The overall goal of this procedure is to evaluate the compressive stiffness and geometry of mouse lenses using a simple and inexpensive approach. This method can answer key questions in the lens field. Such as how different genetic and experimental perturbations affect lens biomechanical properties.
The main advantages of this technique are that it is precise, reproducible, and does not require expensive or specialized equipment. This method provides insight into the properties on mouse lenses, with potential application to larger model organisms such as rats and rabbits. Visualization of the method is critical because lens dissection and coverslip application steps are difficult to learn without demonstration.
Demonstrating the procedure will be Catherine Cheng, a post doc from my laboratory. Begin the dissection using curved forceps to depress the tissue around the eye of a euthanized mouse bringing the eye out of the socket. Then, remove the eye and place it in a dissection dish containing fresh PBS under a dissecting microscope.
Cut off the optic nerve as close to the eyeball as possible. And gently and carefully insert fine, straight tweezers into the eyeball through the hole where the optic nerve exits the posterior of the organ. Carefully make an incision with scissors from the posterior to the edge of the cornea, taking care not to damage the lens.
Continue the incision along the junction between the cornea and the sclera, at least halfway around the eyeball. And gently push on the cornea to remove the lens from the eye through the opening. Then, use fine tipped straight forceps to carefully remove any large debris that is still attached to the lens.
And visually confirm that the lens is free of damage. At least two hours before starting the stiffness measurements, use an analytical balance to weigh at least 10 cover slips from the same box. Determine the average weight of the cover slips.
Then, pre wet the cover slips and a right angle mirror in room temperature PBS. While the materials are being hydrated, fill the measurement chamber with 65 to 75 milliliters of PBS. And place the chamber under a dissecting microscope set to a 30 X magnification, with illumination from the bottom and a fiber optic light source on the left and right sides.
When the cover slips are ready, place the right angle mirror into the chamber at a constant distance from the divot that will be used to hold the lens. Next, using curved forceps, carefully transfer the dissected lenses to the measurement chamber. Set the power optic power supply to 80%of the maximum light intensity, and adjust the power supply output based on the ambient lighting, user preference, and picture quality as needed.
Obtain top view images of the unloaded lenses from directly overhead of the chamber. Then, take a picture of the mirror edge in focus, followed by a side view image of the unloaded lens through the mirror outside of the divot. Then, place a lens into the divot, and confirm that the lens is seated securely and straight within the divot on it's anterior or posterior pole.
Take a photo of the lens in the mirror. Then, gently place a cover slip onto the lens. After two minutes, take another side view image of the loaded lens.
Continue adding cover slips, obtaining side view images two minutes after the addition of each cover slip, until the total of 10 cover slips have been applied. Then, remove all of the cover slips, wait a final two minutes, and obtain side view images of the lens inside and outside of the divot. And a top view image of the lens outside the divot.
To determine the lens nucleus size, place a lens into a clean petri dish filled with PBS and use fine, straight forceps to gently decapsulate the lens. To off the cortical fiber cells, roll the lens between gloved fingers. Next, gently rinse the lens nucleus in the PBS and place the nucleus back into the measurement chamber.
Acquire an image of the lens nucleus through the right angle mirror. Finally, to analyze the images, use the appropriate image analysis software to measure the equatorial and axial diameters of the lenses before and after each loading step. Measure the diameter of each lens nucleus as well.
In this representative experiment, the stiffness and dimensions of two, four, and eight month old mouse lenses were measured and the axial and equatorial strains were calculated. The axial strain is a logarithmic function of applied load with a statistically significant, age dependent decrease in the axial and equatorial strains under the maximum applied load, indicating that the mouse lens stiffens with age. The image data collected during this experiment were also used to determine several other lens morphological characteristics.
As expected, the axial and equatorial diameters, and the lens volume, increased with age. The aspect ratio indicates that the lens has a slightly larger equatorial diameter than axial diameter. And this parameter did not change with age.
The diameter, volume, and fraction of the lens nucleus increase with age, suggesting that the lens nucleus remodels to increase in relative size as the lens ages. Once mastered, this technique can be completed in 30 to 45 minutes per lens, if performed properly. While attempting this procedure, it's important to position the lens evenly inside the divot, and to allow the lens to stress relax for two minutes between each additional cover slip.
After watching this video, you should have a good understanding of how to assess the compressive stiffness, and morphometrics of mouse lenses using the sequential application of cover slips. This precise, simple, and inexpensive technique paves the way for lens researchers to explore the biomechanical effects of gene mutations in mice.
