Tear analytical model based on Raman microspectroscopy for investigation of infectious diseases of the ocular surface.

PURPOSE To establish a tear analytical model by using Raman microspectroscopy to assess ocular surface diseases associated with infectious pathogens. METHODS The authors applied confocal Raman microspectroscopy based on the drop-coating deposition method on Ti/Au-coated glass slides to obtain sample spectra from different types of tears, including simplified synthetic tears (SSTs), SSTs mixed with microbes, and human tears. Raman spectra were sampled by a line-mapping procedure and classified into three groups by three different zones in a dried teardrop. To determine the tear model with optimal discrimination, the spectra of the three zones were compared using spectral morphology and principal component analysis. Finally, the optimal tear model was verified by comparing the Raman spectra of human teardrops of patients with ulcerative keratitis and bacterial infection with those of patients without any identifiable infection. RESULTS Nonhomogeneous intensities of Raman spectra collected by a line-mapping sampling procedure were found in different locations of a dried teardrop. This might have been caused by coffee-ring formation and ferny crystallization phenomena. The normalized spectra in the central zone have better discriminative potential than those in the other zones, ring zone, and transitional zone, tested by pure SSTs or SSTs with microbes. The tear model based on normalized Raman spectra in the central zone was discriminative for patients with ulcerative keratitis in the presence or absence of infection. CONCLUSIONS Raman spectra in the central zone of a dried teardrop may serve as useful spectral fingerprints for investigating ocular surface diseases with or without infection.