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An Improved SEM Preparation Workflow for Plasma Membrane Imaging in HepG2 and IM-9 Cells
Laura Manin1, Antonio Castelliti1, Elena Stuppia1
1Department of Health Sciences, Magna Graecia University of Catanzaro, 88100 Catanzaro, Italy.
Abstract:
Accurate visualization of the plasma membrane is essential for assessing morphological changes induced by experimental perturbations. Scanning electron microscopy (SEM) enables high-resolution imaging of cell surface architecture, but the fixation, dehydration, and drying steps critically affect membrane preservation. Here, we present an improved SEM preparation workflow for two cultured cell models with different growth phenotypes, HepG2 and IM-9, and evaluate the effects of fixation time and drying strategy on plasma membrane preservation. Among the tested conditions, a shorter glutaraldehyde fixation time (15 min) combined with stepwise ethanol-to-HMDS substitution provided the best overall preservation of membrane continuity, cell shape, and surface regularity. Morphometric analysis supported the qualitative SEM observations, and HMDS-processed samples also showed better structural stability during storage under the tested conditions. This workflow provides a simple, reproducible, and cost-effective strategy for SEM-based analysis of cell surface morphology in cultured cell models.
Insights
This study optimized scanning electron microscopy (SEM) sample preparation for better plasma membrane visualization. A shorter glutaraldehyde fixation and HMDS drying best preserved cell surface morphology in cultured cells.
Area of Science:
- Cell Biology
- Microscopy Techniques
Background:
- Accurate visualization of the plasma membrane is crucial for studying cellular responses to experimental conditions.
- Scanning electron microscopy (SEM) offers high-resolution imaging of cell surfaces, but sample preparation significantly impacts membrane integrity.
Purpose of the Study:
- To develop and evaluate an improved SEM preparation workflow for enhanced plasma membrane preservation in cultured cell models.
- To assess the impact of fixation duration and drying methods on cell morphology and membrane structure.
Main Methods:
- Investigated two cell lines (HepG2 and IM-9) with distinct growth phenotypes.
- Optimized glutaraldehyde fixation time and employed stepwise ethanol-to-hexamethyldisilazane (HMDS) dehydration and drying.
- Utilized qualitative SEM imaging and quantitative morphometric analysis.
Main Results:
- A 15-minute glutaraldehyde fixation combined with stepwise ethanol-to-HMDS substitution yielded superior plasma membrane preservation.
- This method maintained cell shape, surface regularity, and membrane continuity effectively.
- HMDS-processed samples demonstrated enhanced structural stability during storage.
Conclusions:
- The optimized SEM workflow offers a simple, reproducible, and cost-effective strategy for analyzing cell surface morphology.
- This improved technique is valuable for researchers studying cellular changes in various experimental contexts.
