Imaging mitochondria in living corneal endothelial cells using autofluorescence microscopy

We report noninvasive autofluoreseence mitochondriall imaging in cultured human corneal endothelial cells (HCECs). HCECs harvested from eye bank corneas were cultured in thin glass-bottom plates. Mitochondria were imaged with an autofluorescence microscope using a DAPI filter set (excitation: G365, emission: band pass 445/50) and then, after fixation with 4% paraformaldehyde, cells were stained with MitoTracker (TM) Green FM (MTG). Both images were aligned using a linear conformal algorithm for image mapping based on manually selected corresponding feature points, and then mathematically compared using two-dimensional spatial image correlation coefficients. Autofluorescence imaging provided highly resolved mitochondrial signals from living HCECs, comparable to those taken with NITG. Both techniques yielded very similar images at high magnification and high resolution, demonstrating the tubular morphology and cytoplasmic distribution that are characteristic of mitochondria. Image registration using a linear conformal mapping technique and cross-correlations showed high correlation of overlapping autofluorescence and NITG images. This study validates the novel use of autofluorescence vital imaging as a noninvasive, inexpensive and functional alternative to the mitochondria-specific dyes in cultured HCEC. This noninvasive mitochondrial imaging technique can be useful in future applications studying mitochondrial biology of ocular cells.