Materials Characterization and Applications in Focused Ion Beam Tomography

To study the fundamental effect of any material’s shape and morphology on its properties, it is essential to know and study its morphology. Focused ion beam (FIB) tomography is a 3D chemical and structural relationship study technique. The instrumentation of the FIB is similar to scanning electron microscopy (SEM), but with a big difference in the beam used for scanning.
For SEM, an electron beam is used with scanning medium, while a highly focused ion beam is used for scanning in FIB, while FIB can be used for lithography and ablation purposes. However, due to the developments and the high energy focused beam, it is used as a tomographic technique today. Tomography is defined as imaging any desired area by dividing it into sectors or cutting it transversely. Spelling the FIB with energy dispersive spectrometry or secondary ion mass spectrometry can give fundamental analysis with very high resolution 3D images for a sample. This technique contributes to the recognition of qualitative and quantitative analysis, 3D volume creations and image processing.

Material Characterization

MaterialsMaterials Characterization and Applications in Focused Ion Beam Tomography
FIB / SEM is one of the newest tools for characterizing materials ranging from micro to nanometer scale. Due to the dual beam setup, FIB / SEM is one of the most modern setups that uses ion beam and SEM to slice the sample for scanning. The same applies to the FIB-TEM and FIB-EDS characterization techniques, where FIB is a source of slicing and milling, while TEM or EDS is a tool for relevant characterization.
Sample preparation
Numerous elemental strengthened alloys of the composite material Al-Si from FIB-SEM and FIB-EDS have been run. The morphological arrangement of these examples depends on the percentage of Si content in the composite, the dopant concentration, as well as the molding procedure, while reinforcements include Ni, Fe, and Mg. All materials were grinded with the help of SiC paper at 300 rpm rotation speed and SiC sizes 320, 500, 1000 and 2400. In addition, the sample surfaces were treated with DP-diamond paste with a particle size of 1, 3 and 6 m. The finishing touches were made using MgO at a spinning speed of 150 rpm to achieve as bright and smooth surface as possible. For this purpose, nickel material was produced after sintering.
The FEI strata DB235 Dual Beam Workstation was used to analyze the sample using the Gallium ion source (Ion Beam) for milling and the electron beam (SEM) for scanning, the angle 0 to 52 set between IB and EB. The area of ​​interest of the sample was selected using SEM and covered with a protective Pt layer by including the FIB-induced decomposition of the precursor gas. This protective layer helps to achieve better cuts during milling. The trench (by grinding process) was dug into the specimen without shadowing the specimen edges on the trench when scanning the specimen.
Sub-image A shows the shadow of the trench over the cell on the XY axis, while sub-image B shows the corrected version. It is better to mill in the edge area, but is only possible for samples with a balanced composition and structure. For other samples, it is recommended to determine the area of ​​interest from the sample and set aside with the aid of a micromanipulator. Whereas, two additional ditches can be dug around the area of ​​interest to prevent the sprayed sample from accumulating. The milling process is done using the existing beam at the 20 nA level. Grinding criteria depend on milling the number of sections required to obtain a clear 3D surface of the smallest possible particle.
The area should be polished under the action of low currents in the pA range to polish the surfaces before analyzing the sample. After polishing, samples should be analyzed using SEM at various solubility ranges to obtain the best needed morphological structures. For example, the AlSi12 sample was analyzed using 300 nm increments and the SEM increments used for AlSi7Sr were 83 nm.


FIB / SEM tomography of compressed cast AlSi7Sr
Materials Characterization and Applications in Focused Ion Beam TomographyThe examples show the example of AlSi7 alloy reinforced with Sr and studied with SEM at 5 keV. This method helps to generate approximately 250 scan images with an overlapping distance of 60 nm, achieving 15 & m of sample in the milling direction. Si in the sample showed fibers and branches ~ 3–5 µm in length from 200-500 nm in diameter. These fibers are mixed together and represented by color difference. The last calculated volume fraction of Si in the coral structure in the 2nd reconstructed region with junction junction sizes 50-100 nm is ~ 5%.
FIB / SEM tomography of porous Ni
SEM’s high voxel resolution has helped architecture of the 3D structure of porous nickel. 3D imaging revealed that approximately 2/3 of the volume of the selected area is porous with a fully interconnected structure. The internal caliber of the channel ranges from 500 to 2000 nm.
FIB / EDS tomography of compressed cast AlSi12
The FIB uses the ion beam as its working source, which is the result of interaction with the sample that produces secondary electrons. However, when equipped with an energy dispersive spectrometer, this can help determine the basic composition of each milled slice. The use of EDS with FIB resulted in the basic composition of the AlSi12 sample with the baseline data for each slice. When dealing with complex chemical structures, this new hyphenation technique has proven to be worthy of generating data on each slicing and giving the exact chemistry of the compound of interest. In the case of the sample, EDS is the use of 8-keV acceleration to analyze each slice with the EDX (EDS) detector.
Biological sample characterization
Since FIB-SEM is the most advanced technique to be used for the analysis of biological samples, various protective measures must be taken to do this, as biological samples are always sensitive to heat, moisture and pressure. Therefore biological specimens must be fixed, stained and embedded in resin. Because biological samples are large, ie micro-scale, they need a lot of time to be processed.
Image processing time is the biggest limiting factor during biological sample passing through FIB-SEM. Because the machine has to scan each block individually to recreate a full 3D image. Therefore, there must be a balance between sample scan time and good resolution and appropriate contrast. New methods such as chemical fixation, high pressure freezing to reduce the time factor and increase the resolution and contrast demonstrate the interaction of Si nanowires with 3 T3 cell lines.
Chemical fixation process
Chemical fixation protocols utilize aldehyde fixation in the presence of uranyl acetate and osmium tetroxide with the inclusion of thiocarbohydrazide osmium tetroxide and tannic acid.
Currently, chemical fixation is the most widely used fixation technique among researchers for FIB-SEM analysis of biological samples, and high resolution images can be recorded from a very small distance. The vast majority of FIB-SEM research is based on chemical fixation. For example, animal liver tissue cell was examined under FIB-SEM, modified with 1.5% K3Fe3 + (CN) 6 and 20% glutaraldehyde. Chemically fixed in phosphate buffer for 120 minutes with a chemical mixture of 2% OsO4. Over-staining was carried out with 2% uranyl acetate (aqueous) at room temperature.
High pressure freezing and freezing substituteMaterials Characterization and Applications in Focused Ion Beam Tomography
Cryoimmobilization of samples for cryomicroscopy offers unique opportunities to examine subcellular structure without chemical fixation and metallic stains. Although biological samples can be examined under FIB-SEM for very clear images at room temperature, freeze substitution (FS) only contributes to the high conductivity of the samples and high contrast images. It also helps protect ultra-small structures when embedded with resin. Because during FS processing, various desired chemicals and metallic substances can be added to the organic solvent to reduce the signal-to-noise ratio. This causes a decrease in the charging effect in the FIB-SEM inspection.
To date, very few high pressure freezing (HPF) and FS studies have been performed for FIB-SEM sample preparation. In this study, 24 different preparation protocols including HPF and FS techniques were used. No significant difference was found in the contrast and structure of the cells. Another study was a comparative study among mouse liver cell samples that were chemically and FS / HPF fixed. For TEM, a mixture of glutaraldehyde and methyl alcohol or acetone containing aqueous or anhydrous uranyl acetate and OsO4 was used.
In a preliminary study, it was suggested that the difference in contrast between SEM and TEM images may be due to fixatives, additive metals and staining agents. As a result, it was concluded that FIB-SEM, FIB-TEM and FIB-EDS are the latest techniques used today for characterization as well as tomographic studies of materials and biological samples.


Author: Ozlem Guvenc Agaoglu

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