Cryo-SEM Investigation of Chlorella Using Filter Paper as Substrate

Area of Science:

• Microscopy
• Cell Biology
• Biophysics

Background:

• Conventional electron microscopy requires dry samples, leading to dehydration artifacts and loss of structural detail.
• Cryo-electron microscopy (cryo-EM) preserves hydrated samples by cryo-fixing them, maintaining native structures.
• Investigating hydrated biological samples like algae requires advanced imaging techniques.

Purpose of the Study:

• To present a detailed, step-by-step protocol for cryo-scanning electron microscopy (cryo-SEM) of hydrated samples.
• To introduce a novel method using filter paper for cryo-fixation and freeze-fracture of water-suspended samples.
• To enable high-resolution imaging of both internal structures and surface morphology of biological specimens.

Main Methods:

• Utilizing filter paper as a substrate to support a thin sample film for efficient cryo-fixation.
• Implementing a freeze-fracture technique on water-suspended samples, including Chlorella sorokiniana.
• Employing cryo-scanning electron microscopy for high-quality imaging of prepared samples.

Main Results:

• Achieved high-quality imaging of Chlorella sorokiniana, resolving both internal structures and surface morphology.
• Demonstrated a reliable and simple method for cryo-fixation and freeze-fracture of hydrated samples.
• Validated the effectiveness of filter paper as a substrate for cryo-SEM sample preparation.

Conclusions:

• The described cryo-SEM protocol provides a versatile and effective approach for studying hydrated biological samples.
• The filter paper substrate method facilitates clean freeze-fracture, crucial for detailed SEM investigation.
• This technique is applicable to various water- or solvent-dispersed samples, including different types of microbial cells.