Abstract
Purpose :
In the field of material science, Atomic Force Microscopy (AFM) is routinely used to investigate the surface and mechanical properties of various materials. Our group has recently shown a link between the biomechanical environment of the retina and neuronal survival. To explore in more detail the biomechanical properties of retinal tissue, a method using AFM was developed.
Methods :
Full-thickness adult porcine retinal sheets (6x6mm) were divided into three groups: freshly dissected retinal tissue, fixed explants, and explants fixed, embedded and cryosectioned at a thickness of 12µm. Samples from all three groups were scanned for elasticity as well as topography using a JPK Nano-Wizard II AFM in contact mode. Cantilevers with either a pyramidal (spring constant of 0.2N/m) or a colloidal (borosilicate glass; 5µm Ø with a spring constant of 0.08 or 0.32N/m) tip were used. For elasticity measurements, non-oscillating force mapping using a Hertz-fit was employed.
Results :
Topographical and elasticity measurements could be obtained using fixed, sectioned retinal explants. A low set point using a stiff cantilever yielded high-detailed topographical images of all retinal layers, with surface variations. Structures including inner and outer segments, Müller cell fibers and cell soma were identified at high resolution. In addition, elasticity measurements of the various layers and cellular structures were obtained. The retina displayed inhomogeneous elastic properties within the layers (inner limiting membrane ∼400 kPa, inner plexiform layer ∼250 kPa, cell soma ∼300 kPa). These values were found to be ∼1000x stiffer than previously reported for unfixed, single cells. The developed AFM protocol for topographical and elasticity measurements was not applicable in unsectioned retinal explants. In both fixed and unfixed explants, the full-thickness sheet was found to be too pliable to generate a cantilever-tissue contact point.
Conclusions :
We have shown that AFM can be used for detailed topographical investigation and elasticity measurements in the adult retina with minimal tissue preparation. This method links retinal architecture with physical tissue properties, opening new avenues of research into biomechanical mechanisms of retinal pathology. Future research will focus on extending the application of this method to full-thickness explants.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.