JPH10123031A - Method for fixing sample to substrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To fix even a thin concentration sample in an aqueous solution firmly to a substrate under a substantially natural state by employing a fixing substrate on which a single molecule film of an organic compound having a hydrophilic group and a hydrophobic group is formed previously. SOLUTION: An LB film is formed such that a hydrophilic group and a hydrophobic group or a functional group reactive to a fixing agent covers the surface of a fixing substrate 3. When the substrate 3 is immersed into a sample solution 6 containing a fixing agent, cells and microorganisms having relatively large size in a suspensible sample precipitates naturally and fixed chemically on the substrate 3. When the size or the specific gravity is low or when a trace sample is employed, a physical action is imparted. More specifically, a field is applied between the solution 6 and the substrate 3 when a positively or negatively charged material is employed as a sample and a magnet or an electromagnet is disposed under the substrate 3 when the sample is magnetized or reacts to magnetism. Consequently, even a suspensible thin sample can be fixed firmly through natural precipitation or further application of electric or magnetic field.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fixing substrate having a monomolecular film or a cumulative film formed thereon for observing an observation sample, for example, a biological sample, a cell using the fixing substrate,
The present invention relates to a method for fixing microorganisms and other biological substances for observation. A specific field of application of the present invention relates to a sample preparation method for observing a biological substance using an electron microscope or SPM.

[0002]

2. Description of the Related Art When observing cells, microorganisms, and other biologically-related substances or organic materials (for example, ion-exchange resins, microcapsules, synthetic resins, etc.) in a sample in an aqueous solution, which are greatly damaged by drying, particularly when observed with an electron microscope. In a scanning electron microscope, after chemical fixation, it is necessary to repeat washing while changing the solvent frequently until the sample is completely dried. Common methods at this time include a method of collecting the pellet by centrifugation each time of washing, a method of adsorbing using a substrate having a good affinity for the sample, and a method of performing pretreatment as it is collected on a filter. Has been taken.

[0003]

[0007] Centrifugation is frequently used to recover a floating sample. However, when a chemically fixed sample is centrifuged, the sample is easily aggregated and the damage is large. Preservation of hair etc. becomes difficult. Even with the method of adsorbing to the substrate, it is often difficult to search for the target sample in the observation field of view because the binding force is not sufficient and it is almost impossible to find the target sample in the observation field of view, and it can not be used when the sample concentration is low . The method using a filter is effective for a slightly large sample such as a cell or a microorganism, but has a problem that it cannot be applied to a sample of 0.1 μm or less. Furthermore, when the sample concentration was very low, the observation was almost impossible and waited for chance by any method. Therefore, there has been a demand for a method of efficiently collecting a sample from a dilute sample solution and firmly fixing the sample to a substrate with little damage. The same can be said for STM and AFM observed in the air.

In the underwater AFM, the pretreatment is simplified because there is no step of drying the sample, but the sample moves when the bonding force with the substrate is weak because the probe traces the sample surface during observation. Image information cannot be obtained. Therefore, in the underwater AFM, it has been a problem to firmly fix the sample to the substrate while maintaining a natural state.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has devised a method for firmly fixing a sample having a low concentration in an aqueous solution to a substrate in a state close to nature. .

That is, the present invention relates to an observation sample on a fixing substrate in which a monomolecular film of an organic compound having at least one hydrophilic group and at least one hydrophobic group in a molecule is previously formed on the surface of the substrate. This is a sample fixing method for fixing a certain bio-related substance or organic material. This fixing means includes a method characterized by applying a physical action simultaneously with chemical fixing and collecting a dilute sample in a solution on a substrate. .

Next, the present invention will be described in more detail.

There are several known techniques for chemically fixing a biological substance in an aqueous solution for an optical microscope or an electron microscope, and these techniques can be applied to the chemical fixation referred to in the present invention. For example most commonly glue available crosslinking reaction between -NH ₂ group if glutaraldehyde is used, the use of paraformaldehyde and polyoxymethylene, are reacted to crosslink double bonds. Cellulose is also crosslinked with formalin. Other known fixing methods include osmium tetroxide and potassium permanganate. There are many immobilization reagents and functional groups that react with them, other than those shown here.In the practice of the present invention, a general chemical immobilization method used for a biological sample may be appropriately selected depending on the sample and the purpose of observation. good. By such chemical fixation, if the cells are cells, the phospholipids forming the cell membrane form a crosslinked structure, the soft sample surface is hardened, and the sample can be dried while maintaining the fine structure.
However, in the case of a buoyant sample, damage and outflow of the sample occur in the repeated washing steps from the chemical fixation to the drying, and when the sample is very small, it is necessary to firmly fix the sample to the support.

The inventor of the present invention has repeatedly studied that the same chemical fixation as that of the sample is performed between the sample and the substrate when the sample is chemically fixed. In this case, a method for fixing a sample to the above has been reached. The means used for this can be applied by forming a monomolecular film or its cumulative film by the Langmuir-Blodgett (LB) method. According to this method, an LB film is formed on a substrate using an organic compound having a hydrophilic group, a hydrophobic group, or another functional group, and the functional group reacting with the fixing agent covers the substrate surface. By simply immersing the substrate in a sample solution containing a fixing agent as a substrate for use, chemical fixation between the sample and the sample-substrate is performed. In addition to this chemical fixation, a means for attracting a floating sample in a medium to a fixation substrate can be combined.

[0010] As the substrate in the present invention, those conventionally used for mounting an observation sample in this field are preferably used. For example, when a biological substance or an organic material is observed with a scanning electron microscope, a conductive substrate is used. Is preferably used, but is not limited thereto.

In the present invention, the material of the LB film covering the substrate is
Amphipathic by having hydrophobic group and hydrophilic group in one molecule
With a structure that causes an immobilization reaction with an immobilization reagent.
Compound. Here is a typical example of a hydrophilic group
Is -COOH, -OH, -NH_Two , -CN, -SO_Two 
H, -NR_Two , -SR_Two , -Si (OR)_Three , -Si
(OR)_Two H, -Si (OR) H_Two (R is an alkyl group
Or an aryl group), -PO _Four , And -SOCH_Three etc
is there. A typical example of a hydrophobic group is a linear alkyl.
Groups, which can be branched alkyl or aromatic
It may be a ring or linear polyacetylene chain. Also
O, NH, S, SO in the alkyl group_Two Etc.
Is also good.

This film forming material (organic substance) is dissolved in a solvent such as chloroform, benzene, acetonitrile, etc.
When developed on the water surface, the molecule forms a monomolecular alignment film with the hydrophobic group exposed on the water surface. The monomolecular film on the water surface has a two-dimensional characteristic. When the molecules are sparsely dispersed, the formula πA = kT of the two-dimensional ideal gas is obtained between the area A per one molecule and the surface pressure π. The result is a gas film. Here, k is the Boltzmann constant, and T is the absolute temperature. If A is made sufficiently small, the intermolecular interaction is strengthened and a two-dimensional solid film is obtained.

The change from the gas film developed on the water surface to the solid film is performed by using a device provided with a partition plate 2 as shown in FIG. You can know by measuring. By keeping the surface pressure constant and raising and lowering the substrate gently vertically, the monomolecular film on the water surface can be transferred to the substrate. The monomolecular film thus transferred is a film in which molecules are arranged in an orderly manner. This method is a method known as the LB method. In the present invention, the LB film is formed such that the functional group that reacts with the fixing agent covers the surface of the fixing substrate. That is, when the functional group is present at the hydrophobic group itself or at the end of the hydrophobic group, as shown in FIG. 2, the final cumulative film ends with the substrate pulled up from water, and the functional group is at the hydrophilic group itself or at the hydrophilic group side. When the substrate is immersed in water as shown in FIG. 3, that is, the film formation is completed in a state in which the hydrophilic group faces the outside of the substrate, and after removing the monomolecular film remaining on the water surface, the substrate is removed. By pulling up, a fixing substrate coated with the hydrophilic group on the surface is prepared. After forming the LB film so that the functional group covers the surface on the substrate in this way to obtain the fixing substrate,
A fixing substrate is immersed in a suspension of a biological substance containing a fixing agent. As described above, the fixing agent may be appropriately selected from known fixing agents and combined as the functional group covering the surface of the fixing substrate and the functional group on the sample side. Moreover,
Of the buoyant samples, relatively large cells, microorganisms, etc., will sediment spontaneously and be chemically fixed on the substrate only by the above operation, but if the sample size is very small, if the specific gravity is small, or if the sample is If the amount is extremely small, some operation for collecting the sample on the substrate is desirable.

The present inventor has conducted intensive studies on the physical action for positively moving a sample floating in a solution toward a substrate. As a result, the present inventors have found that an electrostatic attractive force or a magnetic force acts between the sample solution and the substrate. We have found that we can get great results.

That is, if the target sample is a material charged positively (or negatively), an electric field is applied between the sample solution and the fixing substrate, and the fixing substrate is placed on the negative (or positive) electrode side. Can collect the sample on the substrate. The voltage applied between the sample solution and the fixing substrate varies depending on the viscosity of the sample solution.
0.0 V / cm.

Further, when the sample is magnetized or reacts to magnetism, N
It has been found that it is very effective to provide a magnet or an electromagnetic coil so that the pole or the south pole acts. The strength of the magnet used for this may vary from sample to sample, but is typically 0.3-2000 Gauss on the magnet surface. In this way, even a small amount of a sample existing in the solution can be collected on the substrate without damaging the sample.

The present invention provides an extremely effective means for observing a floating sample in a solution with an electron microscope or SPM. In an electron microscope and SPM, it is important to collect a sample from a dilute solution with little damage. In particular, in the SPM, firmly fixing the sample to a substrate is an indispensable condition for obtaining a high-quality image. According to the present invention, an LB film is provided so that the surface of the substrate is coated with a desired functional group to form a fixing substrate, and a chemical fixing treatment similar to the conventional one is performed thereon, thereby causing no damage to the sample to be observed. It can be firmly fixed without giving, and it can also add a physical action as appropriate according to the properties such as sample concentration, specific gravity, size, sample charge state, magnetism, etc. The problems of the electron microscope for buoyant living body-related substances and organic materials, and the sample pretreatment for SPM were solved, and it became possible to obtain a stable sample for observation.

The substrate fixed to the fixing substrate in this way has the advantage of providing a high-quality background when observed with an electron microscope or SPM because the underlying layer is a high-density two-dimensional crystal film. Have.

[0019]

[Function / Effect] Chemical fixation on the LB film which is a two-dimensional crystal in a state where the sample is integrated on the fixation substrate by allowing the sample to settle naturally or by further applying a physical action such as an electric field or a magnetic field. Thus, even a floating dilute sample can be firmly fixed on a substrate in a natural state with little damage to the sample. When a sample prepared by this method is observed with an electron microscope or an SPM, a high-quality image with little damage can be efficiently observed.

[0020]

The present invention will be described below by way of examples in comparison with comparative examples. (Comparative Example 1) Pseudomonas cepacia
The KK01 strain was transformed into a 2YT medium (Trypton 16 g,
Yeast Ext 10g, NaCl 5g / H ₂ O
(In 1 liter) for 6 hours at 30 ° C. was used as a sample solution. The bacterial concentration at this time was 2.1 × 10 ⁵ . A 4% glutaraldehyde solution (in 0.2 mol phosphate buffer (pH = 7.2)) was mixed with the culture solution at 1:
Mix at a ratio of 1 and pour this mixture into a 3 ml mini-dish, cut out a glass substrate into 5 mm ² as shown in FIG. 4, sink it into the bottom of the mini-dish, and place at about 4 ° C. for 1 hour to fix the sample The reaction was carried out. In this process, the chemically fixed KK01 sediments on the glass substrate.

After the fixing process, the glass substrate was taken out and subjected to a dehydration process according to the following procedure. That is, each of the following solutions 3
A Petri dish containing ml was prepared, a glass substrate on which a sample was placed was sequentially immersed, and this was transferred to perform dehydration.

100% water 5 minutes 3 times 50% ethanol / water 5 minutes 3 times 70% ５ 5 minutes 3 times 80% 〃 5 minutes 3 times 90% １５ 15 minutes 2 times 95% ３０ 30 minutes 2 times 100% ethanol Twice for 30 minutes After the dehydration treatment, 15% in 100% t-butyl alcohol solution
After soaking for 15 minutes, renew the solution and soak again for 15 minutes,
The solution was cooled to 0 ° C. or lower to solidify t-butyl alcohol, and dried by a freeze dryer. The dried sample was coated with gold to a thickness of 80 ° using an ion coater and observed by SEM.

With respect to the number of bacteria fixed on the substrate, the number of bacteria observed in a range of 80 μm × 100 μm at an observation magnification of 1000 was counted for 10 visual fields, and the average value was obtained. As a result, the number of bacteria fixed on the glass substrate by the above method was 0. Example 1 Stearylamine as an organic compound for forming a thin film was dissolved in benzene at a concentration of 1 mg / ml. The water tank of the apparatus shown in FIG. 1 was filled with water, and the water temperature was set at 18 ° C. The glass substrate was mounted so that it could be moved up and down in a direction perpendicular to the water surface, and then lowered below the water surface to prepare. Next, the above benzene solution of stearylamine was dropped on 300 μl of water. Benzene acts as a spreading agent and stearylamine spreads on the water surface. After the benzene was evaporated, the surface pressure was increased to 15 mN / m to form a monomolecular film. While maintaining the surface pressure constant, 3 m
After slowly pulling out the glass substrate completely above the water surface, lowering it again at the same speed below the water surface, removing the stearylamine remaining on the water surface in this state, and then lifting the substrate and drying it. I let it. A monolayer film was formed on a glass substrate. Thus, the bilayer film could be transferred to the glass substrate with the amino group covering the substrate surface, and the fixing substrate was formed.

As an observation sample, Pseudomonas cepa cultured in the same manner as that used in Comparative Example 1 was used.
An equal volume mixture of Cia KK01 culture solution and 4% glutaraldehyde solution was used. This mixed solution was placed in a 3 ml mini-dish, and a glass substrate on which the above-mentioned stearylamine monomolecular film was formed was cut into 5 mm ^2, which was sunk on the bottom of the mini-dish, and subjected to an immobilization reaction at about 4 ° C. for 1 hour. As a result, KK01 precipitates on the substrate while being chemically fixed, and at the same time, stearylamine monomolecular film and KK01
A cross-linking reaction takes place during the period of 01 and is fixed.

Next, dehydration, freeze-drying and gold vapor deposition were performed in the same manner as in Comparative Example 1 in order to use this as a sample for SEM observation.

When the resulting sample was observed by SEM, KK01 was observed in a very well-preserved state such as flagella. The number of bacteria fixed on the substrate was counted under the same conditions as in Comparative Example 1, and the average value was determined. As a result, the number of bacteria fixed on the glass substrate by the above method was 58. (Example 2) A mini dish having electrodes as shown in FIG. 5 was prepared.

A stearylamine two-layer film was formed on a glass substrate in the same manner as in Example 1 to form a fixing substrate, which was set on a minus electrode of a mini dish. The electrode used was a metal mesh of about 1 cm ² as shown in FIG. 3 ml of the same observation sample (equivalent mixture) as in Example 1 was gently placed in a mini dish. A positive electrode was placed above the sample solution so that the distance between the electrodes was 1 cm. Using a dry cell as a power source and applying a voltage of 1.5 V, performing a fixing treatment at 4 ° C. for 1 hour, followed by dehydration treatment and Lyophilization and gold coating were performed in the same manner as in Comparative Example 1.

By SEM observation under the same conditions as in Comparative Example 1,
The number of bacteria was counted and the average was found to be 348. The observed KK01 was very well preserved, such as flagella, as in Example 1. The (Example 3) fixing the substrate, was used to cut the glass substrate stearylamine has accumulated two layers produced in Example 1 to 5 mm ^2. The following liposome solution was used as the observation sample, and the liposome diameter was 0.01 to 0.1 μm.
m, lipid is phosphatidylethanolamine, solvent is 1
0 mmol KCl, pH is adjusted to 10 with NaOH, and the concentration is 5 × 10 ⁶ / ml. Glutaraldehyde was added to the liposome solution to a concentration of 2%, and 3 ml of the mixture was poured into a mini-dish and the substrate for fixation was gently immersed. After fixing treatment at 4 ° C. for 1 hour, dehydration, drying, and gold deposition were performed in the same manner as in Comparative Example 1, and SEM observation was performed.

The observation conditions were the same as in Comparative Example 1 (observation magnification was 10,000 times). The number of liposomes was counted, and the average was obtained. (Example 4) The same fixing substrate and observation sample as in Example 3 were prepared. Using a mini-dish with the same electrode as in Example 2, a plus electrode was set at the bottom of the Petri dish, a glass substrate was placed thereon, and a sample solution adjusted so that glutaraldehyde became 2% was gently placed on the mini-dish at 3 m.
I poured. The distance between the electrodes was set to 1 cm, a voltage of 1.5 V was applied, and a fixing treatment was performed at 4 ° C. for 1 hour, followed by dehydration, freeze-drying, and gold vapor deposition treatment, followed by SEM observation. The number of liposomes was counted under the same observation conditions as in Example 3, and the average thereof was found to be 891. Example 5 An arachidic amide (or arachidamide) as an organic compound for forming a thin film was dissolved in benzene, and 1 ×
A fixing substrate was prepared in the same manner as in Example 1 except that a 10 ^-3 M solution was used. In this case, the surface pressure was increased to 10 mN / m.

As observation samples, magnetotactic bacteria (Magn) were used.
etotic spirillum strai
n MS-1) (medium: 0.1% glutamate / M9;
(Bacterial count: 2 × 10 ⁴ / ml) was prepared, and glutaraldehyde was added thereto to a concentration of 2%. 3 ml of the sample solution was poured into the same mini-dish as in Comparative Example 1, and a glass substrate with arachidic amide (or arachidamide) cut into 5 mm ² was gently immersed.
The fixing treatment was performed at 4 ° C. for 1 hour. Dehydration, freeze-drying, and gold vapor deposition were performed, and SEM observation was performed. The observation conditions were the same as in Example 1, the number of bacteria was counted, and the average was obtained. (Example 6) The fixing substrate (5 mm
² ) was placed in a mini dish, and a magnet (magnetic force on the magnet surface: about 1000 gauss) was set directly below the substrate with the N pole facing the substrate as shown in FIG. 3 ml of the sample solution containing the same glutaraldehyde as in Example 5 was placed in a mini-dish.
The mixture was poured gently and fixed at 4 ° C. for 1 hour. Dehydration, freeze-drying, gold deposition treatment and SEM observation,
The observation conditions were the same as in Example 1, the number of bacteria was counted, and the average was obtained.

[Brief description of the drawings]

FIG. 1 is a diagram of an LB film forming apparatus.

FIG. 2 is a diagram showing the preparation of a fixing substrate coated with a hydrophobic group.

FIG. 3 is a diagram showing the preparation of a fixing substrate coated with a hydrophilic group.

FIG. 4 is an explanatory diagram of an embodiment in Example 1.

FIG. 5 is an explanatory diagram when a voltage is applied between a sample solution and a substrate.

FIG. 6 is a diagram showing electrodes.

FIG. 7 is an explanatory view when a magnet is provided on a fixing substrate.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Water tank 2 Partition plate 3 Substrate 4 Monomolecular film on water surface 5 Mini dish 6 Sample solution containing fixing agent 7, 8 Electrode 9 DC power supply 10 Magnet

Claims (4)

[Claims] An observation characterized in that a monomolecular film of an organic compound having at least one or more hydrophobic groups and at least one or more hydrophilic groups in one molecule or a cumulative film thereof is provided on a substrate. Immobilization of organic materials or biological substances in the presence of substances having an affinity for the hydrophobic, hydrophilic, or other functional groups of the molecules forming the surface of the substrate for immobilizing biological substances A method for fixing an observation sample, comprising forming the sample and fixing the sample on a fixing substrate. 2. The method according to claim 1, wherein the sample is integrated on the substrate by applying a physical action simultaneously with the formation of the chemical bond. 3. The method according to claim 2, wherein the physical effect is an electrostatic attraction. 4. The method according to claim 2, wherein the physical effect is a magnetic force.