Correlative Light and Electron Microscopy Techniques: Challenges and Successes

Abstract

Correlative light and electron microscopy (CLEM) combines the strengths of light microscopy (LM) and electron microscopy (EM) to form a more complete picture of a cellular process. LM allows for a multitude of fluorescent tags and a wide field of view to select rare events, and EM allows for increased resolution and visualization of cellular ultrastructure [1]. We used three methods to examine external and internal morphologies of cells: cell cultures grown on coverslips, resin sections on coverslips, and resin sections on coated slot grids. Coverslip methods used indium-tin-oxide (ITO) coverslips with fiducial markers. The coverslips were coated with a 0.1% (w/v) Poly-L-lysine solution for 30 min, rinsed in water, and dried on filter paper overnight. Cells were grown and labeled on the coverslips depending on the requirements of the projects and imaged in a laser scanning confocal microscope (LSCM). All samples were imaged on a ZEISS Sigma HD VP Scanning Electron Microscope (SEM). Areas of interest on coverslip samples noted in LM were found in the SEM using ZEISS Shuttle and Find software. Slot grids were placed in a STEM holder and imaged using a backscatter detector. Correlation and overlay of images was performed using multiple software packages.