Fluorescence Sectioned Microscopy for Early Diagnosis of Acanthamoeba Keratitis

Abstract

Purpose: : To develop an imaging method for in-vivo diagnosis of Acanthamoeba keratitis by means of the in-situ identification of the fluorescently stained pathogen in the infected cornea.

Methods: : We developed a structured illumination (SI) based corneal microscope with a channel for fluorescence, and applied it to the inspection of Acanthamoeba sp in infected but otherwise intact porcine eyes. A 488 nm CW laser source was used for excitation; fluorescence emission was collected through a 510-590nm filter. The long working-distance dry objective (Olympus LMPFL 50x, NA 0.50, w.d. 10.6) allowed non-contact imaging of 90 µm diameter circular areas of the cornea. Lateral resolution was 0.6 µm and axial resolution was 2.6 µm FWHM. Ocular infection was mimicked transferring 1 SU trophozoites of Acanthamoeba sp to the surface of freshly enucleated porcine eyes. A solution (200 µl, 5 µM) of a fluorescent analogue of the lipid hexadecylphosphocoline¹ (BDP-MT) was topically applied, and the infected eyes were left for incubation for at least 20 minutes at room temperature. As a control experiment, BDP-MT was similarly applied to uninfected eyes. The integrity of the epithelium was tested on control eyes with a live/death fluorescent assay.

Results: : SI fluorescence corneal microscopy allowed the study of the effect of BDP-MT on both healthy and infected porcine corneas. Optically sectioned images demonstrated that the fluorescent compound was not internalised by healthy squamous cells of uninfected corneas. Small specimens (~15 µm) of fluorescently stained trophozoites in the infected corneas were distinctly identified.

Conclusions: : An imaging method based on a structured illumination corneal microscopy together with a fluorescent analogue of miltefosine allows fast, minimally invasive, distinct identification of small Acanthamoeba specimens invading the cornea. This method may be applied to the diagnosis of amoebaean and other keratitis of similar origin.

Keywords: Acanthamoeba • microscopy: light/fluorescence/immunohistochemistry • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)