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Vivian M Sueiras, Vincent T Moy, Noel Marysa Ziebarth; Characterizing the diffusion of molecules in the anterior lens capsule using fluorescence recovery after photobleaching (FRAP). Invest. Ophthalmol. Vis. Sci. 2014;55(13):730.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the size-dependent transport of molecular probes through the lens capsule by means of fluorescence recovery after photobleaching (FRAP).
Experiments were conducted on 4 porcine lens capsules. The eyes were obtained from an abattoir and shipped in saline to the laboratory overnight. A capsulorhexis was performed to separate a portion of the anterior capsule from the lens. Each excised capsule was submerged in a prepared solution of anionic, fluorescein-labeled dextran in PBS: 2 samples in 10kD MW (d-F10) and 2 samples in 40kD MW (d-F40). The samples were soaked overnight, allowing the molecules to diffuse into the capsule and to reach chemical and diffusional equilibrium. The capsules were then removed from the bath, washed 3 times with PBS, plated on a glass bottom dish and hydrated with PBS. A Nikon A1R confocal microscope was used to conduct FRAP experiments. Prior to bleaching, a single image was acquired to establish the baseline fluorescent intensities. A 20µmx20µm square in the center of the field of view (FOV) at a plane within the capsule was bleached for 2 seconds using an argon laser (488nm) set to full power. After bleaching, time-lapse images of the full FOV were continuously acquired each second for 1 minute. From these images, 2 different regions were identified. Changes in fluorescence intensity stemming from the diffusion of the fluorescent tracer were monitored in a 7µmx7µm region within the bleached area. Since the amount of fluorescence decreases during time-lapse imaging, a reference area located at the periphery of the FOV was used for bleaching correction. These experiments were repeated in at least 4 different areas within 10µm of the central depth along the z-axis of the capsule. The Nikon NIS-Elements software was used to determine the time to half maximum recovery for each measurement.
The time to half maximum recovery was 2.87±0.73s and 2.75±0.38s for the d-F10 and 2.92±0.19s and 3.53±0.52s for the d-F40. The time to half maximum recovery was significantly slower (p=0.049) for the larger anionic, fluorescein-labeled dextran.
Our results indicate that diffusion of anionic particles in the lens capsule is size-dependent. Additional experiments with molecules of varied size/charge are needed to further our understanding of diffusion in the capsule and its role in maintaining lens optical clarity.
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