Abstract
Super-resolution microscopy revolutionized biomedical research with significantly improved imaging resolution down to the molecular scale. To date, only limited studies reported multi-color super-resolution imaging of thin tissue slices mainly because of unavailable staining protocols and incompatible imaging techniques. Here, we show the first super-resolution imaging of flat-mounted whole mouse cornea using single-molecule localization microscopy (SMLM). We optimized immunofluorescence staining protocols for β-Tubulin, Vimentin, Peroxisome marker (PMP70), and Histone-H4 in whole mouse corneas. Using the optimized staining protocols, we imaged these four intracellular protein structures in the epithelium and endothelium layers of flat-mounted mouse corneas. We also achieved simultaneous two-color spectroscopic SMLM (sSMLM) imaging of β-Tubulin and Histone-H4 in corneal endothelial cells. The spatial localization precision of sSMLM in these studies was around 20-nm. This work sets the stage for investigating multiple intracellular alterations in corneal diseases at a nanoscopic resolution using whole corneal flat-mount beyond cell cultures.
Original language | English (US) |
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Article number | 108499 |
Journal | Experimental eye research |
Volume | 205 |
DOIs | |
State | Published - Apr 2021 |
Funding
National Science Foundation (NSF) ( CBET-1706642 and EFMA-1830969 ); National Institutes of Health (NIH) ( R01EY026078 , R01EY028304 , R01EY019949 , R01GM139151 , R01GM140478 , and P41GM135018 ).
Keywords
- Corneal endothelium
- Single-molecule localization microscopy
- Super-resolution optical microscopy
ASJC Scopus subject areas
- Ophthalmology
- Sensory Systems
- Cellular and Molecular Neuroscience