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Interference microscopy delineates cellular proliferations on flat mounted internal limiting membrane specimens
A Gandorfer1, R Scheler, R Schumann
1Vitreoretinal and Pathology Unit, University Eye Hospital Munich, Mathildenstr. 8, 80336 Munich, Germany. arnd.gandorfer@med.uni-muenchen.de
Insights
Interference microscopy of internal limiting membrane (ILM) specimens reveals cellular proliferations at the vitreomacular interface. This technique offers a more reliable method for studying cellular distribution compared to conventional microscopy.
Area of Science:
- Ophthalmology
- Cell Biology
- Microscopy
Background:
- The vitreomacular interface is crucial in retinal health.
- Cellular proliferations at this interface can lead to vision impairment.
- Current methods for analyzing these proliferations have limitations.
Purpose of the Study:
- To demonstrate the efficacy of interference microscopy for visualizing cellular proliferations on internal limiting membrane (ILM) specimens.
- To establish a reliable method for assessing cellular distribution at the vitreoretinal interface.
Main Methods:
- Internal limiting membrane (ILM) specimens were obtained during vitrectomy.
- Specimens underwent fixation and were analyzed using interference microscopy.
- Immunocytochemistry and DAPI staining were employed for cellular and nuclear identification.
Main Results:
- Interference microscopy clearly delineated cellular proliferations on the ILM.
- Cellular proliferation areas were distinguishable from acellular ILM regions.
- Immunocytochemistry protocols remained compatible with the microscopy technique.
Conclusions:
- Interference microscopy provides novel insights into cellular proliferation distribution at the vitreomacular interface.
- This method allows for accurate determination of cell density on the ILM.
- The en face visualization of the entire ILM offers a more reliable assessment of the vitreoretinal interface compared to conventional microscopy.
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 phosphate-buffered 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 4',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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