The use of atomic force microscopy for the observation of corneal epithelium surface (original) (raw)
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Atomic force microscopy analysis of normal and photoablated porcine corneas
Journal of Biomechanics, 2006
We showed the capabilities and accuracy of atomic force microscopy (AFM) techniques for imaging and analyzing the corneal epithelium and the photoablated corneal stroma. Eight normal porcine corneas, half of which were ablated using a scanning-spot excimer laser, were examined. All the corneas were imaged in balanced salt solution after fixation in glutaraldehyde. In the normal untreated corneas we observed the epithelial surface showing the typical polygonal cells and presenting numerous microprojections. The superficial epithelial cells were classified in three types as a result of the anterior-surface roughness measurements. AFM images of the photoablated corneal specimens showed undulations and granule-like features on the ablated stromal surface, specific to 193nm ArF laser irradiation. Nevertheless, the quantitative analysis confirmed the precision of excimer laser surgery in removing submicrometric amounts of tissue. AFM showed to be a high-resolved imaging tool for the scanning of both native as well as photoablated corneal specimens. Also, this technique permits precise topographic analysis of the corneal plane, in the nanometric scale, of which smoothness is an important physical characteristic and necessary to achieve an optimal optical quality of the eye. r
Atomic force imaging of ocular tissues: morphological study of healthy and cataract lenses
… Microscopy. Badajoz, …, 2007
This study focuses the recent application of Atomic Force Microscopy (AFM) to determine structural details of crystalline lenses. We are interested in the application of AFM to distinguish structural and physical properties changes in ocular tissues and associated diseases. Crystalline lenses have its welldefined structure, however, details about pathological stages are still to be determined. In this investigation we evaluated semithin sections and rudimentary incise of crystalline canine lens. AFM showed to be a high-resolved imaging tool for the scanning of both healthy and diseased lenses. Threedimensional images of tissue sections with resolution on a nanometer scale were obtained. In addiction, some results were compared between histological optical microscopy analysis and AFM. The promising of AFM applications for characterizing healthy and diseased ocular tissues is discussed.
Analysis of the healthy rabbit lens surface using MAC Mode atomic force microscopy
Micron, 2007
In this investigation healthy rabbit crystalline lenses were characterized by atomic force microscopy (AFM). The lenses were cut in slices with thickness with 1 mm and thus, put after cortex distinct regions of nucleus and cortex for AFM examination. AFM analysis were carried out using a PicoSPM I operating in Mac Mode. We obtained topographic images of rabbit lenses and a quantitative analysis of the width and height of fibers for nucleus and cortex regions. The longitudinal section analysis of fibers in the nucleus region indicated structures with an average width of 200 nm and average height of 200 nm. The intershells distance was determined as 4 mm. Fiber cell cross-section dimensions, longitudinal and transverse widths, could be estimated in these regions from the AFM images. Structures with average widths as small as 1.0 mm are observed in the nucleus; the intershell distance is 4.0 mm. In cortical regions, hexagonal structures with average longitudinal and transverse widths of 5.0 mm and 3.0 mm, respectively, were identified. Three-dimensional images of tissue sections with resolution on a nanometer scale were obtained. The potential of AFM analysis for characterizing healthy and pathologic lens tissues is discussed. #
Biomechanics of the Anterior Human Corneal Tissue Investigated with Atomic Force Microscopy
Investigative Ophthalmology & Visual Science, 2012
To investigate the biomechanics of the anterior human corneal stroma using atomic force microscopy (AFM). METHODS. AFM measurements were performed in liquid on the anterior stroma of human corneas, after gently removing the epithelium, using an atomic force microscope in the force spectroscopy mode. Rectangular silicon cantilevers with tip radius of 10 nm and spring elastic constants of 25-and 33-N/m were used. Each specimen was subjected to increasing loads up to a maximum of 2.7 N with scan speeds ranging between 3-and 95-m/s. The anterior stromal hysteresis during the extension-retraction cycle was quantified as a function of the application load and scan rate. The elastic modulus of the anterior stroma was determined by fitting force curve data to the Sneddon model. RESULTS. The anterior stroma exhibited significant viscoelasticity at micrometric level: asymmetry in the curve loadingunloading response with considerable hysteresis dependent both on the application load and scan rate (P Ͻ 0.01). The mean elastic modulus ranged between 1.14 and 2.63 MPa and was constant over the range of indentation depths between 1.0 and 2.7 m in the stroma. CONCLUSIONS. At microscale level, the mechanical response of the most anterior stroma is complex and nonlinear. The microstructure (fibers' packing, number of cross-links, water content) and the combination of elastic (collagen fibers) and viscous (matrix) components of the tissue influence the type of viscoelastic response. Efforts in modeling the biomechanics of human corneal tissue at micrometric level are needed. (Invest
Compliance profile of the human cornea as measured by atomic force microscopy
Micron, 2012
The ability to accurately determine the elastic modulus of each layer of the human cornea is a crucial step in the design of better corneal prosthetics. In addition, knowledge of the elastic modulus will allow design of substrates with relevant mechanical properties for in vitro investigations of cellular behavior. Previously, we have reported elastic modulus values for the anterior basement membrane and Descemet's membrane of the human cornea, the surfaces in contact with the epithelial and endothelial cells, respectively. We have completed the compliance profile of the stromal elements of the human cornea by obtaining elastic modulus values for Bowman's layer and the anterior stroma. Atomic force microscopy (AFM) was used to determine the elastic modulus, which is a measure of the tissue stiffness and is inversely proportional to the compliance. The elastic response of the tissue allows analysis with the Hertz equation, a model that provides a relationship between the indentation force and depth and is a function of the tip radius and the modulus of the substrate. The elastic modulus values for each layer of the cornea are: 7.5 ± 4.2 kPa (anterior basement membrane), 109.8 ± 13.2 kPa (Bowman's layer), 33.1 ± 6.1 kPa (anterior stroma), and 50 ± 17.8 kPa (Descemet's membrane). These results indicate that the biophysical properties, including elastic modulus, of each layer of the human cornea are unique and may play a role in the maintenance of homeostasis as well as in the response to therapeutic agents and disease states. The data will also inform the design and fabrication of improved corneal prosthetics.
Lens capsule structure assessed with atomic force microscopy
Molecular Vision, 2015
Purpose To image the ultrastructure of the anterior lens capsule at the nanoscale level using atomic force microscopy (AFM). Methods Experiments were performed on anterior lens capsules maintained in their in situ location surrounding the lens from six human cadavers (donor age range: 44–88 years), four cynomolgus monkeys (Macaca fascicularis age range: 4.83–8.92 years), and seven pigs (<6 months). Hydration of all samples was maintained using Dulbecco’s Modified Eagle Medium (DMEM). Whole lenses were removed from the eye and placed anterior side up in agarose gel before gel hardening where only the posterior half of the lens was contained within the gel. After the gel hardened, the Petri dish was filled with DMEM until the point where the intact lens was fully submerged. AFM was used to image the anterior lens surface in contact mode. An integrated analysis program was used to calculate the interfibrillar spacing, fiber diameter, and surface roughness of the samples. Results The...
Microscopy Research and Technique, 2006
Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 3 10 À9 and 0.1-0.7 3 10 À9 (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality. Microsc. Res. Tech. 69:757-765, 2006. in Wiley InterScience (www.interscience.wiley.com).