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Yinhong Qu, Karin E. Thomas, Maria Eugenia Vola, Anne-Catherine Roch-Levecq, Yi-Kai Wu, Tracy L. Purcell, Josef F. Bille, David J. Schanzlin; Rapid Identification of Microorganisms Using the Two-Photon Ophthalmoscope. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6175.
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To demonstrate the capacity of a new two-photon ophthalmoscope prototype to reliably identify a sample of various types of microorganisms previously identified as causing infectious keratitis with the goal of improving the speed and accuracy of diagnosis.
The device is a nonlinear scanning laser ophthalmoscope which includes a two-photon excited fluorescence/second harmonic generation imaging modality. Microorganisms such as Streptococcus, Pseudomonas, Acanthamoeba, Aspergillus fungus, Candida albicans, and Microsporidium were viewed at different laser powers (from 1mW-250mW) @ 780nm, 250fs. Each type of microorganism was determined on the basis of its autofluorescence intensity and morphological characteristics.
We found that Candida albicans could be reliably identified at a later power of 30mw, Acanthamoeba at 40mW, and Streptococcus pneumoniae at 50mW. Microsporidium was best viewed at a mid-range laser power between 50 and 100mW. Due to less autofluorescence intensity, other organisms including Pseudomonas Aeruginosa and Aspergilllus fungus were identified at higher laser power (> 100mW). The weaker autofluorescence intensity of the corneal structures was so low that it would not interfere with the autofluorescence imaging of the microorganisms.
We demonstrated the feasibility and reliability of this new two-photon laser ophthalmoscope prototype in identifying various microorganisms within the recommended safety limits of laser power for the cornea. With sensitivity/specificity established on a larger sample size, this device might provide in the future a faster, more accurate and less expensive method than Gram stain and cultures to diagnose microorganisms causing keratitis, pain, and vision loss.
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