Recent advances in high-pressure freezing: equipment- and specimen-loading methods

Area of Science:

• Cryo-electron microscopy
• Cell biology
• Microscopy techniques

Background:

• High-pressure freezing (HPF) is crucial for preserving cellular structures.
• Previous HPF technology, like the BAL-TEC HPM 010, had limitations.
• Advancements in HPF technology are essential for high-resolution imaging.

Purpose of the Study:

• To provide an updated overview of high-pressure freezing (HPF) technology and methodology.
• To introduce the Leica EM PACT2 and its Rapid Transfer System (RTS).
• To detail critical specimen loading procedures for HPF.

Main Methods:

• Review of advancements in HPF instrumentation, focusing on the Leica EM PACT2.
• Description of the Rapid Transfer System (RTS) for sample loading.
• Detailed protocols for HPF of various biological samples (cells in suspension, attached cells, small and large organisms/tissues).

Main Results:

• The Leica EM PACT2 offers a portable and advanced HPF solution.
• The RTS attachment facilitates reproducible sample loading and correlative microscopy.
• Specific procedures are outlined for successful HPF across diverse sample types.

Conclusions:

• The Leica EM PACT2 represents a significant technological improvement in HPF.
• Optimized specimen loading is key to successful HPF.
• Updated HPF methods enable high-quality sample preparation for advanced microscopy.