Notes when we prepare samples for the SEM:
When using the SEM microscope, we must absolutely comply with the requirements for samples and vacuum based on the following principles:
- Samples must be as clean as possible. Any contamination from the sample can damage the vacuum system of the microscope, leading to reduced effectiveness later.
- Non-conductive samples must be observed in low vacuum or ESEM mode. High vacuum observation is only allowed when the sample is coated. Incorrect usage can contaminate the vacuum chamber.
- Wet samples, dirty samples, organic samples (oil/fat), samples that can generate gas or evaporate should only be used in ESEM mode and must adhere to strict sample preparation requirements as below.
- Powder samples must be securely attached with adhesive, completely removing any free particles before placing them in the sample chamber.
- Always use clean gloves and clean tools when handling samples and accessories in the SEM sample chamber.
- Do not touch with bare hands, blow on samples with your mouth, or blow/breathe into the sample chamber as organic substances will contaminate the sample and the chamber.
- Do not take the sample holder out of the microscope chamber to prepare the sample. Preparation should be done on a separate sample holder outside.
- Contact tools should be regularly cleaned with ultrasound and dried when stored.
- Samples must be securely attached to prevent the vacuum chamber from sucking them away, which could harm the microscope.
- In addition to vacuum, the choice of substrate, adhesive for the sample, sample preparation method, and acceleration voltage are very important.
SAMPLE PREPARATION
Mẫu cho SEM bao gồm tất cả các loại, hình dạng và kích cỡ khác nhau. Kỹ thuật chuẩn bị do đó phải được đặt ra để thực hiện được với tất cả sự khác nhau từ các mẫu hoàn hảo đến các mẫu phức tạp. Các vấn đề chúng ta phải thường xuyên phải đối mặt là các yếu tố sau:
Sample creation
Most samples require cutting/destroying the sample. Therefore, it is necessary to find a technique compatible with the properties of the sample material. First, we can divide them into two groups: metallic materials and non-metallic materials.
Metal sample
Metals are usually cut into small pieces using precision mechanical cutters. Each type of material needs to be cut in different modes. (See the section on cutter selection, cutting blades in the separate presentation file for preparing metal samples).
The cross-sections reflect the nature of the sample, so it needs to be prepared very carefully to create a detailed cross-section, characteristic of the research sample.
In the laboratory, a small sample can be cut from steel with a precise cut, sometimes it can be broken after being frozen with liquid nitrogen. Alloys with high elasticity may need to be cooled in liquid nitrogen before shock processing to create samples.
Non-metallic sample
This group includes plastics, ceramics, and organic materials. There are many different techniques for this complex material stream, but a common principle is that the best sampling should be done at low temperatures.
Plastics can often be easily broken by soaking in liquid nitrogen and breaking at low temperatures.
Some ceramic/porcelain samples are not easily broken at low temperatures. They have high elasticity, so after soaking in liquid nitrogen, a force impact is still needed to break them. The rest can be broken with smaller impacts and may not require low temperatures, such as silicon sheets.
Organic materials need to be prepared more carefully and pay attention to the details of the impacts that may cause damage to the format and structures of the sample. Therefore, special low-temperature sample preparation equipment called Cryo-SEM may be required. This is a method of freezing samples in a liquid nitrogen chamber and then transferring them to a sample holder attached to the port of the SEM chamber. Here, the sample can be fractured, and, or even coated before being placed into the observation sample chamber. Typical samples include: food, plant and animal tissues, semi-solids (fats/oils).
Sample cutting
According to this method, it is necessary to divide into hard and soft materials to distinguish the differences between polishing or cutting methods to create a surface that reflects the internal structural details of the sample.
Hard samples:
- Cross section
Hard materials are usually composite materials, that is, commercial products or specialized materials, such as metals, stones, ceramics, and glass. Some can be cast in resin (resin, epoxy, or Bakelite), for example, metals for easier handling when cutting and polishing. Casting samples in resin is a common method for rock samples to ensure the complete removal of air, water, or oil from the sample before cutting and polishing.
- Surface preparation
Cutting methods can be used with cutting discs or diamond blades. Metal samples usually only need to have their surfaces polished from rough to a smooth flat surface using polishing pads with diamond particles down to 1/4 micron. Rock samples require a grinding process after cutting and before polishing with glass cloths and carborundum powder. Then polished again with 1 or 1/4 micron diamond powder. Although all of this can be done manually, automatic machines help reduce labor and often provide a more precise flatness than manual polishing.
Electrolytic polishing of metals has another advantage, as this is a technique that corrodes certain metals to create a surface with atomic-level distributions, clean and clearly displaying phase distributions on it.
Ion polishing can also provide atomic-level detailed surfaces, clean but importantly the equipment must capture the small details on it.
The necessity for a flat surface is often for WDS analysis rather than EDX, and then it is necessary for analyzing the different phase components on the surface by BSE signal. Some samples are required for cathodoluminescence (CL) analysis with luminescent white elements.
Composite materials have very complex and precise compositions. The best method discovered so far that ensures the composition is not disturbed is using diamond cutting fibers. This is a tungsten wire with diamond coated on the outside. The diamond fiber is evenly rotated by two tubes at both ends. The diamond wire will be cut across the sample at the cutting position. Cooling water is used to lubricate and also clean the wire. This method can be very well used for some materials without the need for grinding or further polishing.
Soft sample:
Cross-sectioning or thin slicing of the sample before cutting requires providing complete information on the two following requirements to ensure success. These are hardness and rigidity to select the metal cutting blade or diamond cutting blade. Otherwise, the cut surface may be contaminated with particles from the casting material or the cutting blade material. Therefore, knowing the hardness of the sample in advance is necessary to ensure the sample does not undergo surface alteration or contamination with other components on the surface, especially when going for X-ray analysis.
These cutting techniques are commonly referred to as microtomes. The macro level uses metal or diamond cutting blades, while the micro level uses diamond knives. The macro cutting method can be used on synthetic materials, embedding materials, plastics, paints, rubber, wood, and paper.
Ultra-microtomy is a technique used for TEM, which can also be used for SEM in the case of preparing heavy metal samples (Os/Pd/U) that are attached to biological samples.
The conventional cryo-cutting method is rarely used in TEM except for polymer materials that have components with different hardness that need to be examined. With this technique, the cut surface is quite flat, less rugged, and does not affect the results.
Another cutting method, although somewhat crude but effective, is the use of a scalpel. This method can be used when the analysis and imaging results do not completely depend on the surface or composition on the BSE signal.
Dispersed samples.
This is the most challenging type of sample preparation to ensure high accuracy on a substrate or metal coating. The observations obtained often need to exclude the possibility of charging of the sample embedding resin, distinguishing bright and dark areas due to surface charging as well as image drift.
This type of sample can be simply divided into two categories including wet dispersions (in suspension) and dry dispersions (powder/fiber).
- Wet dispersion: Suspension
Polymers, paints, and biological components are the main samples in this group. The nature of the technique will depend on the density of the suspension, the size of the materials within it, and importantly, the separation of individual particles, avoiding overlap. There are specific methods to prepare biological samples, depending on the importance of the detailed structure to be considered, as these techniques involve drying the sample to a critical point, which is not as simple as regular drying because it can distort the cellular structure.
- Dilute solution.
Accurate dilution of the suspension sample can be tested at various levels and the most suitable dilution concentration selected. The solvents commonly used for dilution are:
- Distilled water or deionized water
- Absolute alcohol, Ethanol
- Acetone
- Isopropanol
The choice depends on the chemical properties of the sample material, and/or the solvents available in the suspension. Acetone can damage the equipment used with this method, so caution is needed when using this solvent.
After each dilution step, the sample needs to be placed in an ultrasonic bath for at least 10 minutes to ensure complete separation of the particles.
Test by using a small pipette to apply the suspension in clean alcohol onto clean glass slides. Dry under infrared light, in a drying oven, on an electric stove, or in a vacuum to ensure the sample is dry.
Then use a sample stub with conductive carbon tape or copper pressed onto the sample surface to ensure the sample adheres well to the stub. Use a clean air spray to blow off any free particles sticking to the surface to ensure the vacuum chamber remains clean.
If necessary, a metal coating (Au, Au/Pd) or conductive carbon can be applied. At that point, glass slides can also be attached to the sample stub to cover the sample.
Note: do not cover the entire surface of the sample holder with particles to avoid overlapping particles, which makes observation difficult. The easiest observation is when a maximum of 2-3 particles cluster together, while the rest are individual particles. Therefore, preliminary tests are necessary to determine the appropriate dilution.
Another way to keep the particles separate is to take one end of a toothpick (clean toothpick for sampling) and dip it into the dried powder, then touch it to a corner of the carbon tape on the holder. Use the back of the toothpick to spread it evenly to one side (in one direction) to the opposite corner. The particles will be distributed from dense to sparse. This makes it easier to choose the appropriate observation density. Another method is to use a spray bottle to evenly disperse the particles.
The difference between choosing pipetting or an atomizing spray bottle is the particle size and uniform dispersion. Pipetting is considered suitable for small particles ranging from sub-micron to tens of microns, but the dispersion is uneven. Meanwhile, the spray bottle is limited as it is only suitable for particles over 100 microns but provides uniform dispersion.
Note that samples dissolved using an ultrasonic bath or ultrasonic treatment at high frequencies and for long durations may alter their multi-structure. When using a spray bottle, it is mandatory to dissolve the sample using ultrasound first.
- Using a Pipette
Used to drop 1-2 drops of the diluted and ultrasonically dissolved sample onto the surface of the carbon/copper sample holder. Use a new tip each time. Allow the sample to dry before observation or coating. The result is that the particles will disperse into concentric circles, with larger particles typically in the center and smaller particles gradually dispersed into the outer rings.
- Fogger
This technique involves a group of devices that are more artistic than scientific.
Spray guns or airbrushes that are commonly used in painting can be used. The advantage is that these tools allow for changing the dispersion size at the nozzle (aperture), there will be a specific range of sizes of particles that can be sprayed.
After the suspension is dispersed and placed into the spray bottle, it will be sprayed directly onto the surface of the carbon film. Spray at a 450 angle through a tube with a diameter of about 10cm, about 30cm long.
The number of sprays and the spraying time depend on experience, however, it is usually about 2-3 sprays. It is crucial to thoroughly clean the spray nozzles when changing samples. Additionally, the sample can also be loaded into a separate bottle attached to the spray gun/spray bottle. Acetone should not be used with this technique.
- Sandwich joint technique
Some suspension samples are very difficult to observe due to surface charging even in ESEM mode. In that case, the sandwich technique can be used to sandwich the sample between two conductive layers.
First, a glass/quartz slide (figure 3) is cleaned with alcohol, allowed to dry, and then attached to the sample holder. A thick layer of metal is then deposited, with high deposition intensity and short time. At that point, the surface of the metal coating will develop cracks. The suspension sample is then dropped onto the newly coated metal layer using the corresponding method and allowed to dry. Capillary action will cause the particles to settle into the cracks of the metal coating. A second coating with any target will be applied longer, with lower intensity to ensure it is smooth and thin. The particles will be tightly sandwiched by the conductive layers, ensuring no surface charging around, providing a clear and easily observable image.
- Size separation by seed separator
Separating particles of different sizes from the suspension is simpler than when they have been fixed on the sample holder.
When you want to separate, a simple device is a seed separation device. It is a device with multiple spray positions for particles starting from the largest to the smallest particles thanks to different apertures. Different particle sizes can be sprayed onto different sample stubs for observation.
Dry dispersed sample: Powder / fiber
For non-metallic materials in powder form, a technique is needed to ensure that at least some particles are firmly attached to the sample stub for SEM. Choosing a good adhesion method from the start will limit contamination of the particles in the vacuum chamber. Meanwhile, choosing a good adhesive will eliminate surface charging. This is extremely important when analyzing EDX.
Metal powder samples usually do not have complex issues, but double-sided tape adhesives often contain trace amounts of zinc carbonate that can be expressed during EDX analysis.
Fiber samples often cause more difficulties due to their loose adhesion to substrates. The operation involves attaching one end of the fiber to the sample stub and waiting for it to dry, then pulling the fiber evenly across the surface of the sample stub. Excess fibers can be cut off. Special stubs can be used for fiber samples. When attaching fibers to the stub, silver adhesive or carbon can be used to ensure a secure bond. The choice of substrate is very important as all fibers have substructures.
Organic fibers are suitable for substrates with low Z backgrounds such as carbon. Metal coating should be done carefully as damage can occur to organic fibers during thermal radiation.
Choosing an adhesive
Using carbon tape to attach dry particles is actually much easier. First, disperse the powder on a clean surface (glass slide) and press the carbon-coated stub down onto the powder, using a clean air spray to blow away the free particles.
When necessary, samples can be coated. Using carbon glue is better in cases where particles need to be held firmly in the substrate. After using clean gas to blow, samples can be coated if necessary.
- Filter paper
Used to soak samples into filter paper, dry them, and then observe under low vacuum or ESEM mode. Collecting powder or fiber samples using filter paper is not the best method for SEM as it creates holes/voids that will have gas where electrical conduction cannot occur. Using cellulose acetate filter paper is better than fiber filter paper. When necessary, samples can be coated to create a more uniform surface. In cases of high surface charge, a carbon coating before applying a top metal layer is necessary.
Live biological sample.
- Insect model
Insect samples need to have organic fat removed before being placed in a vacuum. The samples should be soaked in ethyl alcohol for at least 1 - 2 weeks to dissolve the fats. After that, the samples are rinsed in clean ethyl alcohol to remove surface fats and dried on filter paper. If fats are not completely removed, the observed image will show fats on the surface of the insect. In the worst case, the insect may explode in the SEM chamber.
Insects when observed are attached to tape or carbon glue and can then be directly observed under ESEM mode.
If observing under high vacuum, it is necessary to coat the sample. The first coating is with carbon to fix the surface. The second coating is with Au or Pd.
- Plant sample
Fresh plant samples should be prepared by the deep freezing method (cryo-SEM) to ensure detailed structure observation without being destroyed. For observation, the maximum time is only 15 minutes and with a voltage below 1kV. Additionally, samples can also be observed with a cold stage in ESEM mode.
- So when is it necessary?
A replica is necessary when the sample is too large and the sample cannot be destroyed or when materials are harmful to the vacuum or cannot be exposed to electron beams.
Additionally, some geological standard samples are often not allowed to be coated. However, this can also be remedied with low vacuum or environmental vacuum (ESEM).
There are two methods for creating replicas: using cellulose acetate sheets and using silicone rubber sheets.
- Cellulose acetate sheet
This sheet is primarily used in polishing and etching metals and ceramic surfaces. It can be used for X-ray analysis such as determining grain boundaries. The surface to be replicated must be able to withstand acetone while the cellulose flows around all structures. After replication, it can be cut to size or the area of interest and then coated with a layer of metal or carbon. This is a highly regarded, detailed type of replica used at high magnification.
- Silicone rubber
Silicone rubber can be used to create replicas on most surfaces; it only slightly emits heat but does not affect plastics or painted surfaces. It is very effective in geological research for reproducing micro fossils and paleontology.
This method uses a two-component silicone rubber, silicone rubber with a plasticizer and a liquid catalyst. First, the sample or surface is completely cleaned of dust or free particles, then a thin layer of silicone is applied using a paintbrush. Next, mix the two components according to the manufacturer's recommendations for those products and then apply it to the previously wet surface. Allow to dry for a time as recommended by the manufacturer. A thin layer of silicone rubber of the replica will be formed. Cut the area of interest to make a sample. A layer of carbon needs to be applied before covering the metal sample on the surface. If not coated, it can be observed under low vacuum or environmental scanning electron microscopy (ESEM). Although this method provides high detail, it is often only used at low magnification.