-Universität MünchenAim: 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.
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