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P. E. King-Smith, J. J. Nichols, K. K. Nichols, R. J. Braun; A High Resolution Microscope for Imaging the Lipid Layer of the Tear Film. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4162.
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Previous microscopy of the lipid layer used systems designed for other purposes, e.g., for non-contact confocal microscopy of the cornea. The current microscope was designed specifically for high resolution imaging of the lipid layer.
The video microscope images a circular area of 200 µm (400 pixels) diameter. The following features are incorporated. 1/. To reduce blur caused by eye movements and movement of the lipid layer, a stroboscopic light source is used with a flash duration of less than 40 µs. 2/. The edge of the image is used as a focusing aid - the image is in focus when the circular edge of the image is in focus. 3/. To ensure good focus throughout the image, the cornea at the center of the image should be perpendicular to the axis of the microscope objective. This is achieved by aligning the center of an "X" with the center of a "+" on part of the video display. 4/. For a high resolution microscope, the depth of focus is very limited - about 1 µm. Given that axial movements of the eye are much greater than this depth of focus, 2000 images are recorded over a 20 s period, with the expectation that a few of them will be in focus. 5/. Reflectance, and hence lipid thickness can be derived for any point in the image, so that a histogram of lipid thickness can be determined.
Resolution was measured to be 1.0 µm (distance between the 10% and 90% points of the edge spread function). The Figure is a contrast-enhanced image of a human lipid layer. Lipid thickness was (mean ± SD) 26 ± 6 nm.
The microscope has higher resolution than previous systems, so it can provide more accurate estimates of lipid thickness distributions. Correlation of lipid structure with tear thinning rate and biochemical composition is planned.
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