FIB-SEM tomography in biology

Area of Science:

• Microscopy and Imaging Technologies
• Biological Sciences
• Materials Science

Background:

• Three-dimensional (3D) visualization offers superior comprehension compared to 2D representations.
• Traditional methods for 3D reconstruction, such as serial microtome sectioning, are labor-intensive and technically demanding.
• Advancements in computing power and instrumentation have facilitated more accessible 3D analysis.

Purpose of the Study:

• To detail the application of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) for high-resolution 3D imaging of biological samples.
• To highlight the advantages of FIB-SEM over conventional 2D imaging techniques.
• To present an improved method for site-specific analysis using FIB-SEM tomography.

Main Methods:

• Utilizing a scanning electron microscope (SEM) with an integrated microtome or a FIB-SEM for serial sectioning.
• Employing the "slice and view" technique where sequential slices are removed by an ion beam and imaged with an electron beam.
• Implementing thin layer plastification of biofilms to expose the original sample surface for precise region of interest (ROI) selection.

Main Results:

• FIB-SEM enables high-resolution 3D imaging of biological fine structures within small volumes.
• The "slice and view" process allows for the reconstruction of detailed 3D volumes.
• Site-specific FIB-SEM tomography is facilitated by identifying ROIs based on topographic features after sample preparation.

Conclusions:

• FIB-SEM significantly simplifies and accelerates 3D analysis compared to older serial sectioning methods.
• Accurate identification of the region of interest is critical for successful and meaningful 3D imaging with FIB-SEM.
• Thin layer plastification combined with FIB-SEM tomography provides an effective approach for site-specific high-resolution 3D analysis of samples like biofilms.