Kumar Sinniah1, James Paauw, John Ubels. 1. Departments of Chemistry & Biochemistry, Calvin College, Grand Rapids, MI 49546, USA. ksinniah@calvin.edu
Abstract
PURPOSE: To investigate the use of atomic force microscopy (AFM) to image live and fixed cell in culture. Rabbit corneal fibroblasts, Chang conjunctival cells, and transformed human corneal epithelial cells were chosen so that AFM parameters could be set for future use in toxicologic and pharmacologic studies of ocular cells. METHODS: Contact mode AFM was performed under air and in balanced salt solution (BSS) using live and fixed cells. All cell lines were imaged in the height mode for optimal resolution of cellular features. RESULTS: Images of fixed cells showed no discernible differences in surface features when visualized in air or under physiologic solution. Structural differences were observed, however, between fixed and live cells in BSS. Although the AFM technique provides high quality images of live cells under BSS, sub-membrane features of live cells are more well-defined compared to fixed cells. It was also possible to image live cells in air if imaging was completed within 10 minutes of removal of the cells from culture medium. Images of cytoskeletal features under air were similar to those obtained under BSS. CONCLUSION: The atomic force microscopy technique can be used to study cells and provide sub-cellular details at resolution equal to or in some situations better than the scanning electron microscopy technique. However, parameters for imaging have to be tailored for individual experimental goals.
PURPOSE: To investigate the use of atomic force microscopy (AFM) to image live and fixed cell in culture. Rabbit corneal fibroblasts, Chang conjunctival cells, and transformed human corneal epithelial cells were chosen so that AFM parameters could be set for future use in toxicologic and pharmacologic studies of ocular cells. METHODS: Contact mode AFM was performed under air and in balanced salt solution (BSS) using live and fixed cells. All cell lines were imaged in the height mode for optimal resolution of cellular features. RESULTS: Images of fixed cells showed no discernible differences in surface features when visualized in air or under physiologic solution. Structural differences were observed, however, between fixed and live cells in BSS. Although the AFM technique provides high quality images of live cells under BSS, sub-membrane features of live cells are more well-defined compared to fixed cells. It was also possible to image live cells in air if imaging was completed within 10 minutes of removal of the cells from culture medium. Images of cytoskeletal features under air were similar to those obtained under BSS. CONCLUSION: The atomic force microscopy technique can be used to study cells and provide sub-cellular details at resolution equal to or in some situations better than the scanning electron microscopy technique. However, parameters for imaging have to be tailored for individual experimental goals.
Authors: Filipa D Oliveira; Marco Cavaco; Tiago N Figueira; Javier Valle; Vera Neves; David Andreu; Diana Gaspar; Miguel A R B Castanho Journal: FEBS J Date: 2021-11-07 Impact factor: 5.622