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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 3, Iss. 10 — Oct. 1, 2012
  • pp: 2489–2499

Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy

Susana Marcos, Jose Requejo-Isidro, Jesus Merayo-Lloves, A. Ulises Acuña, Valentin Hornillos, Eugenia Carrillo, Pablo Pérez-Merino, Susana del Olmo-Aguado, Carmen del Aguila, Francisco Amat-Guerri, and Luis Rivas  »View Author Affiliations

Biomedical Optics Express, Vol. 3, Issue 10, pp. 2489-2499 (2012)

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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 OSA

OCIS Codes
(170.4460) Medical optics and biotechnology : Ophthalmic optics and devices
(170.4470) Medical optics and biotechnology : Ophthalmology
(180.0180) Microscopy : Microscopy

ToC Category:
Ophthalmology Applications

Original Manuscript: June 11, 2012
Revised Manuscript: July 31, 2012
Manuscript Accepted: July 31, 2012
Published: September 12, 2012

Susana Marcos, Jose Requejo-Isidro, Jesus Merayo-Lloves, A. Ulises Acuña, Valentin Hornillos, Eugenia Carrillo, Pablo Pérez-Merino, Susana del Olmo-Aguado, Carmen del Aguila, Francisco Amat-Guerri, and Luis Rivas, "Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy," Biomed. Opt. Express 3, 2489-2499 (2012)

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