Locating cellular contents during cryoFIB milling using cellular secondary-electron imaging

Area of Science:

• * Structural biology
• * Cellular imaging
• * Microscopy techniques

Background:

• * Cryo-electron tomography (cryoET) enables in-situ observation of molecular structures within cells and tissues.
• * Cryo-focused ion beam (cryoFIB) milling is crucial for preparing samples for cryoET.
• * Identifying specific regions of interest in large biological samples for cryoFIB milling presents a significant challenge.

Purpose of the Study:

• * To introduce and validate a novel on-the-fly localization method for cryoFIB milling.
• * To demonstrate the utility of cellular secondary electron imaging (CSEI) for high-contrast imaging of cellular contents in frozen-hydrated samples.
• * To establish a streamlined workflow for cryoET sample preparation and analysis.

Main Methods:

• * Development and application of cellular secondary electron imaging (CSEI) integrated with cryoFIB instruments.
• * Optimization of imaging principles and settings for CSEI.
• * Testing CSEI feasibility and reliability across various commercial cryoFIB platforms and milling conditions.
• * Validation of a milling-localization workflow using Chlamydomonas reinhardtii basal bodies.

Main Results:

• * CSEI provides high-contrast imaging of cellular contents in frozen-hydrated biological samples without requiring fluorescent labels or additional equipment.
• * The CSEI method proved feasible and reliable on mainstream cryoFIB instruments under diverse milling conditions.
• * The developed workflow successfully enabled localization and milling for subsequent cryoET analysis.

Conclusions:

• * CSEI is an effective, label-free imaging technique for on-the-fly localization during cryoFIB milling.
• * This method overcomes a key limitation in applying cryoET to arbitrary biological samples.
• * CSEI significantly advances the accessibility and efficiency of in-situ structural studies using cryoET.