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ZHEN CAI, Yang Zhang, Zheyuan Zhang, Lisa Beckmann, Hao Zhang; Super-resolution imaging of flat-mounted whole corneal tissue. Invest. Ophthalmol. Vis. Sci. 2020;61(7):910.
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© ARVO (1962-2015); The Authors (2016-present)
Super-resolution microscopy revolutionizes biomedical research with unprecedented spatial resolutions up to 10 nm, while most of the imaging samples are cell cultures. The access to image whole intact tissue sample allows detecting regional differences in expression patterns within a cell and heterogeneity among individual cells. To date, only a few studies report super-resolution imaging of thin frozen tissue. Here, we show, for the first time, super-resolution imaging of flat-mounted whole corneal tissue using spectroscopic single-molecule localization microscopy (sSMLM).
We developed and optimized immunofluorescence staining method for sSMLM imaging of subcellular structures in flat-mounted whole corneal tissue. We performed immunofluorescence staining of mouse corneas after the freshly collected cornea tissues were fixed. We systematically compared immunofluorescence staining quality for sSMLM imaging at different incubation time, temperature, and antibody concentrations. For sSMLM imaging, the corneal piece was placed on a glass slide, covered with imaging buffer containing an enzymatic oxygen scavenger system to facilitate stochastic single molecule switching (blinking). The single-molecule blinking signals were collected using our sSMLM with electron multiplying charge-coupled device (EMCCD). Finally, super-resolution reconstructions of different samples were rendered using ThunderSTORM software, and imaging quality was assessed.
We systematically screened immunofluorescence staining quality for sSMLM imaging and optimized the imaging conditions (4°C ,2 µg mL-1, 48 and 24 hours for primary and secondary antibodies respectively) for β-tubulin (Fig.1), vimentin and peroxisome marker for both epithelial and endothelial cells. Comparing with conventional fluorescence imaging, subcellular structures can be visualized with much greater details at ~25-nm resolution.
We optimized the immunofluorescence labeling protocol for flat-mount whole cornea tissues and performed sSMLM imaging to visualize intracellular cytoskeleton structures for the first time with ~25-nm resolution.
This is a 2020 ARVO Annual Meeting abstract.
Fig. 1. (a) sSMLM imaging of endothelial cells layer in flat-mount corneal tissue stained with β-tubulin. (b) the magnified view better shows the fine microtubule structures. (c) Histogram of localization precision distribution of ~ 106 single molecules collected to reconstruct the image in (a). Scale bar: 1 µm.
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