CN1182381C - Measuring method of ultrathin slice thickness based on atomic force microscope - Google Patents

Abstract

A method for measuring the thickness of ultra-thin slices based on an atomic force microscope. After preparing the embedding block and performing ultra-thin slices to select the required slices, pick them up with a clean platinum ring and gently dip them in water droplets on the surface of the mica. Take the platinum ring or buckle the platinum ring upside down to make the slice float on the water surface, then dry the mica slice slowly so that the slice is attached to the mica surface flatly, scan the area containing the edge of the slice and the mica with an atomic force microscope in contact mode, The real-time plane adjustment parameter is set to "compensation", the temperature and humidity are controlled, and the area containing the slice is excluded when the obtained image is flattened, and the average relative height between the slice and the mica surface is measured as a cross-section, which is the slice thickness . The method of the invention is simple, the surface scanning and the thickness measurement can be carried out simultaneously, the ultramicrostructure of the sample will not be destroyed, and the obtained data are accurate.

Description

Measuring method of ultrathin slice thickness based on atomic force microscope

Technical field:

The invention relates to a method for measuring the thickness of an ultra-thin slice, in particular to a method of using an atomic force microscope to scan the edge of the slice, processing the image with software, and then measuring the thickness of the slice, which belongs to the technical field of bioengineering.

Background technique:

Ultrathin sectioning is a preparation method of biological tissue samples for transmission electron microscope (transmission electron microscope, TEM) observation. The general steps of using the ultra-thin section method to observe biological tissues include: (1) taking materials, requiring fast operation at low temperature, minimizing mechanical damage, and the volume of materials should be small; (2) fixing the samples, usually using glutaraldehyde (3) Dehydration, gradient dehydration with ethanol or acetone; (4) Embedding, use domestic epoxy resin 618 or imported Epon812 to infiltrate first and then embed; (5) Slice, use ultra- Slice with a thin microtome, select silver-white or lead-white slices, take them out of a copper grid and dry them; (6) Stain, usually double-stained with lead and uranium; (7) Observe and record with TEM.

Correct interpretation of electron micrographs requires precise knowledge of the thickness of the slices. Since the invention of ultrathin section technology, there have been many methods to measure the thickness of slices. When the slice floats on the liquid surface in the knife groove, its thickness can be estimated from the interference color of the reflected light on the slice, that is, the interference color of the slice is used as an approximate index of the slice thickness. Peachy (J.Biophys.Biochem.Cytol.1958, 4:233) was the first to give the relationship between the color of continuous interference color and the thickness of the corresponding slice. However, since the interference color observed with an incandescent lamp is slightly different from that observed with a fluorescent lamp, and the subjective factors of judging the color must also be taken into consideration, it needs to be emphasized that the estimation of the slice thickness according to the interference color is not accurate enough, especially quantitative hour.

Silverman et al. (J.Cell Biol.1969, 40:768) introduced a method using quantitative electron microscopy (quantitativeelectron microscopy) to measure slice thickness. They used a loose anion-exchange resin stained with phosphotungstic acid as a standard for section thickness measurements. The advantage of this method is that it ensures that the thickness measurement is closely related to the material's own structure.

Gillis and Wibo (J. Cell Biol. 1971, 49: 947) invented another interferometry to measure slice thickness, claiming that the accuracy can reach 1nm. This method has shown relatively high accuracy and reliability before exposure to electron beams.

Edie and Karlsson (J. Microscopie 1972, 13:13) provided a method for calculating the thickness of slices under electron microscopy. Slice thickness is determined by the degree of attenuation of the electron beam after passing through the embedding medium.

Many of the methods mentioned above are based on or prepared for electron microscopy. In recent years, with the increasing application of atomic force microscopy (AFM) in the biological field, some of them use ultra-thin slices as the research object, using AFM to study the fine structure of tissue or cell ultra-thin slices, in this Under normal circumstances, the thickness of the slice is also a very critical condition, which directly affects the degree of unevenness of the slice surface. However, it is not accurate enough to estimate the thickness of the slice from the interference color of the reflected light on the slice, and the method of quantitative electron microscopy is not suitable for the requirements of AFM sample preparation. Therefore, the measurement of slice thickness based on AFM is a new research, which is very necessary and critical, but there is no report on the measurement method of ultra-thin slice thickness based on AFM.

Invention content:

The purpose of the present invention is to address the deficiencies of the prior art, to provide a method for measuring the thickness of an ultra-thin slice based on an atomic force microscope, which is easy to operate, does not need to be equipped with other additional devices, does not destroy the surface structure of the sample, and can measure the surface morphology of the sample with AFM simultaneously.

In order to achieve such a goal, the technical solution of the present invention is, after preparing the embedding block and performing ultrathin sectioning to select the required slice, use a clean platinum ring to pick it up and gently immerse it in water droplets on the surface of the mica, and remove it. Platinum ring or turn the platinum ring upside down to make the slice float on the water surface, then dry the mica slice slowly so that the slice is attached to the mica surface flatly, use the atomic force microscope to scan the area containing the edge of the slice and the mica in contact mode, real-time The plane adjustment parameter is set to "compensation", the temperature and humidity are controlled, and the area containing the slice is excluded when the obtained image is flattened, and the average relative height between the slice and the mica surface is measured as a cross section, which is the slice thickness.

Method of the present invention specifically comprises the steps:

Using the existing mature ultra-thin section technology, first prepare the embedding block and the slices required for ultra-thin section selection, and fish them out with a clean platinum ring.

Drop 15 μl of ultrapure water on the clean mica surface, gently dip the platinum ring in the water drop, remove the platinum ring from the side, and make the slice float on the water surface, or buckle the platinum ring upside down on the water surface, and the slice will also float on the surface of the water. on the water.

Place the mica slices on a drying machine to dry slowly, so that the slices can be attached to the mica surface evenly. The temperature of the sheet drying machine is controlled between 40-65°C.

Use AFM to scan the area containing the slice edge and mica, the scanning mode is the contact mode in the air, the realtime plane fit parameter must be set to "offset", the temperature is controlled at 25±1°C, and the humidity is controlled at 40 %the following.

The obtained image is flattened, and the area containing the slice is excluded during the flattening, and the average relative height between the slice and the mica surface is measured as a cross section, which is the thickness of the slice.

The present invention improves on the existing ultra-thin section technology, adds a step of transferring the section to the surface of mica, and creates an original technology of using the tension of water to attach the thin section to the surface of mica evenly. During the AFM imaging operation, the parameters are adjusted to be suitable for scanning the stepped area containing mica and slice edges, and a special method is adopted in the flattening process, which excludes the area containing slices, which greatly improves the feasibility of height measurement sex and accuracy. The method of the invention has simple steps, is designed according to the characteristics of AFM, can measure thickness and scan the surface at the same time, does not destroy the fine structure of the sample, is suitable for ultrathin slices of various thicknesses, and obtains accurate data and small errors.

Description of drawings:

Fig. 1, Fig. 2 are the image that the sample prepared in the embodiment of the present invention is observed under AFM (Nanoscope IIIa from DigitalInstrument, Santa Babara, CA) air contact mode. The bright parts in the image are slices, and the dark parts are mica. The off-line processing software (version number 4.42r4) provided by DI Company used only smoothes the image.

The sample in Figure 1 is a human tongue squamous cell carcinoma tissue section, and the interference color of the section is blue-purple.

The sample in Figure 2 is the cultured human tongue squamous cell carcinoma cell Tca8113, and the interference color of the slice is blue.

Detailed ways:

To implement the method of the present invention, it is necessary to first attach the ultrathin slices to the flat mica surface. First, prepare the embedding block according to the general electron microscope ultra-thin section method. The embedding solution can be domestic epoxy resin 618 or imported Epon812, but there is no need for osmic acid or block staining when the sample is fixed, because heavy metals do not help to improve the resolution. And osmic acid is expensive. Slice with an ultra-thin microtome, use a newly prepared glass knife, after positioning the semi-thin slice, select the required part for ultra-thin slice, the knife groove is ultra-pure water, select the slice you need, and the thickness of the slice can be roughly judged from the interference color . Dip the platinum ring briefly in chloroform and then dry it or burn it in the flame of an alcohol lamp to cool it and use it to slice it. The slice will remain on the water film inside the platinum ring. Prepare freshly split mica flakes in advance, and blow off the surface debris with an ear wash ball. Drop a drop of ultrapure water on the mica surface, gently dip the platinum ring in the water drop, then remove the platinum ring from the side, and the slice will float on the water. Or quickly buckle the ring upside down on the water drop, and then remove the ring, the slice will also float on the water surface, but the direction of the sliced surface is just opposite to the method just now. Adjust the temperature of the drying machine to 45-60°C, place the above-mentioned mica slices on the drying machine, dry slowly, and the slices will be unfolded under the tension of water, and finally attached to the mica surface smoothly. Place the ultra-thin slice attached to the mica surface to scan under AFM, the mode is the contact mode in the air, the realtime plane fit parameter must be set to "offset", the temperature is controlled at 25±1°C, and the humidity is controlled at Below 40%, the scanning range includes the slice edge and mica area. Then, the obtained image is flattened, and the area containing the slice is excluded during the flattening, and the average relative height between the slice and the mica surface is measured as a cross section, which is the thickness of the slice.

Example 1

Human tongue squamous cell carcinoma tissue samples were taken from the oral and maxillofacial surgery of a certain hospital, fixed with 2% glutaraldehyde fixative solution at low temperature for 1 to 2 hours, or refrigerated overnight in the refrigerator. Rinse with PBS buffer, dehydrate with ethanol gradient, replace with ethylene oxide, and embed with epoxy resin 618. LKB-2088 V-type ultra-microtome slices, and the glass knife used is newly prepared by LKB-7800 knife-making machine. Select blue-purple sheets and collect them on the mica surface with the method of the present invention. The temperature of the drying machine was adjusted to 55°C, and the mica was dried slowly. Observe the slice edge in AFM contact mode, see Figure 1A. It shows that the edge of the slice is clear, and the average relative height between the slice and the mica plane is 162.21±0.32nm when the section is measured, as shown in Figure 1B and Figure 1C.

Scanning range: 30μm×30μm.

Example 2

The human tongue squamous cell carcinoma cell line (Tca8113) used in the experiment was established in 1981 by a tumor biology laboratory. The average doubling time of in vitro culture is 38.8±4h. In the culture medium, the culture conditions were 37°C, saturated humidity, 5% CO ₂ and 95% air, 2% glutaraldehyde, fixed, collected by centrifugation after dehydration, and embedded in epoxy resin 618. After slicing, collect the blue slices by the method of the present invention, and put them on a drying machine at 45° C. for slow drying. Observe and image in AFM contact mode. The obtained image is shown in Fig. 2A, and the edge of the slice displayed is clear and smooth. The average relative height between the slice and the mica plane is measured at 211.80±0.34nm after flattening the obtained image, as shown in Fig. 2B and Fig. 2C.

Scanning range: 30μm×30μm.

Claims (1)

1. A method for measuring the thickness of an ultra-thin slice based on an atomic force microscope, which is characterized in that an embedding block is prepared and an ultra-thin slice is carried out. After the desired slice is selected, it is picked up with a clean platinum ring, and 15 μl of ultrapure water is dropped on it. Clean the surface of mica, dip the platinum ring gently in the water drop, remove the platinum ring from the side or buckle the platinum ring upside down, so that the slices float on the water, and then place the mica slices on the drying machine to dry slowly, so that the slices Flatly attached to the mica surface, the temperature of the drying machine is controlled at 40-65°C, and the area including the edge of the slice and the mica is scanned by an atomic force microscope. The scanning mode is the contact mode in the air, and the real-time plane adjustment parameter is set to "compensation". The temperature is controlled at 25±1°C, the humidity is controlled below 40%, and the area containing the slice is excluded when the obtained image is flattened, and the average relative height between the slice and the mica surface is measured as a cross-section, which is the thickness of the slice .

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