If you have biological samples in your hand and need to observe them through TEM, how should they be preprocessed? Sample pretreatment is very critical and determines the effect and quality of the entire TEM observation. If there is a problem in the material collection, fixation, and transportation processes, it is usually impossible to remedy it in the subsequent sample preparation process. Unfortunately, due to limitations of the sampling location and experimental conditions, preprocessing such as sampling and fixation of biological samples is generally completed by researchers independently, and technicians in the electron microscope laboratory cannot help. Therefore, standardization of preprocessing is particularly important.

Types of Biological Samples Submitted for Testing

There are three main types of biological samples used for TEM observation: tissue, centrifuge pellet and particle suspension.

Tissue Sample

• Type: animal liver, kidney, intestine and plant leaves, roots, stems, etc.
• Features: The structure is relatively dense and stable, and the material extraction operation is relatively simple. However, it may be difficult to penetrate the fixative and accurately locate the observation target.

Centrifuge Pellet Sample

• Type: adherent cultured cells, semen, blood or bacteria, prepared into pelleted samples by centrifugation
• Features: The collection operation is relatively cumbersome, but the fixation effect is good and stable.

Microparticle Suspension Samples

• Type: algae, bacteria and cultured cells that cannot tolerate high-speed centrifugation, exosomes with too low density, as well as nanomaterials for negative staining morphological identification
• Features: The material extraction operation is very simple and the fixation effect is good. However, if subsequent ultra-thin sample preparation is required, it may be relatively troublesome.

Preprocessing of Biological Tissue Samples - Principles and Operations

Biological tissue is one of the main sample types observed by TEM and has the highest requirements for post-sample preparation technology. In the pretreatment process, reducing operational damage and improving fixation effects are of decisive significance to the quality of subsequent sample preparation.

Fixation

Chemical fixation is most suitable for tissues. The main chemical fixative component of electron microscope samples is glutaraldehyde, but the penetration rate is much slower than that of formaldehyde. For TEM observation of tissue samples, the maximum thickness is required not to exceed 3 mm (preferably within 2 mm), and they should be immersed in the fixative within 1 minute after being removed from the body. Thickness refers to the dimension in the thinnest direction of the tissue sample, and does not require cutting into 2 to 3 mm square particles. In addition to controlling the size of the sample, minimizing the time from tissue removal to the start of fixation, and ensuring a sufficient amount of fixative is also very important for obtaining excellent TEM images. Generally speaking, the ratio of fixative volume to sample volume should not be less than 20:1, and preferably 50:1.

Material Damage

The magnification and resolution capabilities of TEM are very high. If there is any damage, it will be visible at a glance and interfere with structural observation and analysis. In addition to autolysis of cellular components caused by delayed fixation, the most common forms of damage to materials are crushing injuries, cutting injuries, and injuries caused by improper osmotic pressure. Operations such as extensive pulling, hard cutting (don't "chop", it should be "scratching" or "cutting" in one direction), clamping, etc., will inevitably cause structural damage. The sharper the blade and the thinner the blade thickness, the less damaging the cut will be. Double-sided razor blades, thin surgical blades, disposable paraffin sectioning blades, etc., are all ideal choices.

Centrifugal Fixation Collection Method of Cultured Cells

There are three types of animal cells cultured in vitro: adherent, suspension and three-dimensional culture. Three-dimensionally cultured cells can be cut into thin slices and directly fixed and sent for inspection according to the pretreatment method of tissue samples. The operation of suspension cells is similar to that of adherent cells.

3D Cultured Cells

Three-dimensionally cultured cells can be directly fixed and sent for inspection after being cut into thin slices according to the pretreatment method of tissue samples.

Adherent Cells

Cells can be separated from the culture plane by scraping or digestion. Scraping is more suitable for maintaining the membranous structure of the cells.

After collecting the scraped cells, centrifuge at low speed for a short time in a 1.5 ml conical centrifuge tube, then discard the supernatant, add electron microscope fixative to resuspend the cells, and finally centrifuge at high speed.

The size of the cell cluster obtained after centrifugation should be less than half the size of a mung bean to ensure the quality of the subsequent sample preparation process.

Either a horizontal rotor centrifuge or a fixed-angle rotor centrifuge can be used, but if the cell amount can be controlled well, the quality of the cell pellets prepared by the horizontal rotor centrifuge may be better.

Fig.1 Proper size of centrifuged cell pellets.

Suspension Cultured Cells

The cells cultured in suspension are collected in a 1.5 ml conical centrifuge tube. Start by centrifuging at low speed and discarding the supernatant. The subsequent steps are the same as for adherent cells.

Subsequent Processing

After centrifugation to form a ball, let the centrifuge tube stand for 30 minutes, suck out the liquid in the tube, add new electron microscope fixative, and then send it for inspection or temporary storage.

Processing of Other Cell Types

Rinsed bacteria, fungi, anticoagulated blood cells and washed sperm can also be submitted for inspection for TEM sample preparation and observation by referring to similar processing methods.

Pretreatment of Microparticle Suspension

• Type: cells with long protrusions, nanomedicine materials, viruses, exosome samples, etc.
• Approach: It is more appropriate to fill up the tube with a 0.5 ~ 1 ml small tube and send the particle suspension directly for inspection.

Frequently Asked Questions (FAQ)

How long can samples be stored in electron microscopy fixative?

The time that the sample can remain fixed in the electron microscope fixative depends on the sufficient contact between the fixative and the sample. Generally speaking, the fixation effect is sufficient within 2 hours after the sample comes into contact with the fixative. Taking into account factors such as penetration time and temperature, overnight fixation of samples is usually sufficient.

According to sample preparation experience, if the sample is stored at 4 ~ 8°C and fixed for 2 days to 2 months, the structural difference will be very small, and there will be basically no problems in general structural observation. However, if the sample in the fixative is stored for more than 2 months, especially if the liquid volume is insufficient and the liquid is not changed, the ultrastructure of the sample may become "blackened". It manifests itself as an increase in matrix electron density and a decrease in the distinction between structural boundaries.

Therefore, if samples must be stored for longer than 2 months, they should be transferred to isotonic buffer at the end of the second month and stored until sample preparation begins.

Fig.2 Differences in TEM imaging of mouse liver tissue at different fixation times (Left: Fixed for 2 days; Right: Fixed for 2 months, without changing the fluid).

Is the fixative used at room temperature or at low temperature?

The fixation of most biological samples should follow the principle of starting at room temperature and maintaining at low temperature. It is also recommended to warm the fixative to room temperature before use. After starting the fixation, wait for 1 to 2 hours (centrifuged cells only need to stand for 30 minutes), and then transfer to 4°C for storage. The fixation of some special specimens requires special communication with technicians to determine the optimal fixation temperature.

Do samples sent for inspection need to be kept cold with ice packs?

Ice bags are generally filled with materials with high specific heat, which can absorb a large amount of heat and quickly reduce the temperature of surrounding objects. Once chemically fixed, the sample must never freeze again. When winter is colder, you can choose normal temperature transportation. In hot summer, ice packs are necessary to prevent accidental exposure to the sun during transportation. However, do not let the samples get too cold before packing, and be sure to separate them from the sample containers with sponges, bubble bags, etc. to prevent frostbite.

What should I do if there are clear requirements for the sectioning and observation directions of samples?

The first step is to obtain materials and cut them. It is up to the researchers to conduct quality control themselves and clarify the observation areas.

After being submitted for inspection, the technicians in the electron microscope room will further repair the sample according to the observation requirements and the basic shape of the sample, and repair it into a tissue block with clear orientation information according to the regulations. This is the second step to ensure the direction.

Before the third step is ultrathin sectioning, electron microscopy technicians will first prepare semi-thin sections and observe them under a light microscope to ensure that the area with the correct direction is selected for subsequent production and observation.