Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells

作者： Jue Wang1,2, Anthony V. Sica2, Grant J. Jensen3, Peter D. Dahlberg2,4 1Division of Biology and Biological Engineering,California Institute of Technology, 2SLAC National Accelerator Laboratory, 3Department of Chemistry and Biochemistry,Brigham Young University, 4Department of Photon Science and Structural Biology,Stanford University

简介：

Overview

This workflow enables the production of cryogenic lamellae targeting fluorescently labeled biological structures that are small and rare. The integration of fluorescence microscopy, focused ion beam milling, and scanning electron microscopy at a single focal position allows for simultaneous imaging and milling.

Key Study Components

Area of Science

• Neuroscience
• Biophysics
• Microscopy Techniques

Background

• Optical microscopy methods are essential for preserving cellular features during milling.
• Integration of fluorescence microscopy into CryoFIB-SEM systems is rare.
• Registration-based guidance is necessary to maintain fluorescently labeled targets.
• Understanding native biological organization is crucial for detailed analysis.

Purpose of Study

• To develop methods for preserving cellular features in cryogenic lamellae.
• To enable detailed analysis through cryogenic electron tomography.
• To improve the accuracy of targeting fluorescent structures during milling.

Methods Used

• Manual cooling of the transfer station using liquid nitrogen.
• Integration of fluorescence microscopy with FIB-SEM systems.
• Acquisition of pre-milling fluorescent z-stacks.
• Monitoring fluorescence intensity during milling using a Python toolkit.

Main Results

• Fluorescence intensity increased after removing non-fluorescent bulk material.
• Correlated cryogenic fluorescence and electron microscopy enabled precise targeting.
• Reconstructed tomograms revealed the structure of centrioles.
• Single centriole visualization was achieved in some cases.

Conclusions

• The study demonstrates effective methods for cryogenic lamella production.
• Fluorescence-based targeting improves the accuracy of structural analysis.
• Integration of imaging techniques enhances understanding of biological organization.

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