We aim to determine the molecular mechanisms that establishes the intricate lens architecture and how this established architecture regulates lens function in transparency and lens shape change. To advance the research in our field, we use new imaging methods that allows us to visualize the lens features with high spatial resolution, and this lets us perform quantitative image analysis of lens structures and cellular features. Hormone mount imaging is advantageous over visualization of tissue sections or flat mounting procedures as it enables preservation of the overall 3D tissue structure.
This allows us to perform in-depth morpho metric examination and quantification on native lens structure. The lens is an integrated biological tissue with specialized functions that rely upon localization and depth dependent geometries of the cells and their associated structures. Using the imaging protocols and quantification methods demonstrated will allow for a greater understanding of how lens structures and the complex organization of the lens are established.
Begin the procedure by creating immobilization divots in agarose. To do so, heat and mix 2%agarose in PBS using a microwave until the solution is liquified. Pipette 250 microliters of the liquified agarose into a glass bottomed dish.
And using a flexible plastic cover slip, flatten the agarose across the dish. Once the agarose cools and fully solidifies, use fine tip forceps to remove the cover slip. Then using a three millimeter biopsy punch, create a hole in the agarose at the center of the dish.
With the help of a delicate task wipe remove excess agarose. Store the agarose mold hydrated with PBS at four degrees Celsius until use. Before mounting the isolated Mirion ocular lens, add two milliliters of the appropriate medium to the prepared agarose mold depending on the type of lens.
Using embryo forceps, gently transfer the fix store live lens into the divot in the agarose. Then place the dish on an inverted microscope stage. Confirm that the lens is situated with the anterior region facing the objective by visualizing nuclei staining.
If no nuclei are observed, use curved forceps to gently rotate the lens approximately 180 degrees so that the anterior region faces the objective. Next, proceed to acquire images using a confocal microscope. For visualization of lens capsules, acquire Z stack images using a 40X objective with a step size of 0.3 microns.
Acquire the first image prior to the surface of the lens capsule indicated by WGA staining and the last image after the apical surface of the epithelial cells. To visualize epithelial cells acquire Z-stack images using a 63X objective with a step size of 0.3 microns. Begin the whole mount preparation of the fixed lens by creating immobilization agarose wedges.
To do so, take a glass bottomed dish and pour about five to six milliliters of molten 2%agarose in PBS into it. Wait until the agarose solidifies. Then use a sharp blade to create a triangular divot in the solidified agarose.
Remove the agarose wedge and add one milliliter of PBS to the dish. Aspirate any residual agarose left behind after cutting. Create multiple wedges in a single dish to fit multiple lenses if required.
To store the mold, add one milliliter of PBS into it before keeping it at four degrees Celsius. Using curved tweezers, place the lens into the agarose wedge containing one milliliter of PBS. Adjust the lens so that the equatorial region faces down onto the microscope glass when placed above the confocal objective.
Place the dish on the microscope stage. To confirm that the equatorial region is in focus, visualize the nuclei ensuring they are aligned in rows. Also check the irregularly packed and shaped equatorial epithelial cells and the precisely aligned and hexagonal shaped meridian row cells indicated by affect and staining at the cell membranes.
A random packing of the nuclei in the field of view indicates the lens anterior facing the objective. If nuclei cannot be observed, it likely indicates that the posterior side of the lens is facing the objective. In such cases, use curved tweezers to rotate the lens until the precisely aligned nuclei at the lens equator are observed.
