Purpose: Epiretinal membranes (ERMs) are composed of neural elements and different cellular and extracellular matrix (ECM) components usually visualized using multi-labeling methods. We have used two-photon microscopy to image non-stained human ERMs to analyze the sources of two-photon excitation fluorescence (TPEF) and second harmonic generation signal (SHG). This would help to better understand the organization and structure of ERMs.
Methods: A custom-developed multiphoton microscope equipped with adaptive optics (Bueno et al., J. Biomed. Opt. 2010) was used to record pairs of TPEF-SHG images originated from the ERM structures in a backward configuration. Specimens from ten patients were successfully removed during vitrectomy and ERM peeling, without the use of ophthalmic dyes. Immediately after surgery, ERMs were fixated with a paraformaldehyde solution overnight and flatmounted for two-photon microscopy imaging. Series of images across the specimen and 3D stacks as a function of depth were acquired for the different samples.
Results: Both TPEF and SHG signals were obtained from the ERM’s samples, revealing different structures. SHG signals confirmed the presence of filamentous structures corresponding to collagen fibers and covering the specimen. TPEF signal was significantly higher probably due to the presence of numerous types of cells (Muller, glial…). This cellular autofluorescence is originated from the mitochondrial reduced pyridine nucleotides NAD(P)H and flavin compounds. Moreover, the elastin of the ECM might also contribute to TPEF images.
Conclusions: Two-photon microscopy provides a non-invasive tool to image unstained isolated ERMs. TPEF/SHG signal combination revealed detailed information on the ERM morphology not available with classical imaging techniques. Since staining procedures are not required, the sample manipulation is reduced. Non-linear imaging techniques are useful in the visualization and analysis of healthy and pathological retinal and epiretinal tissues. Farther studies should investigate the dependence between TPEF/SHG signals and ERM etiology (idiopathic, diabetic or inflammatory).