Atomic force microscopy measurements of lens elasticity in monkey eyes

Abstract

Purpose: To demonstrate the feasibility of measuring the elasticity of intact crystalline lenses using atomic force microscopy (AFM).

Methods: AFM elasticity measurements were performed on intact lenses from 18 fresh cynomolgus monkey cadaver eyes (4-10 years old, <1 day postmortem) that had been left attached to their zonule-ciliary body-sclera framework. The eyes were prepared by bonding a plastic ring on the sclera after removal of the conjunctival, adipose, and muscle tissues. The posterior pole was sectioned, with the excess vitreous removed, and the eye's anterior section was placed on a Teflon slide to protect the posterior pole of the lens. The cornea and iris were then sectioned. The lens-zonule-ciliary body-sclera section was then placed in a Petri dish filled with balanced salt solution in an AFM system designed for force measurements. Next, the central pole of the anterior surface of the intact lens was probed with the AFM cantilever tip. The recorded AFM cantilever deflection-indentation curves were used to derive force-indentation curves for the lens after factoring out the deflection of the cantilever on a hard surface. Young's modulus of the lens was calculated from the force-indentation relation using the Hertz model.

Results: Young's modulus was 1,720+/-880 Pa (range: 409-3,210 Pa) in the 18 cynomolgus monkey lenses.

Conclusions: AFM can be used to provide measurements of the elasticity of the whole lens including the capsule. Values obtained using AFM on cynomolgus monkey lenses are similar to published values obtained using dynamic mechanical analysis on young human lenses.

Figure 1

Atomic force microscope for elasticity measurements. The atomic force microscopy (AFM) system for elasticity measurements is a laboratory-made modification of the AFM design used for imaging [15,20]. It is shielded inside an acoustic/vibration isolation chamber. A: The cantilever is moved vertically using a piezoelectric translator that responds to applied voltage. A Petri dish containing the lens is placed below the cantilever, and the cantilever is lowered onto the lens. The cantilever is bent, causing the beam of the laser diode to be deflected. A photodiode monitors these deflections. Custom software controls the piezoelectric translator and times the measurements. B: Shown is a labeled photograph of the AFM used for lens capsule elasticity measurements.

Figure 2

Calibration scans performed to characterize the atomic force microscope cantilever. A: Force scan conducted on a hard surface to determine voltage-deflection relationship. A force scan was conducted on the surface of the Petri dish with no sample to determine the relationship between voltage detected at the photodiode and cantilever deflection. The curve provides the voltage-displacement curve in the absence of indentation. This response is used in the calculation of the force-indentation curves obtained on a samples. B: Recording of natural vibrational frequency of cantilever in fluid. The thermally induced cantilever fluctuations are measured by recording the vibrational frequency of the cantilever in fluid. This response is used to measure the spring constant using equation 1.

Figure 3

Location of the lens sample in the atomic force microscopy system. Location of the lens sample in the atomic force microscopy system. The lens sample is placed in a Petri dish filled with DMEM. The dish with the lens is then placed in the AFM system under a PMMA block that contains the AFM cantilever.

Figure 4

Measurement locations used to validate the visual lens positioning technique. Measurement locations used to validate the visual lens positioning technique. The same lens was measured in five different locations around the lens center. These measurements were used to validate the sample positioning technique and to determine the effect of probe positioning on the variability of the measurements.

Figure 5

Analysis process for atomic force microscopy measurements. Force scans are taken by probing the sample with the cantilever and recording the cantilever deflection (upper panel). Force (in picoNewtons) versus indentation was derived from the cantilever spring constant and slope found during calibration (lower panel). Young's modulus was then calculated using the Hertz model.