April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Corneal stroma collagen order determined by polarization sensitive second harmonic microscopy
Author Affiliations & Notes
  • Giuseppe Lombardo
    IPCF - Unit Support of Cosenza, CNR, Rende (Cosenza), Italy
    Vision Engineering, Rome, Italy
  • David Merino
    ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain
  • Pablo Loza-Alvarez
    ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain
  • Marco Lombardo
    Fondazione G.B. Bietti IRCCS, Rome, Italy
  • Footnotes
    Commercial Relationships Giuseppe Lombardo, None; David Merino, None; Pablo Loza-Alvarez, None; Marco Lombardo, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 4858. doi:
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      Giuseppe Lombardo, David Merino, Pablo Loza-Alvarez, Marco Lombardo, ; Corneal stroma collagen order determined by polarization sensitive second harmonic microscopy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4858.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

To evaluate stromal collagen order distribution across depth in donor human corneas.

 
Methods
 

Eight human donor corneas were imaged via polarization sensitive second harmonic generation microscopy (P-SHG). Each corneal tissue was cut in two lenticules using an eye-bank Moria microkeratome with 200 µm head. A linear polarizer, followed by a zero order λ/2 wave plate placed on a motorized rotational stage, was used to control on the fundamental input polarization at the sample plane. Forward SHG images were collected from anterior and posterior lenticules and used for subsequent analysis of stromal collagen structural organization. Under assumption of cylindrical symmetry of collagen fibers and using mathematical tensorial framework, a biophysical model was developed to quantify the mean average fiber orientation and the degree of fiber order orientation (order parameter, S) across corneal depth. The S parameter indicates how clustered the collagen fibers are, and whether they are clustered along the main average fiber direction (S≥0) or orthogonal to it (S<0). For each pSHG image obtained across the stroma, the percentage of pixels with a negative order parameter value was recorded (inhomogeneity ratio, N). Logistic regression analysis was performed to classify stromal collagen order of the anterior and posterior corneal lenticules based on S and N variables. Receiver operating characteristic (ROC) curves were plotted to obtain critical values that allow classification with maximum accuracy.

 
Results
 

The mean S values of the anterior and posterior stromal lenticules were 0.49 ± 0.03 and 0.57 ± 0.04 respectively. The mean N values of the anterior and posterior stromal lenticules were 21.0 ± 0.8% and 16 ± 1.10% respectively. Regression logistic analysis reached an area under the ROC curve of 1.00, showing a sensitivity and a specificity of 100% to discriminate between anterior and posterior stromal collagen structures.

 
Conclusions
 

P-SHG was shown to be an accurate method to ascertain structural order of stromal collagen across corneal depth. Further studies are needed to understand the reliability of the technique to screen corneal disorders, such as keratoconus, and the efficacy of treatment aiming to manipulate collagen structural organization.

 
 
Figure 1: PSHG Intesity image of posterior cornea.
 
Figure 1: PSHG Intesity image of posterior cornea.
 
 
Figure 2: Fiber order orientation (S) of collagen fibers shown in Figure 1.
 
Figure 2: Fiber order orientation (S) of collagen fibers shown in Figure 1.
 
Keywords: 484 cornea: stroma and keratocytes • 551 imaging/image analysis: non-clinical  
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