Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy

Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 μm/0.4-0.6 μm) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking. © 2012 Optical Society of America.The authors acknowledge funding from a CSIC Proyecto Intramural Frontera PIF80F0171/2/3; Ministerio de Ciencia e Innovación, Spain, FIS2008-02065 (S. M.), FIS2011-25637 (S. M.), RYC2006-2920 (J. R. I.), FIS2009-07966 (J. R. I.), CTQ2010-16457 (A. U. A.), PROFIT CIT-300100-2007-50 (J. M. L.); Instituto de Salud Carlos III ISCIII- (RICET)-FEDER RD 06/0021/0006 (L. R.), and FIS 2009-01928 (L. R.); European Union FP7 HEALTH-2007-223414 (L. R.); EURHORCS-ESF Grant EURYI-05-102-ES (S. M.).Peer Reviewe

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