JP6708327B2 - How to observe fine particles - Google Patents

Description

The present invention relates to a method for observing fine-sized particles on a smooth substrate, and more particularly to a method for fixing fine particles on a silicon substrate while maintaining dispersion of the fine particles and observing the particles under a microscope.

In an atomic force microscope (AFM) and other observation methods for fine particles, fine particles may be fixed on a smooth substrate for observation. Such a substrate is required to have high smoothness according to the size of fine particles such as sub-micron size and nano size, and for example, a cleaved surface of a silicon wafer or mica can be used. Further, in order to observe each particle independently without overlapping the fine particles dispersed in the liquid, the dispersion liquid is dropped onto the smooth substrate as described above from the capillary tube, and the liquid is formed by drying. There has also been proposed a method of selecting a suitable place where fine particles do not overlap each other from a concentric agglomerate structure, a so-called "coffee ring", and performing microscopic observation (see Non-Patent Document 1).

By the way, for example, in a quantitative measurement for obtaining a statistical distribution of particle diameters, it is preferable to be able to measure the diameter of fine particles dispersed over a wide visual field range as much as possible. In such a case, the method using the "coffee ring" as described above lacks stability of dispersion, and a large number of fields of view capable of measuring the particle size are selected, which complicates the work.

Here, a freeze-drying method is known in which the dispersion medium is dried and removed from the frozen dispersion liquid, and the fine particles are taken out without being strongly aggregated. Generally, if the dispersion medium is simply dried from the dispersion liquid, the dispersion of the dispersion medium narrows the intervals between the fine particles and causes aggregation. Therefore, it is possible to take out while maintaining the dispersion of fine particles by freezing the dispersion liquid and then sublimating the dispersion medium and removing it by drying.

For example, Patent Document 1 describes a method of extracting fine particles from a dispersion liquid using a freeze-drying method. As a dispersion medium, an organic solvent having a high vapor pressure such as t-butanol having a melting point in the range of −30 to 30° C. is used to facilitate sublimation during freeze-drying. By freeze-drying such a dispersion liquid, a dried body of fine particles having high redispersibility can be obtained. According to such a freeze-drying method, the dispersion state of the fine particles in the dispersion liquid can be maintained and frozen together with the dispersion medium, and only the dispersion medium can be removed as it is, whereby aggregation of the fine particles can be suppressed.

Further, in Patent Document 2, a method in which a dispersion liquid is dropped onto a cooling base material to be solidified, and the solvent is sublimated while the liquid droplets are in a solidified state to suppress formation of a coffee ring and microscopically observe fine particles. Is disclosed.

International Publication No. WO2014/057564 JP, 2005-141083, A

R.D.Deegan et al.; Nature; vol.389; 23 Oct. 1997

By freeze-drying method, a highly redispersible dry powder can be obtained, and by fixing such a dry powder on a substrate, it is possible to disperse fine particles over a wide range and to observe each individual fine particle under a microscope. An observation substrate can be obtained. Here, in the quantitative measurement for obtaining the statistical distribution of the particle size, a method for producing an observation substrate with good reproducibility and more stable is required.

The present invention has been made in view of the above situation, and an object thereof is to provide a method of microscopically fixing the particles on a silicon substrate while maintaining the dispersion of fine particles. Especially.

The method for observing fine particles according to the present invention is a method of fixing the fine particles on a silicon substrate while observing the dispersion and observing the same under a microscope, and disposing the dispersion liquid in which the fine particles are dispersed in water. The silicon substrate is sandwiched between two silicon substrates, and the silicon substrate is cooled to be rapidly cooled and frozen while maintaining electrostatic repulsion of the fine particles, and then the silicon substrates are separated from each other and depressurized. It is characterized in that the above water is sublimated.

According to such an invention, by reducing the thickness of the dispersion liquid to reduce the temperature gradient during cooling, it is possible to prevent the aggregation of fine particles accompanying the growth of ice crystals during cooling and to reduce the thickness direction of the fine particles. Overlapping can be suppressed, and the fine particles can be fixed on the silicon substrate while maintaining the dispersion of the fine particles, and the fine particles stably dispersed with good reproducibility can be observed under a microscope.

The above invention may be characterized by including a step of sandwiching the dispersion liquid between the silicon substrates and reciprocating the silicon substrates while keeping the silicon substrates parallel to each other. Further, in the above invention, the silicon substrate may have a surface which has been subjected to a hydrophilic treatment. According to such an invention, the thickness of the dispersion liquid can be made thinner, the temperature gradient during cooling can be made smaller, and the overlapping of the fine particles in the thickness direction can be further suppressed, and the dispersion of the fine particles can be maintained. It can be fixed on a silicon substrate as it is, and can be stably observed with a microscope with good reproducibility.

In the above invention, the silicon substrate may be precooled without freezing the water. According to this invention, rapid cooling and freezing can proceed more rapidly, and microscopic observation on a silicon substrate can be performed with good reproducibility and stability.

The above invention may be characterized by including a step of bringing the outer side surface of the silicon substrate into contact with a cooling medium to perform rapid cooling and freezing. According to this invention, the temperature gradient during cooling can be made smaller, and the fine particles can be fixed on the silicon substrate while maintaining the dispersion, and can be microscopically observed with good reproducibility.

It is a flowchart which shows the observation method of the fine particle by this invention. FIG. 3 is a process diagram showing a main part of a method for observing fine particles according to the present invention. It is (a) top view and (b) side view of a freeze-drying bottle. 3A is a reflection image and FIG. 2B is an oblique white light irradiation image by an optical microscope of an example. It is a shape image of fine particles by an atomic force microscope. It is a shape image of fine particles by an atomic force microscope. It is a shape image of fine particles by an atomic force microscope.

[Example 1]
Hereinafter, a method of observing fine particles, which is one embodiment of the present invention, will be described with reference to FIGS. 2 and 3 in addition to FIG.

Referring to FIGS. 2A and 2B together with FIG. 1, first, a droplet of the dispersion liquid is sandwiched between two silicon substrates 1 (hereinafter, referred to as substrate 1) that are arranged to face each other (S1). The substrate 1 has a size of, for example, 1.1 cm square and 0.5 mm thickness, and is prepared by cleaning and drying. On one side of the substrate 1, 0.2 μL of the dispersion liquid 3 of fine particles is dropped by the micropipette 2, and the other side of the substrate 1 is covered from above and sandwiched.

At this time, it is preferable to make the dispersion liquid 3 sandwiched between the substrates 1 as thin as possible. For example, the substrates 1 are pressed against each other, or reciprocated so as to rub each other while keeping the substrates 1 in parallel. It is good to do something. For the same reason, it is preferable that the substrate 1 has a surface that has been subjected to a hydrophilic treatment. As the hydrophilic treatment, for example, ozone surface modification can be used.

As the dispersion liquid 3, for example, nano-sized fine particles dispersed in water is used. However, the concentration is such that it can be easily observed in the observation described later, that is, the fine particles do not overlap in the thickness direction and It is adjusted so as to have an appropriate density while maintaining dispersion in the vertical direction. Here, a dispersion liquid containing polystyrene latex of 50 nm diameter particles at 0.5 g/L and containing 100 nm diameter particles of 1.25 g/L as water was used.

Next, the substrate 1 in which the dispersion liquid 3 is sandwiched is cooled and rapidly cooled and frozen while maintaining the electrostatic repulsion of the fine particles (S2). Referring also to FIG. 2C, here, a disk-shaped copper plate 4 having a size larger than the two substrates 1, for example, a diameter of 25 mm and a thickness of 3 mm is used as a coolant. The copper plate 4 is cooled in advance in a refrigerator at −80° C., and the dispersion liquid 3 is rapidly frozen and frozen while sandwiching the substrate 1 so that the two plates come into contact with the outer surface of the substrate 1.

Here, if a large temperature gradient is generated inside the dispersion liquid 3, the intervals between the fine particles excluded in the liquid phase due to the growth of ice crystals during cooling are narrowed and aggregated. However, in this example, since the dispersion liquid 3 is thin as described above, it is difficult for a temperature gradient to occur inside the dispersion liquid 3 and the electrostatic repulsion of the fine particles is maintained without growing ice crystals during cooling. It is cooled as it is and frozen while maintaining its dispersed state. Further, since the both sides of the substrate 1 are cooled by the copper plate 4 which is the coolant of the same temperature, the temperature gradient is hard to be generated also by this.

Further, it is preferable to pre-cool the dispersion liquid 3 together with the substrate 1 prior to such cooling. For example, if the water is pre-cooled to a temperature around 0° C. so as not to be frozen, the cooling rate of the above-described cooling can be increased, and the temperature gradient of the dispersion liquid 3 during cooling can be reduced.

Next, the substrates 1 are separated from each other (S3). Referring also to FIG. 2D, the tip of tweezers or the like is inserted between the substrates 1 on the lower copper plate 4, and the two substrates 1 are vertically separated. At this time, the frozen film of the dispersion liquid 3 adheres to the upper or lower substrate 1 at each position in the direction along the surface of the substrate 1 without cracking in the thickness direction. Since the adhesion of the frozen film can be identified by the interference color, it is preferable to record the position for later microscopic observation.

The frozen film should not be exposed to the outside air at room temperature to prevent melting. For example, as shown in FIG. 2D, the separated substrates 1 are stacked so that the side to which the frozen film is attached is placed inside and kept in contact with the copper plate 4 until immediately before the next step. Further, the work of rapid cooling and freezing (S2) and separation (S3) may be performed inside the refrigerator in which the copper plate 4 is cooled.

Next, the substrate 1 is depressurized and water, which is a dispersion medium of the frozen dispersion liquid, is sublimated. That is, the frozen film is dried under reduced pressure (S4). Referring also to FIG. 3, an aluminum freeze-drying bottle 10 is used for vacuum drying. The freeze-drying bottle 10 has a substantially cylindrical outer shape, and is provided with a small-diameter portion 12 having a stepped shape in which the inner diameter is made smaller at the lower side with respect to the upper inner diameter having the upper end 11 as an opening, and the periphery thereof The wall 13 is formed thick. Further, the freeze-drying bottle 10 is pre-cooled at -20°C, and the cold insulating agent 9 at -17°C is brought into contact with the bottom thereof. Further, it is preferable that the side surface except the vicinity of the opening and the bottom surface containing the cold insulating agent 9 are wrapped with a heat insulating material or the like to be shielded from the outside air.

Here, the separated substrate 1 is quickly moved into the freeze-drying bottle 10 and placed on the bottom of the small-diameter portion 12 with the frozen film facing upward. The freeze-drying bottle 10 has its opening connected to a decompressor (not shown) via a rubber stopper or the like, and the inside thereof is depressurized to about 10 Pa, for example. Here, in the freeze-drying bottle 10, the surrounding wall 13 serves as a refrigerant to keep the temperature of the substrate 1 inside the small-diameter portion 12 low, preventing the temperature rise due to radiant heat from a rubber stopper or the like, Keep the temperature below about ℃ to prevent the frozen film from melting. The temperature is maintained as it is for about 1 hour, and water is sublimated from the frozen film under reduced pressure and dried.

After freeze-drying, in order to prevent dew condensation on the substrate 1, the freeze-drying bottle 10 is removed from the cold-retaining agent 9 while the inside thereof is depressurized, and the bottom is brought into contact with a heat source to raise the temperature of the substrate 1 to room temperature or higher. After that, the internal pressure is returned to normal pressure, and the substrate 1 can be taken out.

The taken-out substrate 1 is subjected to microscopic observation (S5).

As described above, by sandwiching the dispersion liquid 3 between the substrates 1, the thickness of the dispersion liquid 3 can be reduced and the temperature gradient during cooling can be reduced. That is, even if the substrate 1 is made of silicon having a relatively small thermal conductivity or heat capacity, the dispersion can be made thin so that it can be rapidly cooled and frozen even with respect to water having a large heat capacity by reducing the temperature gradient. Therefore, even if fine ice crystals are generated during freezing of the dispersion, the growth of ice crystals can be suppressed and the aggregation of fine particles can be prevented. In addition, by reducing the thickness of the dispersion liquid 3, it is possible to suppress overlapping of the fine particles in the thickness direction. That is, it is possible to rapidly freeze and freeze the fine particles while maintaining the electrostatic repulsion of the fine particles, to fix the fine particles having the dispersion maintained on the substrate 1, and to stably observe the fine particles with a microscope.

Next, the results of microscopic observation of fine particles using the substrate 1 prepared by the above method will be described with reference to FIGS. 4 to 7.

Here, first, the image was observed with an optical microscope, and its reflection image is shown in FIG. 4(a) and the oblique white light irradiation image is shown in FIG. 4(b). As shown in the figure, the distribution of the fine particles is greatly changed in the direction along the surface of the substrate 1, and it is understood that the frozen film adheres to one of the upper and lower substrates 1 depending on the position as described above. .. For example, a frozen film absent portion 21 in which almost no fine particles are present, an aggregate 22 in which white fine particles are aggregated, and a dispersion portion 23 in which the fine particles are dispersed around the frozen body are observed. That is, the fine particles can be observed in more detail in the dispersion section 23.

Next, a shape image of the fine particles observed by an atomic force microscope (AFM) is shown in FIG. As shown in the figure, the above-mentioned polystyrene latex particles of 50 nm diameter and 100 nm diameter are dispersed and observed. It can be seen that the particle size distribution in the dispersion is also maintained.

[Example 2]
As another example, silica particles having a particle diameter of 50 nm were fixed on the substrate 1 by the same procedure as above and observed by an atomic force microscope, and the shape image thereof is shown in FIG. It can be seen that the silica particles are also dispersed and observed here.

[Comparative example]
As a comparative example, a shape image of a sample obtained by dripping a dispersion liquid in which silica particles having a particle diameter of 100 nm are dispersed in water on a silicon substrate cooled to −80° C. and lyophilizing the dispersion liquid with an atomic force microscope. Is shown in FIG. As shown in the figure, dispersed and isolated particles were not observed at all, and huge particles having a particle size of about 1 μm, which were considered to be aggregates of fine particles, were observed at a low density. That is, it is difficult to maintain the dispersion of fine particles on the silicon substrate if the droplets are thick, even if they are simply freeze-dried.

Although the embodiment according to the present invention and the modification based on the embodiment have been described above, the present invention is not necessarily limited to this, and a person skilled in the art deviates from the gist of the present invention or the scope of the appended claims. Without doing so, various alternatives and modifications could be found.

1 substrate (silicon substrate)
3 Dispersion 4 Copper Plate 10 Freeze Dry Bottle

Claims (5)

A method of observing with a microscope by fixing it on a silicon substrate while maintaining the dispersion of fine particles,
After the dispersion liquid in which the fine particles are dispersed in water is sandwiched between the two silicon substrates arranged opposite to each other, the silicon substrate is cooled and rapidly frozen while the electrostatic repulsion of the fine particles is maintained and then frozen. In separating the silicon substrates from each other and reducing the pressure to sublimate the water on the silicon substrate , the step of sandwiching the fine particles is dispersed and observed on the silicon substrate after the sublimation is observed independently. A method for observing fine particles, characterized in that the thickness of the dispersion is thinly sandwiched between . The method for observing fine particles according to claim 1, further comprising a step of sandwiching the dispersion liquid between the silicon substrates and reciprocating the silicon substrates while keeping the silicon substrates parallel to each other. The method for observing fine particles according to claim 1 or 2, wherein the silicon substrate has a surface that has been subjected to a hydrophilic treatment. 4. The method for observing fine particles according to claim 1, wherein the silicon substrate is precooled without freezing the water. 5. The method for observing fine particles according to claim 1, further comprising a step of bringing an outer surface of the silicon substrate into contact with a cooling medium to perform rapid cooling and freezing.

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