July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Full assessment of cornea structure with a combined confocal Mueller Matrix and non-linear microscope
Author Affiliations & Notes
  • Jessica C Ramella-Roman
    Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States
  • Joseph Chue-Sang
    Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States
  • Megan Laughrey
    Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States
  • Natalia Lopez
    Florida International University, Florida, United States
  • Ilyas Saytashev
    Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States
  • Footnotes
    Commercial Relationships   Jessica Ramella-Roman, None; Joseph Chue-Sang, None; Megan Laughrey, None; Natalia Lopez, None; Ilyas Saytashev, None
  • Footnotes
    Support  Herbert and Nicole Wertheim Professorship Endowment, NSF STROBE
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2137. doi:
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      Jessica C Ramella-Roman, Joseph Chue-Sang, Megan Laughrey, Natalia Lopez, Ilyas Saytashev; Full assessment of cornea structure with a combined confocal Mueller Matrix and non-linear microscope. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2137.

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

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Abstract

Purpose : Visualization of three dimensional arrangement of the cornea extracellular structure is relevant in the study of corneal disease. For example in Keratoconus progressive structural and cellular changes in the cornea compromise its integrity leading to scarring and compromised vision. Non-linear microscopy (NLM) has been used to visualize collagen fibrils and loss of crosslinking in Keratoconus, unfortunately the technique is costly and offers a very low field of view that may not be representative of the disease status in the full cornea. We propose the use of Mueller Matrix confocal polarimetry as an alternative to NLM. In this paper we demonstrate the correlation between NLM and MMCP in the study of the cornea.

Methods : The home-built laser scanning microscope utilizes two photon multiplier tubes for the collection of Two Photon Excitation Fluorescence (TPEF) and Second Harmonic Generation (SHG) while a CMOS camera combined with a polarizer and two liquid crystal retarders is used for retrieving the polarization information. A virtual pin strategy is used to achieve confocal imaging. The laser excitation polarization is altered with a polarizer and two liquid crystal retarders.
Excised rat cornea were imaged in this study.

Results : First we imaged rat cornea stained with DAPI (Fig. 1) in order to mark the presence of epithelial cells and keratocytes. Depolarization maps of stromal anterior layer exhibit higher values than posterior stroma or epithelial depolarization showing clear difference in imaged layers. The presence of NLM signal in 0 µm layer imaging suggests that imaging plane was at the Bowman’s layer, therefore containing signals from epithelium cells TPEF and stromal collagen SHG.

Conclusions :
We have developed an imaging system that provides depth-resolved TPEF and SHG imaging to achieve 3D reconstruction of cellular and collagen distribution and confocal MMP to measure polarization properties. Further MM decomposition allowed us to measure diattenuation, linear retardation and orientation of the sample. We demonstrated co-registered linear and nonlinear imaging modalities in excised rat corneas and directly compared polarization properties of the rat cornea with its fibrillar collagen microstructure.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Depth-resolved two-photon and confocal MMP imaging of a DAPI-stained rat cornea. SHG and TPEF signals are square-rooted for visualization purpose. 120x120 µm2 imaging area.

Depth-resolved two-photon and confocal MMP imaging of a DAPI-stained rat cornea. SHG and TPEF signals are square-rooted for visualization purpose. 120x120 µm2 imaging area.

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