Confocal microscopy reveals zones of membrane remodeling in the outer cortex of the human lens.
OBJECTIVE
To optimize fixation, sectioning, and immunolabeling protocols to map the morphology of the human lens with confocal microscopy.


METHODS
Transparent human lenses were fixed in 0.75% paraformaldehyde for 24 hours, cut in half, and fixed for another 24 hours. Lenses were cryoprotected, sectioned, and labeled with wheat germ agglutinin, aquaporin-0 antibodies, Hoechst, or toluidine blue. Before fixation, some lenses were incubated in an extracellular marker dye, Texas Red-dextran. Labeled sections were imaged with a confocal microscope. Overlapping images were tiled together to form a continuous image montage of fiber cell morphology from the periphery to the lens center.


RESULTS
Fiber cell morphologies were identical with those previously described by electron microscopy and allowed immunohistochemistry to be performed for a representative membrane protein, aquaporin-0. Sectioning protocols enabled the epithelium and outer cortex to be retained, leading to the identification of two unique morphologic zones. In the first zone, an age-independent compaction of nucleated fiber cells and the initiation of extensive membrane remodeling occur. In the second zone, fiber cells retain their interdigitations but lose their nuclei, exhibit a distorted shape, and are less compressed. These zones are followed by the adult nucleus, which is marked by extensive compaction and a restriction of the extracellular space to the diffusion of Texas Red-dextran.


CONCLUSIONS
The authors have developed sectioning and imaging protocols to capture differentiation-dependent changes in fiber cell morphology and protein expression throughout the human lens. Results reveal that differentiating fiber cells undergo extensive membrane remodeling before their internalization into the adult nucleus.
Department of Physiology, New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand.
Julie C
JC
Lim
Kerry L
KL
Walker
Trevor
T
Sherwin
Kevin L
KL
Schey
Paul J
PJ
Donaldson
D000293:Adolescent; D000368:Aged; D020346:Aquaporins / Q000378:metabolism; D002454:Cell Differentiation; D002462:Cell Membrane / Q000502:physiology*; D004396:Coloring Agents; D004847:Epithelial Cells / Q000166:cytology*; D005136:Eye Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007150:Immunohistochemistry; D007904:Lens Cortex, Crystalline / Q000166:cytology*; D018613:Microscopy, Confocal; D008867:Microtomy; D008875:Middle Aged; D016707:Tissue Fixation; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D020346:Aquaporins; D004396:Coloring Agents; D005136:Eye Proteins; D014909:Wheat Germ Agglutinins; C030328:aquaporin 0