Atomic force microscopy (AFM) and fluorescence imaging of ARPE-19 cells subjected to sub-lethal oxidative stress

摘要

Purpose: Retinal pigment epithelium (RPE), being exposed to intense visible light from focal irradiation and high oxygen tension, is at risk of oxidative stress, exacerbated by accumulation of the age pigment lipofuscin. It has been postulated that chronic oxidative stress in this retinal tissue could contribute to the pathogenesis of age-related macular degeneration (AMD). To monitor sub-lethal oxidative stress in ARPE-19 cells, subjected to photodynamic action mediated by phagocytized human RPE lipofuscin or selected photosensitizers, atomic force microscopy (AFM) and fluorescence imaging were employed. Methods: Purified lipofuscin granules obtained from RPE of human donors were introduced into ARPE-19 cells by phagocytosis. Control cells, lipofuscin loaded cells or cells with added rose Bengal (rB) or Merocyanine 540 (MC 540) were exposed, for selected time intervals, to blue or green light, respectively. Agilent 5500 atomic force microscope working in either ac mode or force spectroscopy mode, was used for obtaining cell images and for analyzing mechanical properties of the cells. Standard immunofluorescence staining was employed to visualize actin fibers and microtubule filaments of control cells and cells subjected to photodynamic treatment. Results: Immunofluorescence imaging revealed that early changes associated with photodynamic stress comprised disorganization of the architecture of the cell cytoskeleton. Simultaneous examination of the cells by AFM showed that lipofuscin-mediated photodynamic stress brought about significant decrease in the formation of actin stress fibers. Importantly, such cells exhibited substantially modified nanomechanical properties, as demonstrated by the measured changes in the distribution of the cell Younga??s modulus. Similar results were obtained with cells subjected to photodynamic treatment mediated by rB and MC 540. Conclusions: Our results indicate that AFM can be used for sensitive early detection of changes of cultured RPE cells subjected to sub-lethal oxidative stress.