Cell observation method under near-living conditions by scanning electron microscopy

Area of Science:

• Cell Biology
• Scanning Electron Microscopy (SEM)
• Biotechnology

Background:

• Primary cilia are crucial sensory organelles in multicellular organisms, yet their structure and function in dermal papilla (DP) cells remain unclear.
• Conventional SEM specimen preparation involves harsh chemical treatments and dehydration, potentially damaging delicate cellular structures like primary cilia.
• DP cells regulate hair follicle development, making their detailed structural and functional analysis important for understanding hair biology.

Purpose of the Study:

• To develop and validate a novel, gentler method for preparing DP cell samples for SEM observation.
• To investigate the structure of primary cilia on DP cells under near-living conditions.
• To compare the effectiveness of ionic liquid-based preparation with conventional methods in preserving cellular morphology.

Main Methods:

• Specimen preparation using ionic liquid as a substitute for traditional ethanol dehydration steps.
• Fixation of cultured DP cells (2D and clumps) with glutaraldehyde, followed by ionic liquid immersion.
• Observation of prepared specimens using scanning electron microscopy (SEM) after gold coating.

Main Results:

• The ionic liquid method significantly reduced specimen preparation time and minimized structural damage, particularly avoiding artifacts from drying.
• SEM imaging revealed primary cilia-like structures on 2D cultured DP cells with improved preservation of length and diameter compared to conventional methods.
• While cilia were not definitively identified in DP cell clumps using this method, cells in clumps exhibited better attachment and preserved structures like filopodia.

Conclusions:

• Ionic liquid-based specimen preparation offers a promising approach for observing DP cells and their primary cilia with enhanced structural integrity.
• This method reduces preparation-induced damage, providing clearer insights into the near-living state of these cells.
• Further optimization and complementary techniques like correlative light and electron microscopy (CLEM) are being explored for comprehensive analysis.