Compared to conventional scanning electron microscopy (SEM) imaging, FIB-SEM 3D nano-tomography adds a “cutting” tool in the form of a focused ion beam (FIB). Due to its heavy ionic mass, FIB removes a small amount of material, exposing the surface underneath. Repetitive FIB milling simultaneous with SEM imaging produces a stack of images which can be reconstructed into a 3D volume. The combination of these techniques, focused ion beam scanning electron microscopy, serves as a powerful tool to analyze and quantify internal microstructures.
Below, a cross section of the polymer membrane of a drug pellet sample was prepared using two methods: conventional freeze cleave and FIB polishing. The resulting cross-sectioned SEM images are dramatically different. Due to mechanical fracturing, residual surface topology remains on the cleaved sample, and obscures the internal microstructure. In comparison, the FIB cross section reveals microporosity accurately, and allows for both qualitative and quantitative assessment.
FIB-SEM typically runs with a resolution of 3–50 nm. Assuming an image produced has 2000x2000 pixels along X and Y directions, its field of view might be about 6 – 100 micrometers in size. This size is very small compared to other imaging techniques. Hence, it is critical to ensure that imaged volumes are representative.
For microscopic imaging, sample representativeness is critical, yet often difficult to verify. A larger sample can be as unrepresentative as a high-resolution small sample. When using FIB-SEM microscopy, we work closely with our clients to guarantee that a representative sample volume will be imaged at the most appropriate resolution. The sample will be prepared to ensure both optimal image quality and budgetary responsibility.
Regardless of successful an imaging experiment is, there are always artifacts. FIB-SEM imaging can take a few hours to a few days. A sample with gas or liquid contents can react to electron and ion beams, which can cause undesirable effects.
DigiM manages imaging artifacts in three ways:
Example of DigiM’s artifact corrections are shown below.