Comprehensive Compilation and Comparison of TEM Sample Preparation Methods! (Part 2)

Sample Preparation Method for Bulk Samples

Resin Embedding Method

Figure 1:Embedding Tissue Samples in Paraffin Image

Sample Requirements: For larger bulk materials, it is necessary to cut them into small pieces and then process them through subsequent thinning processes. For micrometer-scale large particles where mechanical grinding and crushing may damage the sample, making it impossible to obtain an ideal specimen, the embedding and thinning method can be tried.

The ideal embedding agent should have high strength, high temperature stability, and not react with various solvents and chemicals. Currently, commonly used domestic epoxy resins include Epoxy Resin 618, Epon 812 epoxy resin, and low-viscosity embedding Spurr. The specific sample preparation steps are as follows:

1. Place the sample block to be observed into a suitable mold (such as a capsule) filled with embedding agent. Heat and cure in a constant temperature oven. Spurr can be cured at 70°C for 8 hours in an oven, while domestic resins Epoxy Resin 618 and Epon 812 need to be cured at 37°C overnight, followed by curing at 45°C for 12 hours and 60°C for 24 hours.

2. Remove the cured and embedded sample, and cut thin sections using an ultramicrotome or other thinning methods to achieve dimensions smaller than 3 mm in the two-dimensional direction perpendicular to the electron beam. This ensures the sample is compatible with the size requirements for transmission electron microscopy specimen rods.

3. Disperse the obtained thin sample sections on a support grid to prepare samples suitable for transmission observation.

Precautions for Embedding Agent Preparation and Use:

1. All containers and glass rods should be clean and dry.

2. Stir the embedding agent evenly during the preparation process, and avoid the introduction of foreign substances, especially water, ethanol, acetone, etc.

3. Seal and store the prepared embedding agent to prevent moisture absorption. The remaining embedding agent can be sealed and stored in a refrigerator at -10 to -20°C to extend its shelf life.

Mechanical Thinning Method

Figure 2:The Machine Used for Mechanical Thinning Image

1. Use sandpaper to manually grind and thin the sample sections parallel to the direction of the electron beam. During this process, increase the grit size of the sandpaper in sequence, meaning a decrease in particle size. When the sandpaper reaches the desired thickness, the mechanical damage and scratches caused by the sandpaper are minimized. When the sample thickness reaches approximately 5 μm, the thinning process is stopped.

2. Use a dimpler to continuously thin the sample until reaching a thickness of 1 μm.

3. Use a polishing machine to remove surface scratches and damage.

4. Utilize an ion milling machine to further thin the sample to a final thickness of tens of nanometers with a specific area.

Ultrathin Sectioning Method

Figure 3:Ultrathin Sectioning Machine Image

The ultrathin sectioning method is commonly used for biological tissues, polymers, and inorganic powder materials. In comparison to mechanical thinning after embedding, the ultrathin sectioning method can rapidly and accurately obtain samples with specific positions, orientations, and thicknesses.

Sample Requirements: This method is advantageous for preparing samples with specific orientations but is not suitable for highly ductile materials like metals. It is often used for biological tissues, polymers, and inorganic powder materials.

1. Embed and fix the sample. If there are specific crystal orientations or position requirements, carefully place the sample during embedding so that the desired observation direction is perpendicular to the diamond knife of the microtome.

2. Select a specific step size (sample thickness) and cutting speed, and directly cut out samples suitable for transmission electron microscope observation.

Notes:

Operation should be swift: Complete sampling in the shortest time and quickly immerse it in a fixative.The sampled volume should not exceed 1 mm³.Operate delicately: Handle samples gently, and avoid violent actions such as pulling, sawing, or pressing when using sharp tools.Low temperature: Operate within the range of 0 to 4°C.

Ion Thinning Method

Figure 4:TEM Mill Precision Ion Thinning Instrument Image

Ion thinning is commonly used for the final thinning of samples that have already undergone mechanical thinning. It is also widely applied to clean damaged layers on sample surfaces, amorphous layers, and layers with contaminants. Generally, it is used in the last step of TEM sample preparation, and the entire process is completed by instruments, making it relatively straightforward.

Sample Requirements: Since the thinning induced by ion bombardment occurs on a relatively microscopic scale, the sample itself needs to be thin enough for the effects to be noticeable. Ion thinning is not influenced by the electrical properties of materials; thus, it can be used for both conductive and non-conductive materials, metals, non-metals, or their mixtures, regardless of the complexity of the material structure.

Sample Requirements: Since the thinning induced by ion bombardment occurs on a relatively microscopic scale, the sample itself needs to be thin enough for the effects to be noticeable. Ion thinning is not influenced by the electrical properties of materials; thus, it can be used for both conductive and non-conductive materials, metals, non-metals, or their mixtures, regardless of the complexity of the material structure.

1. Prepare the sample into thin slices .

2. Place the sample into the ion thinning instrument. Choose an appropriate working voltage (usually in the kV range) and working current (usually in the mA range) based on the thickness of the thin slices and TEM testing requirements. For very thin samples, ion thinning methods such as PIPS, Nano Mill, and Ion Mill can be used. Another type of thinning with higher intensity, such as Ion Slicer, can directly thin 100 μm thick samples to obtain samples suitable for TEM observation.

Electrolytic Polishing and Thinning Method

Figure 5:Electrolytic Twin-Jet Thinning Instrument Image

Electrolytic polishing relies on electrochemical action to make the sample surface smooth and polished. It is characterized by its simplicity, speed, and low cost. Electrolytic polishing is often the preferred method for bulk electron backscatter diffraction (EBSD) samples.

Sample Requirements: The working principle of the instrument relies on the electrical properties of the material; therefore, it can only be used for metallic samples. Samples placed in the electrolytic polishing device need to be uniformly polished first to avoid deviations in perforation positions. Additionally, sample cleanliness must be ensured.

1. Connect the power supply, place the fixed sample into the electrolytic solution, adjust the polishing current to the rated value, and simultaneously stir and cool or heat the electrolyte sufficiently to maintain its temperature at the specified value. Carefully select the polishing agent, power supply, and cathode plate. The sample is connected to the positive electrode, the electrolyte to the negative electrode, and the electrolyte is sprayed onto the sample from both sides. The cathode plate is divided into vertical and L-shaped types, with the L-shaped electrode improving the success rate of electrolytic polishing samples.

2. Complete the polishing operation, remove the sample from the electrolyte, cut off the power, and then quickly rinse it in clean water. Alternatively, rinse first and then clean with ultrasonic waves to remove electrolyte from the sample surface to prevent chemical reactions with the sample.

Notes:

The polished sample should not be too large. Although the current density can be adjusted, operational experience shows that smaller samples have a higher success rate. Different materials have different electrolytic polishing processes, so it is necessary to combine literature and extensive experimentation to find suitable polishing agents and parameters (e.g., reagent formulation, polishing time, temperature, electrolyte concentration, stirring speed, etc.).

Focused Ion Beam Milling Method

Figure 6:Focused Ion Beam Scanning Electron Microscope and Its Structural Principles Image

Focused ion beam (FIB) milling is a technique commonly used to obtain TEM samples. It is a dual-beam system that adds a focused ion beam column to a scanning electron microscope (SEM). FIB-SEM is versatile; it enables dual-beam imaging during sample preparation, facilitating precise positioning for cutting. The focused ion beam can also be used to thin the sample directly for TEM observation.

Focused ion beam (FIB) milling is a technique commonly used to obtain TEM samples. It is a dual-beam system that adds a focused ion beam column to a scanning electron microscope (SEM). FIB-SEM is versatile; it enables dual-beam imaging during sample preparation, facilitating precise positioning for cutting. The focused ion beam can also be used to thin the sample directly for TEM observation.

Sample Requirements: Sample size should be 5×5×1 cm, and if the sample is too large, cutting and sampling are required. The sample needs to be conductive, and if it is non-conductive, a conductive coating such as gold must be sprayed to enhance conductivity. The cutting depth must be less than 10 μm.

1. Prepare the sample and place it into the FIB-SEM.

2. Conduct thinning and milling on the sample within the FIB-SEM.

3. Remove the thin sample from the FIB-SEM. Optionally, thin the sample surface with ion milling, then transfer it to TEM for observation.

Replica Technique

The replica technique is an indirect sample preparation method that uses thin films (carbon, plastic, oxide films) transparent to the electron beam to replicate the morphology of the material surface or fracture. The replica method includes primary plastic replication, primary carbon replication, plastic-carbon secondary replication, extraction replication, etc.

Sample Requirements: Suitable for samples prone to changes under the electron beam and challenging to make into films. The sample needs to be amorphous, have small molecular dimensions, good conductivity, thermal conductivity, resistance to electron beam bombardment, and sufficient strength and stiffness.

1. Primary Plastic Replication: Operating Steps: Drop a specific solution (cotton cellulose acetate solution or cellulose acetate acetone solution) onto the sample. Let the solution spread on the sample surface, absorb excess with filter paper after the solution is flattened, and leave a layer of plastic film of about 100 nm on the sample surface after solvent evaporation.

Sample Requirements: It is a negative replication concerning the sample surface.

2. Primary Carbon Replication:

Operating Steps: Use a vacuum coating device to vapor-deposit a layer of carbon film on the sample surface. Place the sample in the vacuum coating device, immerse it in a prepared separation liquid for electrolysis or chemical separation, and separate the deposited carbon film for analysis.

Carbon film replication is a positive replication. It differs from plastic replication in the following ways:

a. The thickness of the carbon film replica is basically the same, while the thickness of the plastic replica varies with the sample position.

b. Plastic replication does not damage the sample; carbon film replication damages the sample (the sample needs to be electrochemically corroded during separation).

c. Due to the larger size of plastic molecules, the resolution of plastic replication is lower (10-20 nm); carbon ions have a small diameter, and the resolution of carbon film replication is high (2 nm).

3. Plastic-Carbon Secondary Replication:

Operating Steps: Simply put, make a carbon replica on the plastic primary replica, and then dissolve the primary replica to obtain the secondary replica. To increase contrast, a layer of heavy metal, such as Cr or Au, can be sprayed in the direction inclined at 15-45 degrees.

Technical Features: The secondary replica retains the original surface of the sample without damage, has a heavy metal projection, does not damage the sample, is resistant to electron beam irradiation, has high contrast, stability, good thermal and electrical conductivity, low resolution comparable to the primary plastic replica, and thin film thickness.

4. Extraction Replication:

Sample Requirements: Used to analyze the shape, size, distribution of second-phase particles, as well as the analysis of phase and crystal structure.

a: Deeply corrode the sample containing the second-phase precipitation phase to expose the second-phase.

b: Deposit a layer of carbon film on the sample.

c: Use electrolytic corrosion to remove the sample matrix. The obtained extraction replica sample only consists of a carbon film and second-phase particles.

Technical Features: Extraction replicas allow for the observation of the matrix tissue morphology while observing the size, shape, distribution, and crystal structure analysis of second-phase particles.