Abstract
PurposeTo investigate the use of atomic force microscopy (AFM) to image the three groups of corneal epithelial cells fractionated by a novel rapid centrifugation isolation technique.MethodsEpithelial cells harvested from primary cultures of rabbit limbal rings were centrifuged onto uncoated dishes, first at 1400 rpm and then at 1800 rpm. The adherent cells after centrifugation at 1400 rpm (ATC1), the adherent cells at 1800 rpm (ATC2) and the non-adherent cells at 1800 rpm (NAC) were investigated for BrdU retention and were subjected to contact mode AFM and Transmission Electron Microscopy (TEM).ResultsCompared with unfractionated cells, the ATC1 group, accounting for about 10% of the whole population, was enriched in BrdU label-retaining cells. There were dramatic overall shape, surface membrane and intra-cellular ultrastructure differences noted among ATC1, ATC2 and NAC populations. The whole cell roughness measurements were 21.1±1.5 nm, 79.5±3.4 nm and 103±4.6 nm for the ATC1, ATC2 and NAC groups, respectively. The mero-nucleus roughness measurements were 34.2±1.7 nm, 13.0±0.8 nm and 8.5±0.5 nm in the ATC1, ATC2 and NAC populations, respectively.ConclusionsAFM was found to be a good tool for distinguishing among the three groups of cells. BrdU label retention, the AFM parameters and TEM together suggest that the ATC1, ATC2 and NAC populations may be progenitor corneal epithelial cells, transit amplifying cells and terminal differentiation cells, respectively.
Highlights
Atomic force microscopy (AFM) is a powerful technique established as a surface science method that is capable of investigating material surfaces from near atomic resolution to mesoscales
We previously developed a centrifugal cell seeding method for rapid and efficient reconstruction of the rabbit ocular surface with limbal stem cell deficiency (LSCD) in rabbits
ATC1 Cells are Enriched for BrdU Label-retaining Cells As shown in Fig. 2, after 72 hr labeling with BrdU followed by a chase for 18 days, the ATC1 population contained the highest number of label-retaining cells (LRCs)
Summary
Atomic force microscopy (AFM) is a powerful technique established as a surface science method that is capable of investigating material surfaces from near atomic resolution to mesoscales. [1] AFM allows for the noninvasive examination of specimens under natural conditions and with minimal preparation, and enables the imaging of living cells in vitro and in vivo. [2] The greatest advantage of atomic force microscopy is its ability to obtain topographic information from the surface of a specimen in nonaqueous, aqueous, or dry conditions without staining, coating or freezing. [3] This allows for the observation of the specimen in conditions close to its natural environment. Marco Lombardo et al [4] showed that AFM is capable of imaging and analyzing the corneal epithelium and the photoablated corneal stroma In this experiment, AFM proved to be a high-resolution imaging tool for the scanning of both native as well as photoablated corneal specimens, and it permits precise topographic analysis of the corneal plane on the nanoscale. Chang conjunctival cells, and transformed human corneal epithelial cells were studied by AFM These authors found that atomic force microscopy can be used to study cells and provide sub-cellular details at a resolution equal to or in some situations better than the scanning electron microscopy technique. Their work defined the AFM parameters appropriate for corneal epithelium imaging in a physiological medium They concluded that AFM represents a new powerful tool for corneal epithelium imaging, and its application in this field warrants further investigation
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