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J. E. Morgan, D. Tudor, B. Povazay, B. Hofer, A. Unterhuber, B. Hermann, W. Drexler; In vitro Detection of Neuronal Programmed Cell Death by Ultra High Resolution Optical Coherence Tomography. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2877.
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© ARVO (1962-2015); The Authors (2016-present)
Major advances have been made in the understanding of the way in which cells undergo cell death. Neurons undergo prolonged periods of compromise and structural change prior to the activation of cell death pathways, if this period of cell compromise could be identified, interventions can be timed to coincide with a therapeutic window that could lead to possible reversal in neuronal damage.The development of ultrahigh resolution optical coherence tomography (UHR-OCT) allows tissue to be imaged in vitro with an image resolution better than 2 µm. In this project we aim for the first time to image single cells and detect pre-apoptotic signatures using UHR-OCT.
RGC-5 cells were cultured in growth medium of DMEM with 10% fetal calf serum (FCS), 4 mM Glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37°C. Cells were seeded onto glass coverslips and cultured overnight. Cells were then washed with serum-free medium before incubation in DMEM growth medium containing 0% or 10% FCS for 24, 48 and 72 hours. Cells were imaged using UHR-OCT or incubated with 10 µg/ml JC-1 in culture medium for 15 minutes at 37 °C. Live cell images were obtained at a sampling 1024x466x1024 voxel at 20 Mvx/s, at 800 nm central wavelength and a bandwidth of 230 nm, enabling visualization of sub-cellular structures.
All cells were stained with JC-1, cells cultured with 10% serum and without serum for 24 hours showed both green and red fluorescence, however, cells serum deprived for 48 and 72 hours were devoid of red fluorescence indicating loss of mitochondrial membrane potential. Non serum deprived cells displayed a uniform intensity distribution throughout the cell body and proximal neuritic processes. By contrast, serum deprived cells showed a reduced soma volume with greater variance in scattering density of the order of 20% (P<0.05). These changes correlated with changes seen in JC1 staining in parallel cultures.
Changes in the optical properties of RGC5 serum deprived cells can be detected by UHR-OCT. Profiling the intensity distribution for cell populations may be a useful non-invasive technique for the quantification of neuronal health.
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