Rapid Identification of Microorganisms Using the Two-Photon Ophthalmoscope

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: keratitis • microscopy: confocal/tunneling • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)