Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution

Area of Science:

• Neuroscience
• Cell Biology
• Microscopy

Background:

• Volume electron microscopy techniques like ultramicrotomy (ATUM, ssTEM) offer large fields of view but limited z-resolution.
• Focused ion beam-SEM (FIB-SEM) provides isotropic voxels for high resolution but is limited in volume size.
• Existing methods present a trade-off between field of view and resolution for ultrastructural analysis.

Purpose of the Study:

• To introduce a hybrid ATUM-FIB method combining ATUM and FIB-SEM.
• To overcome the limitations of existing volume electron microscopy techniques.
• To enable high-resolution 3D ultrastructural analysis of large biological tissue volumes.

Main Methods:

• Developed ATUM-FIB, a hybrid approach integrating serial sectioning with FIB-SEM.
• Collected "semithick" (2-10 µm) serial sections of tissue onto tape.
• Utilized serial light and electron microscopy for region of interest identification, followed by targeted FIB-SEM.

Main Results:

• The ATUM-FIB method creates a tissue "library" offering 3D context for on-demand high-resolution analysis.
• Demonstrated the technique's capability to reveal ultrastructure of rare pathological events.
• Successfully identified microglia contact sites with amyloid plaques in a mouse model of familial Alzheimer's disease.

Conclusions:

• The ATUM-FIB technique successfully integrates the strengths of ATUM and FIB-SEM.
• This hybrid approach provides a powerful tool for detailed ultrastructural analysis of biological tissues.
• Facilitates the study of complex cellular interactions in disease models, such as Alzheimer's disease.