Purchase this article with an account.
Rahul Yadav, Donna Shannon, Jeffrey M. Harder, Richard Libby, Geunyoung Yoon; Optical Coherence Microscope for Cellular Imaging in Cornea. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3127.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To develop an optical coherence microscope that is capable of visualizing and quantifying cellular structures in the cornea.
The system is based on a spectral domain optical coherence tomography (OCT) which, to achieve high axial resolution, uses a broadband (600 to 1000nm) light of a supercontinuum source (Leukos SM-20). The system was assembled in free space to avoid axial resolution degradation due to dispersion. To achieve high lateral resolution with such a large bandwidth, chromatic aberration was minimized by constructing the system mostly using mirrors (an off-axis parabolic mirror for collimation and two concave mirrors for beam expansion) with the exception of the objective lens which was apochromatic for near infrared (correction range: 480 nm to 1800 nm) and has NA of 0.26. The objective has a working distance of 30.5 mm which allows for non-invasive measurement. The axial resolution of the system was estimated by measuring the full width at half maximum of the point spread function obtained by keeping a mirror in the sample arm. The lateral resolution was first predicted by optical ray tracing and then experimentally evaluated by imaging individual pixels (2.2 μm square) of a CMOS sensor. The imaging performance of the system was evaluated by imaging the cornea in two enucleated mouse eyes.
The axial resolution of the system was measured experimentally to be 1.1 μm in corneal tissue. The lateral resolution of the system was estimated to be 1.7 μm by ray tracing and was limited only by diffraction. The 2.2 μm pixels of the CMOS sensor were successfully resolved by the system. In the mouse eyes, the individual keratocytes were visualized over the entire depth of the cornea in a single image acquisition. The keratocyte density was measured to be 88,960 cells/mm3 and 66,080 cells/mm3 in the two eyes respectively. Individual endothelial cells were also visualized and the cell density was measured to be 2819 cells/mm2 and 2176 cells/mm2. The cell densities measured were within the range of values previously reported.
Our prototype optical coherence microscope has demonstrated the feasibility of imaging cellular structures in the mouse cornea non-invasively. This system can be a valuable tool in providing new insights into corneal disease progression and the efficacy of therapeutic interventions.
This PDF is available to Subscribers Only