Correlative Organelle Microscopy: Fluorescence Guided Volume Electron Microscopy of Intracellular Processes

Area of Science:

• Cellular and Molecular Biology
• Microscopy Techniques
• Biophysics

Background:

• Cellular functions rely on precise spatial and temporal organization of proteins and organelles.
• Understanding cellular physiology requires linking molecular information to nanoscale ultrastructure.
• Existing volume correlative light and electron microscopy (volume-CLEM) methods face throughput and correlation efficiency limitations.

Purpose of the Study:

• To develop a novel, high-throughput pipeline for volume-CLEM.
• To achieve high-precision (<100 nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) datasets.
• To enable targeted imaging of small cellular volumes and live-cell dynamics.

Main Methods:

• Utilized multi-modal fiducial nanoparticles for robust FM-EM registration.
• Integrated a 3D confocal fluorescence microscope with a Focused Ion Beam Scanning Electron Microscope (FIB.SEM).
• Developed a targeted volume-CLEM approach for imaging specific organelles and live-cell dynamics.

Main Results:

• Achieved significantly increased throughput and high-precision (<100 nm) registration for volume-CLEM.
• Enabled rapid, targeted imaging of rare and transient cellular structures, such as ER-lysosome contact sites, within hours.
• Demonstrated that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility.

Conclusions:

• The novel integrated CLEM pipeline effectively links molecular dynamics to high-resolution ultrastructural detail in 3D.
• This approach overcomes previous limitations in volume-CLEM throughput and efficiency.
• Provides a powerful tool for investigating cellular physiology and organelle interactions with unprecedented speed and precision.