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Judith Birkenfeld, Jorge Lamela, Sergio Ortiz, Susana Marcos; Imaging crystalline lens microscopic structures of intact in vitro mammal lenses using confocal microscopy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3783.
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© ARVO (1962-2015); The Authors (2016-present)
To image microscopic structures of the intact in vitro mammal crystalline lens using confocal microscopy.
Rabbit eyes were obtained from a local slaughterhouse and transported at a temperature around 4°C. The cornea was removed from the eye 2-24h post-mortem, and the eye was placed in a cuvette and used immediately for imaging. Measurements were done with a custom made optical microscope, which can operate alternately or simultaneously as a confocal microscope or a multiphoton microscope. The microscope is equipped with two lasers, one diode laser at 488 nm and a Ti: Sapphire femtosecond laser tunable over a range of wavelengths between 670 nm and 1040 nm, and two detection channels. Images were obtained with an air objective (MPLAN, 50x, NA 0.75, Olympus). The anterior pole of the lens was imaged, and volumes of images were obtained around the lens apex. All measurements were done on intact lenses with the capsule still attached to the lens zonulae.
A z-scan through the lens allowed identifying distinct regions of the intact crystalline lens: the lens capsule, a thin epithelium layer, and the lens fibers. The lens capsule was seen as a striated structure with an estimated thickness of 10 μm. The structures were oriented, and usually parallel to each other, with an average inter distance of 1.6 μm square. The lens epithelium appeared as a thin cell layer below the lens capsule, with cells of approximately 9 μm in diameter. The lens fibers appeared as elongated, tightly packed fibers with an estimated thickness of 2-3 μm, and a predominant orientation, with all fibers located parallel to each other within the imaged volume.
The potential of confocal light microscopy (CLM) in the anterior pole of the eye lenses was demonstrated by performing an in vitro study of in eye intact rabbit lenses. This method is suitable for quantifying the lens structures in the intact crystalline lens, holding promise for applications in vivo and for microscopic analysis of the lens under accommodative forces.
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