JPH08112099A - Method for analyzing base sequence of nucleic acid, molecular structure or the like of protein and analytical apparatus - Google Patents

Abstract

PURPOSE: To provide both a method for analysis by which the base sequence of a DNA or an RNA of nucleic acid, an amino acid sequence, etc., of a protein can be analyzed in a simpler manner for a shorter period than those of a conventional method and an apparatus therefor. CONSTITUTION: A molecular reagent prepared by respectively binding a fluorescent substance molecule of an intrinsic color or a molecule in a shape discriminable with a present electron microscope, etc., to a molecule specifically bindable to various bases or amino acids constituting a molecule of a nucleic acid or a protein which is a sample is bound thereto and the base sequence of the nucleic acid, molecular structure, etc., of the protein are analyzed from the fluorescent color emitted from the resultant reagent molecule by collision of an electron beam or shape of the labeled molecule while detecting the shape of the sample molecule with the electron microscope, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus for directly visualizing and analyzing a base sequence of a nucleic acid, a molecular structure of a protein and the like.

[0002]

2. Description of the Related Art Conventionally, in order to analyze the base sequence of a kind of nucleic acid, DNA, first of all, a large amount of the same DNA molecule is constructed by the PCR method, etc. Cleavage with an enzyme, electrophoresis, obtaining data by segmenting the fragments by size, then cleaving another site with a different restriction enzyme, and obtaining similar data was repeated. Infer and analyze sequences. Therefore, it has taken a long time to analyze several tens of nucleotide sequences. Also when analyzing the structure of a protein molecule, after crystallization, an X-ray diffraction image is taken, and a long time is taken.
Analysis of the structure has been performed.

Recently, studies have been conducted to visualize DNA molecules and protein molecules using a high-magnification electron microscope or atomic force microscope and analyze their base sequences or atomic sequences in a short period of time. Due to lack of, etc., it has not succeeded in analyzing accurately.

[0004]

DISCLOSURE OF THE INVENTION The present invention uses a fluorescence discrimination type reagent and a morphology discrimination type reagent together, and displays an image of a molecule using an electron microscope or an atomic force microscope with a resolution of about 0.4 nm at this stage. The objective is to obtain a method and device for clarifying the base sequence and other existing positions of the atomic groups while taking the image directly, and also to obtain the applied system thereof.

[0005]

[Means for Solving the Problems] DNA has four types of nucleobases, adenine (A), guanine (G), and thymine (T) in the sugar moiety of a chain in which phosphate and pentose (deoxyribose) are alternately linked. ) And cytosine (C) are bound to each other to form a long molecule. In the present invention, there are four types of bases capable of binding complementarily to each base in the molecule, or their derivatives, etc. Fluorescent substance that emits fluorescence of different colors is combined with a fluorescent discriminating type molecular reagent, and a fine electron beam or a fine electric current is generated while taking a fine image with a scanning electron microscope or tunnel scanning electron microscope. Fluorescence generated each time it collides with a molecular reagent is detected by a photoelectric conversion device, and the type of reagent, that is, the type of base in DNA is determined from the color, or four types of base molecules can be detected with the current electron microscope. But identifiable shape Morphology-identifying molecular reagents that combine molecules of different sizes and sizes are combined, visualized together with DNA molecules by various electron microscopes, atomic force microscopes, etc., and the data is judged by image analysis by a computer to determine the bases of DNA. Parse the sequence.

When the molecule to be measured is a protein or the like, the same reagent is bound to each part of the surface, and which part of the molecule and what kind of atomic group exist are determined using an electron microscope or the like. .. The present invention is based on these methods, and various variations are used.

[0007]

EXAMPLES FIG. 1 is a plan view of a sample preparation device for a single-stranded DNA / reagent to be investigated using the present invention. FIG. 2 is a vertical sectional view thereof. 1 is a box-shaped UV irradiation device. 2 is a small water tank made of transparent synthetic resin placed in front of it. 3 is a metal plate electrode mounted on the bottom surface. 4 is a nitrocellulose membrane placed on it.
5 is a water supply pipe connected to the left wall of the aquarium. 6 is a drain cock that hangs down from the right side of the bottom wall of the aquarium. 7 is a glass tube with a tip of about 0.1 μm pierced into the hole on the left wall of the water tank. 8 is one chromosome attached to the tip. 9 is a glass sample plate placed on the inner surface of the rear wall of the water tank. Reference numeral 10 denotes a top and bottom width of 1 mm, which is formed by applying a gold plating layer having a thickness of about 5 nm on the surface.
It is a transparent electrode.

The glass tube 7 is filled with water and the like, and the white blood cell and other cell nuclei are stabbed by using a micromanipulator or the like,
Negative pressure is applied to the inside of the tube, and one chromosome is aspirated and pulled out of the cell to attach the chromosome 8 to the tip of the glass tube 7,
Puncture into the hole of the water tank 2 and make the state shown in the figure. A tube of a water absorbing device (not shown) is connected to the water supply pipe 5, and an aqueous solution containing a proteolytic enzyme is gradually fed into the water tank 2 to dissolve and wash away proteins such as histones, which are nuclear proteins constituting chromosomes. At this time, drainage goes out of the water tank through the cock 6.

Then, an aqueous solution containing an alkali having the function of denatured the double-stranded DNA to divide it into two single-strands and a urea having the function of linearizing the single-stranded DNA is gradually introduced from the water supply pipe 5 into the water tank 2. To a dark field, and irradiate ultraviolet light from the UV irradiation device 1 into the water tank to observe the fluorescence emitted from the DNA with a slightly magnified microscope, using a thin dissecting needle, etc.
Twist the DNA strand of the book and leave only one in the aquarium.

When the remaining single chain is attached to the tip of the glass tube 7, one end thereof is attached to the tip of the glass tube by using a dissecting needle or the like. At this time, water may be spouted or sucked from the glass tube to attempt desorption of the DNA chain. Then, an aqueous solution containing only urea is caused to flow from the water absorption tube 5 until the glass tube 7 is contacted with the nitrocellulose membrane 4.
The tip of the DNA is lowered, the straight chain of DNA is directed in the flow direction, and the DNA is attached (blotted) to the nitrocellulose membrane.

Next, pure water is made to flow from the water supply pipe 5 to remove alkali.
The urea solution is washed away, and then an aqueous solution containing the reagent described below is fed. DNA chains are four types of DNA: adenine (A), guanine (G), thymine (T), and cytosine (C).
One molecule of any of the bases is a nucleoside linked to one 5-carbon sugar (deoxyribose), and many nucleotide molecules are linked to a single phosphate group. It is a long molecule formed by connecting them, and the width between each nucleotide is about 0.5 nm.

The reagent molecule that binds to each base of this DNA must also be no larger than that. To selectively bind to any of the four bases. To be able to identify what the reagent is by emitting different colors of fluorescence (or in a distinguishable form). Any substance may be used as long as the above conditions are satisfied (a nucleic acid base derivative is easy to use). Here, the following substances are used.

Any of these bases on the DNA can be complementarily and selectively bound to T (or uracil / U of RNA base), C to G, A to T, and G to C. Either one molecule, a nucleoside molecule with one 5-carbon sugar attached to it, or a nucleotide molecule with a phosphate group is used, and at the end of the molecule or somewhere in the molecule, the color corresponding to the type of base is used. The thing which couple | bonded the fluorescent substance molecule which emits fluorescence is used.

Here, one hydrogen attached to the carbon skeleton of the sugar of the nucleotide is replaced, and the substance of base A emits red fluorescence (rhodamine-isothiocyanate, etc.),
A reagent in which a fluorescent substance of yellow is used for G, green for T, and blue for C is used. An aqueous solution containing these four types of reagents is supplied to the water supply pipe 5
And bind the reagent to the sample DNA.

Then, pure water is sent from the water supply pipe 5 to wash away excess reagent, a lead wire connected to a negative electrode of a power source (not shown) is brought into contact with the electrode 3, and a positive electrode is brought into contact with the transparent electrode 10 to form a dialysis membrane or the like. A weak current flows through the nitrocellulose membrane 4 having micropores which is also used and is attracted by the electric field between the electrodes, and the negatively charged sample molecules reach the electrode 10 by electrophoresis while being linearized by the water flow. Adhere to.

At that time, according to the movement of DNA, the glass tube 7 is rotated around the hole in the left wall of the water tank 2 as a fulcrum to move the tip of the glass tube, and when the electrode 10 is reached, the water in the tube is removed. The sample DNA is released by jetting or the like. If you extend the DNA of one chromosome without cutting it, it will reach several cm or more, so divide it into several mm with an appropriate restriction enzyme, and use short DNA or RNA that matches the nucleotide sequence of each cut end. It is also possible to use a large number of glass tubes or metal needles to which the probes of (1) are attached, connect the DNA fragments to each, dye with a reagent, pull up from the water tank, and attach each one to a separate glass sample plate 9 with a transparent electrode. (A probe such as RNA may be attached to this transparent electrode.) In addition, DNA emits weak fluorescence upon irradiation with ultraviolet light, but it may be a fluorescent dye or the like that binds to the phosphate group portion of the DNA chain that is easily released later. After dyeing and adhering to the transparent electrode, the dye may be separated from the DNA chain by reacting with an acid or alkaline aqueous solution.

When the reagent is bound to the sample DNA, the DNA
Synthetic enzyme and ATP may also be made to exist so that the nucleotide type reagent complementarily pairs with the sample DNA and further polymerizes to form a single chain. It is desirable to perform these operations with an automatic device including a television camera with a microscope, a manipulator with a servomotor, a pipeline with a solenoid valve, a chemical solution feeder, a computer and the like.

At the time of electrophoresis, another electrode to which a slightly higher positive voltage is applied is provided near the left end and the right end of the transparent electrode 10,
Electrophoresis may be performed while pulling and stretching both ends of the DNA chain (separated from the glass tube 7 and using urea if necessary). At that time, if the electrode is too close to one of the electrodes, the voltage is controlled to be weakened. From time to time, reverse voltage may be applied to push back the part that is too close.

The sample plate 9 is applied to a system including an ordinary scanning electron microscope or a scanning tunneling electron microscope and a fluorescence detector to analyze the DNA base sequence of the sample.
FIG. 3 is a scanning electron microscope (SEM) type D embodying the present invention.
The top view of NA base sequence analyzer. FIG. 4 is a vertical sectional front view of the electron microscope section covered with a vacuum container. FIG. 5 is a vertical left side view of a computer or the like.

11 is a fixed base. Reference numeral 12 is a sample plate driving device mounted thereon, in which the sample plate 9 is inserted, and a mechanism such as a pulse motor for driving the sample plate 9 in the X, Y, and Z axis directions and a circuit for applying a voltage to the transparent electrode 10 are provided. It is built in. Reference numeral 13 is the sample DNA with the above-mentioned fluorescent discrimination type molecular reagent attached on the transparent electrode 10 of the sample plate. 14 is a column mounted on a fixed base. Reference numeral 15 is a scanning electron microscope attached thereto, which has an electron lens for narrowing the electron beam emitted from the electron gun to a fine focus of about 0.4 mm and a deflection electrode for driving the focus in the X and Y axis directions. There is. Reference numeral 16 is a television camera with an optical microscope fitted on the right side of the sample plate driving device 12. 17 is the attached light source. 18 is a color photon counter on the right side. 19 is a lens attached to the upper surface. 20 and 21 are internal dichroic mirrors. 22,
23 and 24 are high-sensitivity photomultiplier tubes corresponding to blue, green, and red light, respectively. 25 is a vacuum container that covers them. 26
Is a cable that connects the fixed base and the computer.

27 is a control computer. 28 is the main switch. 29 is a liquid crystal display placed on a computer. Reference numeral 30 designates a synthetic resin grooved transparent plate placed on it, and a large number of lateral grooves having a depth of 0.2 mm and a front-rear width of about 0.2 mm are formed at a constant pitch on the surface (corrugated section, The wavelength and amplitude can be selected arbitrarily.) 31 is a handy image scanner made of transparent synthetic resin placed on it. 32 is a click switch using a transparent electrode or the like mounted on it. A cable 33 connects the scanner and the computer.

FIG. 6 shows a handy image scanner 31.
FIG. 3 is an enlarged vertical cross-sectional view of a stereoscopic image input device and the like. Three
Reference numerals 4 and 35 are rotary shafts made of a transparent material, and 34 is connected to an encoder (not shown) for converting the rotary angle into a digital signal. Reference numeral 36 is a transparent endless belt which is made of a synthetic resin and is stretched around both shafts. Reference numeral 37 denotes a television camera, which can receive light from the display 29 as non-refractive / non-scattered light through a portion of the corrugated transparent plate 30 and the belt 36, which are made into parallel transparent plates. Reference numeral 38 denotes a transparent disc, the lower surface of which is in contact with the transparent plate 30. 39 is an encoder connected to the axis.

Reference numeral 40 is a stereoscopic image input device attached to the front of the computer 27. 41 is a TV camera stored in it. 42 is a light source that emits light obliquely toward the upper front. 43 is a light source that emits light obliquely upward and rearward. 44
Is a transparent plate that covers the top of the device 40. 45 is the depth 1 of the upper surface
Black ink diluted with water stored in a shallow recess of about mm. Reference numeral 46 is a flexible white rubber film having a thickness of about 0.3 mm, which covers the above.

Next, the usage and operation of this system will be described. First, using a vacuum pump (not shown), the air inside the container 25 is evacuated to a high vacuum, and the main switch 28 is turned on.
When the computer 27 is started, the following operations are performed. Illuminated from below by white light including ultraviolet rays emitted from the light source 17, the sample DNA 13 fluoresces, and its image is captured by the television camera 16 and displayed on the left half of the display 29 via the computer 27.

If the right end of the sample DNA 13 is displaced from the center of the screen to the left and right, the disk 38 is rotated by moving the image scanner 32 in the left and right direction, and the encoder 39 is used to rotate the disk 38.
A signal proportional to the rotation angle enters the computer 27, the motor in the sample plate driving device 12 moves, and the sample plate 9
Moves left and right, and the deviation is corrected. If it is shifted back and forth, the scanner 31 is moved back and forth, the belt 36 is rotated, the shafts 34 and 35 are interlocked, and a signal proportional to the rotation angle is output from the encoder attached to the shaft 34 to the computer 27.
Then, the motor in the drive unit 12 rotates, the sample plate 9 moves back and forth, and the deviation is corrected.

Then, when the click switch 32 is pressed once, the computer 32 displays the letters "start" and "stop" in two lines below the corrugated transparent plate 30 in the right half of the display 29 in the next mode. . These characters are almost visible to the naked eye through the transparent plate 29 and the scanner 31 which is mostly made of a transparent material (a liquid crystal display for displaying an image captured by the camera 37 may be attached to the upper surface of the scanner 31. .) Lift the scanner 31 a few centimeters, place it on the letters "Start", move it from left to right, read it with the TV camera 37,
Input to the computer 27.

The fixed base 11 is instructed by the computer.
A circuit inside or the like works, a positive high voltage is applied to the transparent electrode 10, electricity to the camera 16 and the light source 17 is stopped, and the micro focus of the electron beam emitted from the scanning electron microscope 15 becomes a sample DN.
Electrons scattered at the sample due to the height of A13 are absorbed by the positive electrode at the lower end of the electron microscope, and non-scattered electrons are absorbed by the transparent electrode 10.

The focus of the electron beam having a diameter of 0.4 nm moves from the front to the back with a period of 10 KHz and an amplitude of 40 nm, and when one Y-axis scanning is completed, the next Y-axis scanning is performed 0.2 nm to the left. (While making the overlapping part), 200
With 0 times Y-axis scan (1 time X-axis scan, cycle 0.2 seconds),
Create one screen with an area of 40 × 400 nm and 200 × 2000 = 400,000 pixels. (The effective resolution is about 0.4 nm.) The intensity change of scattered electrons becomes light and dark, and it is displayed on the left half of the display 29 while moving to the right, and the sample DNA molecule is shaped like a ladder cut in half. Appears.

When one X-axis scan is completed, the sample plate driving device 12 is activated and the sample plate 9 is fed to the right side by 400 nm, which is repeated. During this time, when the image of the sample DNA shifts back and forth from the center of the screen, the computer 27 moves to the left, the deflection voltage of the electron microscope 15 is changed, and the driving device 12 causes the sample plate 9 to move.
Adjust by moving back and forth.

When an electron beam collides with a reagent molecule in the sample DNA during imaging in this way, it emits fluorescence and enters the transparent electrode 10 → the sample plate 9 → the lens 19 of the photon counter, and the blue component is dichroic. The green light is reflected by the mirror 20 and enters the photomultiplier tube 22, and the green light is reflected by the mirror 2.
The red light is reflected by 1 and enters the photomultiplier tube 23, and the red light enters the photomultiplier tube 24 and is converted into current pulses proportional to the amount of light, which are input to the computer 27, whose color is determined, and , The type of reagent, that is, the type of base in the DNA chain with the reagent molecule is specified.

The computer 27 stores the data of the type of the base in the memory together with the data of the number of the base from the beginning of reading by analyzing the image information, and if the data is stored in a certain amount, it is attached. It is repeated to transfer to a hard disk etc., and the base sequence analysis of the sample DNA is advanced. As described above, if the electron beam is scanned in the Y axis at 10 KHz and 0.01 seconds is required for switching the X axis scanning, the time required to analyze a sample DNA having a length of 1 cm is 10500 seconds (about It will take 3 hours).

When the analysis is completed to the left end of the sample DNA, the computer 27 automatically judges and displays "Analysis completed". Therefore, the scanner 31 is placed on the corrugated transparent plate 30 where the characters "Stop" can be seen, and the right side. If you move it to
The electron microscope 15 is turned off, the solenoid valve in the fixed base 1 is opened, and the outside air enters the container 25. Therefore, remove the container,
The sample plate 9 is replaced with the next one, the container 25 is again covered, a vacuum is applied, and the same operation as before is repeated.

Assuming that the length of human DNA having 3 billion base pairs is 1.5 m, it takes about 18.2 days to read the whole DNA (genome analysis). In the above case, using a normal transmission electron microscope,
Since electrons collide with a large number of reagent molecules at the same time and emit fluorescence all at once, the resolution is insufficient and the sources cannot be separated and identified not only by using a one-pixel photon counter but also by using a television camera with an optical microscope.

When a scanning tunneling electron microscope is used, since the applied voltage of the tunnel current is low, it is necessary to use a fluorescent reagent that emits fluorescence with low-energy electrons and a photon counter that can catch the fluorescence.
(Alternatively, apply a strong magnetic field in the vertical direction and apply a high applied voltage that can generate fluorescence between the electrode needle and the sample.
The diameter of the tunnel current or the discharge current flowing from the electrode needle may be prevented from increasing. ) With an atomic force microscope, it is not possible to apply a voltage or current to generate fluorescence in a molecular reagent.

Next, the case of using the morphological discrimination type molecular reagent will be described. Even with the current high-resolution electron microscope, it is difficult to distinguish the type of base in DNA from the shape because of the influence of oxygen and other impurity molecules adhering to the sample support membrane and other factors and the lack of resolution. In the present invention, a molecular reagent whose shape and size can be clearly identified by an electron microscope at the present stage is used.

The reagent molecule is capable of selectively binding to any of the four DNA bases. The width of the molecule should not be too large. The labeled atomic group can be identified by the current electron microscope or atomic force microscope. If the conditions such as
As the selective binding atomic group, a nucleoside, a nucleotide, a derivative thereof, or the like can be easily used as the selective binding atomic group.

As the form-labeling atomic group, carboxylic acid, alcohol, a substance in which hydrogen in the molecule is replaced by a halogen atom, or the like is easily used. For example, it is a product in which nothing is bound to the nucleotide containing adenine, and each of the nucleotides containing guanine, thymine, and cytosine is bound to a carboxylic acid having 5, 10, or 15 carbon chains.

Similar to the above-mentioned fluorescence discrimination type reagent, these four kinds of reagents are bonded to the sample plate 9 to prepare the sample DNA 13, which is set in the sample plate drive device 12, the container 25 is covered, and the inside is evacuated. . Double-sided adhesive tape is pasted on a rubber sheet, on which a small ball of an atom model made of metal, synthetic resin, rubber, etc. with a diameter of about 2 mm is pasted, and four types of image models of reagent molecules are made. The upper rubber film 46 is turned upside down and pressed one by one.

When the scanner 31 is double-clicked,
The word "Molecular image input" appears on the bottom of the transparent plate 30.
The image of the model is stored in the memory of. That is, the rubber film 46 is recessed into a hemispherical shape everywhere with a small sphere, and the lower end of the hemisphere is in contact with the surface of the transparent plate 44, and is reflected by the camera 41 as a white image. In addition, the thickness of the layer of the diluted ink 45 gradually becomes thicker around them, and becomes closer to black, and becomes black at the portions other than the dents, and is stored in the computer as an image close to the image of the reagent molecule attached to the sample DNA. To be done. (For one kind of reagent molecule, several kinds of deformed models are created and input respectively.) Alternatively, the rubber film 46 and the ink 45 are removed, and the molecular model in which a small ball is attached to the rubber sheet is directly applied to the transparent plate. 44,
You may input into the camera 41.

When the color of the small ball is similar to that of the rubber sheet, only the light source 42 or 43 is turned on, the shadow of the small ball is projected on the rubber sheet, and the shadow is mainly input to the camera. (The light source of a commercially available image scanner was modified to illuminate the paper diagonally.
May be used instead of. ) It should be noted that by the electron microscope 15, only the images of the four kinds of reagent molecules are displayed on the display 29 and photographed, placed on the transparent plate 44 and inputted, or directly inputted into the memory of the computer without taking a photograph. May be.

In this way, the forms of the four kinds of molecular reagents are stored in the computer 27, the molecular model and the like are removed from the input device 40, and then the characters of "morphological analysis mode" appearing under the transparent plate 30. When the scanner 31 is read by the scanner 31, a "morphological analysis start" with an arrow indicating the left half of the rubber film 46 and a "morphological analysis stop" with an arrow indicating the right half are displayed below the transparent plate 30. Appears.

When the left half portion of the rubber film 46 is pressed with a finger, the video camera 41 presses the rubber film with the fingertip and detects a white spot on the rubber film that has entered the left half portion of the visual field. Enter the computer 27, and the computer will use the sample DNA.
Begins morphological analysis. The electron microscope 15 visualizes from the right end of the sample DNA 13, the computer 27 determines what number base is, the length and shape of the reagent molecule attached to it, and each reagent input from the camera 41. The type of base in the sample DNA is determined by referring to the shape and size of.

At this time, since the sizes of the respective reagent molecules are remarkably different from each other, the identification can be easily performed. Sample DNA 1
When the analysis is completed up to the left end of 3, the display 29 displays “Analysis completed”, so if you press the right half of the rubber film 46 with your finger, the electron microscope 15 will stop and air will enter the container 25. Move on to the next sample plate analysis. Hereinafter, the same operation is repeated.

When the morphological discrimination type molecular reagent is used, the image can be visualized by using an ordinary transmission electron microscope, scanning tunneling electron microscope or atomic force microscope to analyze the base sequence. A fluorescent substance may be bound to the morphological discrimination type molecular reagent, and the reagent may be determined from both sides by the above system using a scanning electron microscope.

Nucleotide sequence analysis may be performed on a sample obtained by transcribing DNA into RNA using a ribonucleotide, a deoxyribonucleotide, a fluorescent substance, or a reagent in which a substance having a markedly different shape or size is bound. Fluorescent substances are linked to substances with a clear molecular structure such as synthesized DNA and RNA fragments, synthetic drugs, etc.
You can find out where it joins.

It is also possible to analyze the codon sequences of RNA and DNA by binding to RNA transcribed from DNA using a reagent in which different fluorescent dye molecules are bound to 20 kinds of amino acids. Next, the structural analysis of protein molecules will be described. A protein is composed by polymerizing any of 20 kinds of amino acid molecules. Its intracellular synthesis is DNA.
R with a nucleotide sequence transcribed from a part of
NA serves as a template to polymerize the amino acids carried by the transfer RNA that binds only to specific amino acids.

Amino acids (atomic groups) in such proteins
It is also possible to obtain a labeled molecule reagent by binding various fluorescent substances or morphologically discriminating labeled molecules to transfer RNA or a derivative thereof capable of selectively binding to. (The properties of free amino acid molecules change when they are incorporated into protein molecules, so various selections and processings are required.) Alternatively, oligonucleotides / antibodies that selectively bind to several amino acid sequences In addition to the enzyme or the like, a fluorescent substance or a morphotype identification labeling molecule may be bound.

An aqueous solution containing such various single or molecular reagents corresponding to several amino acid sequences is shown in FIGS.
In a water tank 2 of, and add a dilute solution of the protein to be sampled,
When a product having reagent molecules bound to one surface is made on the surface of a protein and the sample plate 9 is pulled up from the liquid, a state in which protein molecules with reagents are attached to the surface is obtained. This sample plate is applied to the device shown in FIGS. 3 to 5, and the shape and the kind and arrangement of the reagent molecule of one protein molecule attached on the transparent electrode 10 are recorded in the computer 27 or the like.
The amino acid sequence in the protein molecule is analyzed.

Even when the structural analysis of one kind of protein is performed, the analysis may be repeated several times using only molecular reagents corresponding to several kinds of amino acids. In that case, a molecular reagent corresponding to a certain amino acid (eg, glycerin may be always added and used as a landmark in one molecule. A unique protein in a cancer cell (clarify the structure of a cancer antigen and Alternatively, DNA or RNA for synthesis may be synthesized, an antibody corresponding to the protein produced by using the DNA or RNA may be produced, and used for treating cancer.

In addition to the distribution of the neurotransmitter contained in each synapse in the nervous system, it may be used for structural analysis of proteins involved in its production. DNA as above
In addition to proteins and proteins, one kind of nucleic acid, RNA, and other macromolecules are bound with a fluorescent discrimination type or shape discrimination type molecular reagent that selectively binds to various atomic groups on each surface,
Similar analysis can be performed.

It is also possible to bind a reagent to the surface of a living cell membrane and observe it with a scanning tunneling electron microscope, or freeze-dry and slice it, attach it to the sample plate 9 and stain it with a fluorescent type molecular reagent for examination. The presence of the corrugated transparent plate 30 and the belt 36 that meshes with the corrugated transparent plate 30 allows the scanner 31 to be accurately moved in the lateral direction by hand, the displayed characters to be accurately input to the computer 27, and the operation in a dark room can be facilitated. However, place this transparent plate and scanner on the paper surface of books etc.,
Move the scanner and input to the computer 27
Characters are judged by CR software, and further voiced.
As an analysis device for the visually impaired and the blind, as well as a reading machine,
You may use it.

(The corrugated transparent belt 36 may be fixed.) Braille is usually composed of a large number of small projections with a minimum distance between two points of 2 mm on the surface of the paper, which is difficult for elderly blind people to read. Although it is an object, a light source for irradiating light obliquely with respect to the paper surface is built in the scanner 31, a shadow of a Braille projection is formed, and a black and white contrast is added.
It may be used for a Braille reading device by inputting into a voice and displaying it on a Braille display with a minimum point distance of about 3 mm.

Alternatively, the Braille paper surface is placed on the rubber film 46 and is deformed by the protrusions, and the camera 41 is used by the computer 27.
May be input to the computer 27, the ink layer 45 and the rubber film 46 may be removed, the Braille characters may be placed, and the light source 42 alone may be used to illuminate the shadow generated by the fixed camera 41 or the drive-operated camera. . In place of the ink layer 45, a gray urethane foam film (a non-woven fabric made of transparent resin fibers having a small apparent specific gravity may be used) is placed, and in the pressurizing section, the bubbles in the urethane home disappear and the resins adhere to each other.
Since the number of times light is scattered by air bubbles is reduced or disappears, the white color of the rubber film 46 may be displayed on the camera 41.
(The urethane foam layer may be white and the rubber film 46 may be black, or only the white urethane foam film having its upper surface painted in black may be used. Instead of the urethane foam layer, a hard transparent plate is placed and the surrounding area thereof is used. It may be possible to use a phenomenon in which light in the horizontal direction is input from the cross-section, and the near-horizontal light leaking from the upper surface illuminates brighter the closer to the tip of the upper protrusion.) When performing DNA or RNA base sequence analysis PCR
A molecular reagent may be bound after the number of molecules is amplified by a method or the like.

When using a tunnel scanning electron microscope or an atomic force microscope, the sample may be suspended on the water surface.
When an ordinary electron microscope is used, it takes a considerable time to bring the electron beam passage into a high vacuum, but the time may be eliminated as follows. When a thick sample piece is placed on the sample plate 9, data of various depths is obtained by Z-axis scanning, and at that time, the sample plate drive device 12 is operated to raise or lower the sample plate or to scan electronically. Microscope 15
The acceleration height of the electron beam may be increased or decreased, the focal length of the electron lens may be changed, and the height of the focus of the electron beam formed in the sample piece may be changed.

X-axis scanning and Y-axis scanning are also performed on the sample plate driving device 12
, But in that case, place a light shield plate with a pinhole at the window of the photomultiplier tubes 22-24,
Light from other than the focal portion of the electron beam may be blocked as much as possible. In this case, a piezoelectric element is inserted between the lens 19 and the container 18, and the lens 19 is driven up and down in conjunction with the rise and fall of the focus of the electron beam so that the fluorescent image is always formed at the pinhole position. You may

When performing Y-axis scanning in which the electron beam itself is moved back and forth, slits may be used instead of pinholes. (When using the X-axis scanning which moves the electron beam itself to the left and right, the light shielding plate is interlocked or the lens 19 is interlocked left and right in the direction perpendicular to the slit and parallel to the surface of the light shielding plate. , The fluorescent image may be matched on the slit.) FIG. 7 is a vertical cross-sectional view of the closed vacuum vessel type electron microscope.

47 is a fixed base. 48 is a pillar standing on it.
Numeral 49 is a vacuum container of a normal type electron microscope which is magnified by an electron lens attached to it. 50 is an electronic lens in it. 5
1 is a fluorescent plate in which a glass plate is coated with a fluorescent substance that closes the upper end of the container. 52 is a video camera that captures the image. Reference numeral 53 is a metal disk attached to the lower end of the container, and a truncated cone-shaped electron beam passage hole having a diameter of the lower end narrowed to about 100 nm is opened in the center thereof. Reference numeral 54 denotes a carbon material such as diamond structure, graphite structure, or other carbon, silicon carbide, silicon nitride, silicon oxide alumina, titanium, or any other tough material having a thickness of 10 nm or less attached to its lower surface. Air barrier membrane consisting of. Reference numeral 55 is a sample film that is placed under the sample film, and sandwiches a sample DNA or the like having a molecular reagent bonded between two silicon oxide thin films. 56 is a screw drive type jack mounted on the fixed base. 57 is a vacuum container for a scanning electron microscope mounted on it. Reference numeral 58 is an electron gun with an electron lens mounted on the bottom wall to obtain a micro focus. 5
9 and 60 are X-axis deflection electrodes. 61 is a Y-axis deflection electrode. Reference numeral 62 denotes a metal disk that closes the upper end of the container 57, and has a truncated cone-shaped electron beam passage hole with a diameter of the upper end narrowed to about 100 nm in the center thereof. 63 is pasted on it 54
Air blocking membrane similar to. 64 is water accumulated in the recess of the upper surface of the container 57. Reference numeral 65 is a color photon counter attached to a column.

The electron beam emitted from the electron gun 58 converges and rises, passes through the air blocking film 63, and is focused at the position of the sample in the sample film 55, where some of the scattered electrons have a positive voltage. It is absorbed by the metal disks 53, 62, the air blocking films 54, 63 made of a conductive material, and is input to a computer similar to the computer 27 to drive the electron beam back and forth. The voltage values of the X-axis deflection electrodes 59 and 60 driven to are also input, and a scanning electron microscope image is obtained.

Fluorescence generated when the electron beam hits the sample enters the photon counter 65, the kind of the molecular reagent is specified, and the same data as those shown in FIGS. 3 to 5 are obtained.
Further, the electrons that have passed through the sample film 55 and the air blocking film 54 are
An enlarged image is formed by the electron lens 50, is reflected on the fluorescent screen, is input to the computer via the video camera 52, and becomes detailed image information from the scanning electron microscope portion.

At the end of one X-axis scan, the jack 56 is activated, the container 57 is lowered a little, and the micromanipulator (not shown) moves the sample film 55 slightly to the right,
Analyze the following part. The holes of the metal disks 53 and 62 are smaller on the side of the sample film, and the conical electron beam does not come into contact with them, and the diameter of the portion to which the atmospheric pressure is applied is small, so the air blocking film thinner than that diameter is used. It is possible to block the air, block the air, and transmit the electron beam.

Water or oil may be attached to the upper and lower surfaces of the sample film 55, and the sample film 55 may be moved horizontally without pulling down the container 57. In order to remove the gas emitted from the materials of the containers 49 and 57 and the like, it is desirable to constantly operate a small vacuum pump to maintain the degree of vacuum in both containers. In order to cope with the deterioration of the air blocking film, it is desirable to replace the metal disks 53 and 62 with new ones from time to time.

The metal disks 53 and 62 may be replaced with insulating disks. The upper electron microscope part consisting of the container 49 and the like is removed, and the transparent electrode is attached to the lower surface on the sample film 55, and the glass plate to which a positive voltage is applied is covered on the sample film 55, and only the lower scanning electron microscope part is formed. You may use it. Figure 3-
Using a scanning electron microscope as shown in 5 or a tunneling scanning electron microscope, cut the human brain into multiple sections, attach them to a transparent continuous synthetic resin film with transparent electrodes, dye with a fluorescent identification type reagent, and collide with a minute electron beam. The generated fluorescent light is captured by a color photon counter and input to a computer together with image information, and the wiring structure of neurons can be automatically analyzed at the same time as the fine distribution structure of substances in each part of the brain.

At this time, in order to easily understand the correspondence between the positions of the respective sections, a flexible multicolored line made of rubber, polyurethane, gelatin, or the like having a diameter of about 0.1 mm is provided at the adjacent portion. If you make a knitted net with different colors, put it on the brain, put it in the embedding solution, freeze-fix it, slice it, stick it on a transparent film, dye it with a reagent, and perform automatic neuron analysis by computer It will be easier to align the. (Such a line may be joined to the rear end of an elongated sewing needle and passed through a large number in the parenchyma of the brain.) The above device can be used for genetic diagnosis of a patient with a genetic disease, etc. It may be used to investigate whether or not there is any difference in the nucleotide sequence of DNA in the process in which the organism described above develops from an individual fertilized egg into an individual. The sample cells may be collected from naturally occurring embryos, but cells obtained by experimental induction as described below may be used.

FIG. 8 is a longitudinal sectional view of an embryo differentiation induction experimental apparatus. 66 is a fixed base. 67 is a petri dish placed on it. 68 is an embryo of one fertilized egg in the process of amphibian and other development. 69 is a disc-shaped synthetic resin capsule with a diameter of about 1 mm, which is attached by using a small amount of adhesive or the like. 70 is a fine hole having a diameter of about 0.1 mm opened on the lower surface thereof. 71 is a sustained release drug particle formed by kneading noradrenaline, dopamine, acetylcholine, growth hormone, and any other drug filled in a capsule with polyvinyl alcohol, gelatin, calcium alginate, epoxy resin, starch, and other bases. . 72 is a jack mounted on the fixed base. 73 is a synthetic resin container mounted on it. 74 is a soft synthetic resin tube connected to it with an inner diameter of about 0.1 mm. 75 is the aqueous drug solution contained in them.

In the process of developing a fertilized egg, if a part of the small tissue piece is transplanted to another part, the graft is differentiated into a tissue related to the surrounding tissue, or conversely, the surrounding tissue is related to the graft. It is known in experimental embryology that "induction" such as differentiation into a certain tissue occurs. It is considered that the signaling substances composed of amines, proteins, RNAs, and others that are produced from the woven pieces or surrounding tissues interact with each other to induce differentiation.

It has been discovered that several tens or more kinds of nerve-conducting substances are present in the brain or the like, but they are directed toward cells or tissues that emit those substances and move away from them in the course of development. It is also said that self-organization may occur due to chemotactic growth of nerve fibers or migration of nerve cells from tissues or tissues. If the sustained-release drug particles 71 in the capsule 69 now contain noradrenaline, the noradrenaline gradually acts on the embryo 68 locally through the micropores 70 to induce cells in that part or to self-assemble. Sometimes it happens.

Further, the acetylcholine liquid is put in the container 73, the jack 72 is driven, the height of the container is adjusted, the liquid pressure is changed, and the flow velocity of the liquid is adjusted, and the tube 74 is used to control the embryo 6
It is gradually released to 8 and induces differentiation of that part or self-organizes. When conducting such an experiment on a large number of cases and observing how embryos affected by noradrenaline, acetylcholine, and other substances differentiate until the end of development, sections should be prepared at each of the preceding stages. Or, take out DNA, RNA, etc. from inside the cell and bind the molecular reagent as described above,
Analyze with an electron microscope.

The capsule 69 and the tube 74 may be attached to the lower surface of a nitrocellulose membrane or an acetate membrane that allows oxygen to pass over the embryo 68. The capsule 69 is colored with a color corresponding to the kind of the drug inside, and many capsules are used, or the capsule is made of a dialysis membrane such as nitrocellulose, and the pores 70
, The capsule 69 is removed, and only the drug particles 71 are used, or the adhesive that cures when water or oxygen is contacted and the drug is adhered to the embryo, and the adhesive is cured. May be.

For diagnosing human diseases, when cells are taken out from the liver, spleen, kidney, or other deep organs and examined for DNA or the like, the cells must be collected using a long and thick puncture needle, Must suffer from pain (because thin bends tend to bend). Since the puncture needle made by the following manufacturing method is thin and difficult to bend, such a pain can be reduced.

FIG. 9 is a plan view of the manufacturing process of the rigid thin needle. 76 is tungsten, cobalt, and other heat-resistant metal wire with a diameter of 100 μm. 77 to 79 have a thickness of about 1 μm on the surface of carbon, silicon carbide, boron nitride, alumina, various whiskers and other mineral fibers having a diameter of about 5 μm.
A metal-coated mineral fiber that surrounds the metal wire 76 and is composed of several hundreds of metal plated with chromium, nickel, stainless steel, and other metal layers having a melting point lower than that of the wire 76. 80, 81 are a large number of similar metal-coated mineral fibers not yet attached to the metal wire 76. Reference numeral 82 is a diamond die having a truncated conical hole with a rear end having a diameter of 500 μm and a front end having a diameter of 200 μm. 83 is a gas burner nozzle. Reference numerals 84 and 85 are rollers having grooves on the periphery. Reference numeral 86 is a gas burner nozzle.

First, several hundreds of metal-coated mineral fibers 77 to 79 are attached to the periphery of the front end of the metal wire 76 using an adhesive (the outer diameter of the front part is 100 μm or less), and the die 82 is passed through the rear end of the roller. It is sandwiched between 84 and 85, and then a slightly reducing flame made by adding acid to hydrogen methane or the like is discharged from a nozzle 83, and the die 82 and the die 82 are heated to a temperature slightly lower than the melting point of the coating metal of the fibers 77 to 81. Heat the wires and fibers passing through.

The tips of a large number of metal-coated mineral fibers 80, 81 are pushed into the periphery of the metal wire 76 and the fibers 77 to 79, the rollers 84 and 85 are driven, and a few c from the die 82.
When it is fed forward, the coating metals of the fibers 77 to 81 are plastically deformed and pressed against each other to form a tube of a unitary body with no gap. (The total cross-sectional area of them is made to match the minimum diameter of the die.) Here, the flame is ejected from the nozzle 86, and the wire 76 and the fiber 77 to
The adhesive that connects 79 and 79 is burned, the flame is extinguished, and the rear end of the wire 76 is clamped and fixed by a stopper (not shown). (After extinguishing the flame, only the front end of the wire 76 may be pulled forward by 1.2 cm to securely disconnect from the surroundings.) Thereafter, the rollers 84 and 85 are continuously driven, and the fibers 77 to 8 are continued.
Only the tube consisting of 1 is pulled forward several thousand meters.

The drawn tube is wound on a drum (not shown). This tube is cut into a length of about 15 cm, and the first half is 20 cm long and 50 μm in diameter, and the second half is 20 cm long and 100 μm in diameter, and a metal wire or an integrated metal-coated mineral fiber is inserted into the tube from the front end. Then, if the front end of the thick portion and the front end of the tube match, the thin portion is cut off, the remaining front ends of both are ground and sharpened to obtain a puncture needle. (The inclined surface may be plated with metal and the front end may be smoothed by electrolytic polishing etc. to reduce pain and frictional resistance at the time of needle insertion.) Since this needle has a large amount of mineral fibers with a large Young's modulus, It is hard to bend, it can pierce deep even if it is thin, the patient's pain is less, and it can pierce deep organs, and after reaching it, pull out the needle inside or apply negative pressure with a pump to put cells in the tube The tube is pulled out, the tube is removed from the body, the needle is pushed into the tube to push out the cells, and the cells are put on the device shown in FIGS.

Further reduce the pain when piercing the needle, and
In order to allow easy puncture, a metal having a good affinity for mercury such as gold or silver may be plated on the outer surface of the tube, and mercury, which is excellent as an antifriction material, may be applied and punctured. (Mercury rarely remains in the body. The amount of mercury that adheres to the skin is less than the amount of mercury in conventional mercury ointments, but if you stick a bandage on the skin and pierce through it, you can reduce the amount of mercury that adheres to the skin. The diameter can be further reduced.) A metal wire with a diameter of 0.1 mm is placed in a hard metal tube with an outer diameter of 1 mm, an inner diameter of 0.1 mm, and a length of 15 cm, and the front end to the rear end are ground with a uniform slope to form a cone. If you make a puncture needle in the shape of a needle, it will be less likely to bend, puncture more easily, and the holes in the skin will gradually expand, making it a painless needle.

A conical needle having a needle tip of about 10 cm was put into a cylindrical tube (may be an elliptical tube) having a length of 20 cm and an outer diameter of several mm, and passed through a through hole in the needle to While ejecting an anesthetic from the vicinity of the tip, an anti-friction material containing proteolytic enzymes such as trypsin, hyaluronidase, etc. into the muscle, puncture two gallbladder, remove the needle inside and put a fiberscope in one, and in the other A gallstone crusher equipped with an ultrasonic vibrator may be inserted, and gallstones may be crushed while looking inside.

Instead of the metal-coated mineral fiber, a mineral fiber coated with a thermoplastic or thermosetting synthetic resin intermediate polymer is used, and while heating to about 200 ° C., the metal fiber 76 and the die 82 are integrated with each other. It may be made into a tube and used as a puncture needle. Such a tube or needle made of an integrated body of metal-coated mineral fibers, or a tube made of an integrated body of synthetic resin-coated mineral fibers may be used for acupuncture and moxibustion needles and other applications.

A conductive core wire made of such a material is surrounded by an insulating tube made of alumina or other ceramics, and further surrounded by such a material so that the entire outer diameter is 0.1.
It may be used as a guide needle for electromyography, which is about mm and has a sharp tip, which is thin and has less pain. DNA in the human brain is R
In order to examine the distribution of NA, proteins, and other substances, the difference in base content depending on location, the wiring structure of nerve fibers, etc., the whole human brain is sliced into many sections of about 1 μm. May be used.

FIG. 10 is a vertical sectional front view of an apparatus for serially cutting a whole human brain. 87 is a fixed base. 88 is a metal sample container mounted on it, and the front and rear width and the left and right width of the inner space are 1
The height is about 5 cm and the height is about 30 cm, and the upper edge is polished flat. 89 is a screw drive type jack provided in the lower part.
Reference numerals 90 and 91 denote piezoelectric bodies which are attached to the upper surface of the piezoelectric body and which can expand and contract in the vertical direction. 92 is 15c attached to the upper surface
M-square movable plate. Numerals 93 and 94 are high-precision distance meters provided in large numbers in the gap between the periphery of the jack and the inner surface of the container, and measure the distance between the fixed base 87 and the movable plate 92 using a laser interferometer or the like. Reference numeral 95 is a sample block placed on the movable plate. 96 is the whole human brain contained in it. Reference numeral 97 denotes a knife having a front-rear length of about 20 cm, the lower surface of which is in contact with the upper surface of the container 88. Reference numeral 98 is a front-back length 2 made of metal, sintered metal, ceramics, etc., whose lower surface is in contact with the sample mass 95 and the upper surface of the container 89
The sample pressing plate is about 0 cm, and the distance between its lower left edge and the blade edge of the knife is about 1 mm (may be further narrowed).
Numerals 99 and 100 are grooves formed on the lower surface of the net for guiding the lubricant and the like. Reference numeral 101 denotes a connecting plate that connects the knife and the rear edge of the pressing plate (a connecting plate that connects the front edge is also present (not shown)). Reference numeral 102 is a transparent glass film having a transparent electrode made of a gold-plated layer on the lower surface and a transparent silicone resin coating with a thickness of about 3 μm baked on the upper surface, the front-back width is about 20 cm, the thickness is about 5 μm, and the glass fiber is As described above, it can be freely bent, and both ends (not shown) are wound around a drum, and the total length is more than 3000 m. (A transparent synthetic resin film may be used.) First, a film of polyethylene or the like is attached to the inner surface of a box having the same volume as the container 88, liquid nitrogen is put therein, human brain is put therein, and it is frozen to surround it. Cover with a net bag made of monofilament made of fibers made of polyurethane, gelatin, etc. dyed with various fluorescent dyes for use as a marker in the section (the net bag may be covered first). Filling the surroundings with a subcooled gelatin liquid or the like, freezing, taking out the polyethylene film from the box, and putting it in a container 88,
A human brain fixed with formalin or the like and embedded with celloidin is placed in a container 88 to form a sample mass 95.

A knife 97, a pressing plate 98, and a connecting plate 101 are simultaneously reciprocated in an anteroposterior direction with an amplitude of about 1 cm by a driving device (not shown), and at the same time, they are moved to the right at a slower speed to make a sample mass 95. Slice into 1 cm thick slices. At that time, a small amount of glycerin or other lubricant is fed into the grooves 99, 100, etc. by a pump (not shown),
Friction between the sample mass 95 and the pressing plate 98 is reduced. Further, in the case of frozen samples, these are placed in a low temperature environment, but if they come into contact with glycerin, they will dissolve in the water in the sample mass, lower their freezing points, and soften the sample mass. (A fluorescence identification type reagent or the like may be mixed in the lubricant.) If there is no pressing plate, the surface of the sample lump will be deformed with the movement of the knife, and will have a large area such as 15 cm 2 It becomes impossible to cut out a uniform section having a thickness of about 1 μm.
(The connecting plate 101 may be pulled downward by a spring so that the knife and the pressing plate do not float up from the upper edge of the container 88.) The container is attached to one pole of a DC power supply of several thousand volts (not shown). 88 is connected, the section is charged through a knife 97, etc., the other electrode is connected to the transparent electrode on the lower surface of the glass film 102, and the charged section is attached to the transparent electrode surface. (When the sample mass contains a flammable or volatile organic solvent, it is necessary to send a nonflammable gas such as nitrogen into the enclosure to prevent ignition due to discharge.) The knife is on the right end of the sample mass. If one piece adheres to the glass film, the glass film is immediately moved to 10 cm.
It is sent to the right, the knife and pressing plate are returned to their original positions, the jack 89 is driven, and the movable plate 92 is raised by 1 μm.

Since the jack is driven by a screw and a considerable error may occur, if there is an error, it is detected by the range finder 93, 94 or the like, and a positive or negative compensation voltage is applied to each piezoelectric body 90, 91 or the like. To expand and contract to compensate for the rising distance of the movable plate. The distance between the movable plate 92 and the pressing plate 98 is about 15 cm, but if the movable plate moves up by 1 μm, it will be compressed by 1 / 50,000 (a foamed resin layer having a thickness of about 1 mm on the movable plate 92). May be placed to absorb the compression.) From the lower left edge of the pressing plate, the surface of the sample mass is tangent 10
Lifts to the left and right with a gentle inclination of 1/00.

Then, as before, drive the knife and pressure plate,
Slice the second layer, lift the movable plate by 1 μm,
Furthermore, the third layer and below are also sliced, and a total of 15,000 pieces (15 c
m minutes). If it takes an average of 1 minute to slice one sheet, the time required to slice 15,000 sheets is 10 days and 10 hours.

The glass film with the section attached is wound around a drum. At this time, the silicone layer on the upper surface of the film comes into contact with the section, and when unwinding, the glass surface has a stronger adhesive force. It leaves and remains on the glass surface. This glass film is automatically dyed with a fluorescent type molecular reagent solution, washed with water, dried by an apparatus such as an automatic developing machine for movie films, and then applied to the apparatus shown in FIG.

On the other hand, on the next glass film, the following 15,
Thousands of pieces are attached, and finally, a total of 150,000 pieces cut out from the sample block are attached to 10 glass films. In each section, the cross section of the thread of the mesh bag surrounding the human brain 96 appears in a dotted line, and by repeating the four colors of red, yellow, green, and blue, landmarks of continuous sections in each section are displayed. Can be used as

Whether the thickness of the section is increased or decreased,
The driving distance of the jack 89 may be changed, but at that time, since it is not necessary to raise and lower the pressing plate 98, adjustment is easy.
The lateral width of the pressing plate may be 2.3 mm. ) However, widen the front-back width of the upper edge of the container 88 to about 5 cm,
The front and rear edges of the pressing plate 98 of about 1 cm are left as they are, the lower part between them is separated from the upper part, a piezoelectric body is inserted between them, and the piezoelectric body is moved up and down by a voltage applied to the upper and lower pressing plates. The height of the lower surface of the pressing plate may be expanded and contracted so that the height can be controlled precisely and arbitrarily.

An adjusting device for adjusting the distance between the blade edge of the knife and the pressing plate may be provided. The knife 97 may be disk-shaped and the pressing plate 98 may be ring-shaped. Only this knife may be rotated, and the pressing plate may be rotated together. When targeting an animal, a fluorescence identification type reagent or the like may be placed in the blood vessel to reach the brain or other tissues.

When any chemical reaction occurs, a fluorescent reagent system that emits fluorescence is applied to a frozen sample, and an electron beam pulse is irradiated to raise a temperature in a very narrow range so that a chemical reaction can occur. Alternatively, it may be detected whether a chemical reaction has occurred. FIG. 11 is a plan view of an apparatus for reading the data of the sample piece attached to the glass film 102 at high speed. FIG. 12 is a vertical sectional front view thereof.

103 is a fixed base. Reference numeral 104 is a vacuum container attached thereon. Reference numeral 105 is a diamond thin film having a thickness of about 0.1 μm, which closes a frustoconical hole having an upper end diameter of about 2 μm, which is opened in its upper wall. Reference numeral 106 denotes a perforated accelerating electrode plate for closing a window opened on the rear wall of the container, which has 10 small holes each having a diameter of about 0.1 mm and is separated from each other by 0.3 mm or more. , Are arranged side by side in the X-axis direction at a constant pitch (10 holes are within a range of 2 mm or less in diameter), and a positive voltage of about 100 KV is applied. 107 is 0.01μ thick attached to the rear surface
An electron beam scattering plate made of aluminum of about m. 1
08 is an electron gun. Deflection coils 109 and 110 are attached to the side wall of the container. 111 is an electronic lens provided in the upper part of the container. Reference numeral 112 is an optical microscope provided below the optical microscope. Reference numeral 113 denotes a light-receiving element provided below it. A large number of red, green, and blue light-receiving surfaces are arranged in a mosaic pattern on the upper surface of the silicon chip, and their output voltage is led to an external terminal through a 30-channel DC amplifier. It has an output circuit.

Although not shown, both ends of the glass film 102 with a sample piece having a total length of 3000 m or more are wound around a drum with a rotating device using a separate pulse motor,
The drum is mounted on a drive device in the front-rear direction, and the drive device is mounted on a fixed base 103. An electron beam with a diameter of about 2 mm emitted from the electron gun 108 is connected to the accelerating electrode 106, collides with the scattering plate 107 having a positive voltage of about 100 KV, is weakly scattered, passes through the plate, and passes through the metal of the accelerating electrode 106. The matter that collides with the material is absorbed by it, and the matter that reaches the ten holes passes through it, is ejected forward, and when passing through the magnetic field lines generated between the deflection coils 109 and 110, 90 ° upward. When it is directed and passes through the electron lens 111, it becomes a truncated cone beam with a diameter of the focal portion reduced to 1/1000 and having a diameter of 0.1 μm, and passes through the diamond thin film 105 (the mass of carbon atoms forming diamond). Is small, the electron beam is not scattered so much) that it focuses on the sample piece with a thickness of 1 μm attached to the lower surface of the glass film 102, and the transparent metal on the lower surface of the glass film 102 is focused. Drawn 100KV more positive voltage applied via the contact (not shown), electrons flows to the power supply.

The focal points of the 10 electron beams fall within the range of 2 μm in diameter in the horizontal plane of the section, electrons collide with the fluorescence identification type reagent in the section, fluorescence is generated, and the light passes through the diamond thin film 105. , Enter the optical microscope 112, 30
It is magnified 00 times and forms an image on the light receiving element 113. Each focal point of the electron beam in the slice has a diameter of 0.1 μm, and if it is magnified 3000 times, it becomes 0.3 mm, but actually, it is about 1-2 mm, which is 3000 times the wavelength of light. spread.

However, since the distance between the holes formed in the accelerating electrode 106 has some allowance, the images of the respective focal points on the light receiving element surface do not almost overlap. (Electrode 106
It is also possible to cover the light receiving element with a perforated plate having the same diameter as that to block the crossed light. ) The output voltage of the minute vertical section under each focus image is grouped into three colors of red, green, and blue for each focus (the output of the light-receiving surface is connected in parallel or in series, in addition to the amplifier), and 30 channels Is output as an analog voltage, and is recorded in the memory through an AD conversion circuit of a computer (not shown). The 10 electron beams that enter the section have two vertically-symmetrical truncated cones that exist vertically above and below the focal point. At the maximum, the fluorescence from the focal portion causes a large output to the light receiving element. Also, by computer processing,
You may make it extract above the focus part.

At a cycle of about 1 MHz, the voltage applied to the acceleration electrode 106 and the deflection coils 109 and 110 is slightly increased or decreased in a saw-tooth waveform, or the voltage applied to the electron lens is similarly increased or decreased, and the electron beam in the intercept is changed. Move the focus up and down, scan the Z-axis, and cut the slice in 1 μm increments.
Data is obtained by dividing into 0 layers. Further, the glass film 102 is moved to 10 cm / se by the above-mentioned drive device (not shown).
When it is moved forward at a speed of c (Y-axis scanning), and the vicinity of the trailing edge of the slice reaches the center of the diamond thin film 105, the glass film driving device works to move the film 1 μm to the right (X-axis scanning). After that, 10c with the film facing backwards
The film is moved at a speed of m / sec, and after 1.5 seconds, the film is moved to the right by 1 μm, and the Y-axis scanning is changed to the forward direction again.

In this way, while scanning the X, Y and Z axes, the data of the section is recorded in the computer and
Take data on all sections attached to the glass film of the book. In this example, the accelerating electrode plate 106 having ten holes is used, and ten electron beams are used. Data processing is performed at a speed 10 times faster than when one electron beam is used. To increase the speed.

The cross section of the fiber of the mesh bag containing the fluorescent dye, which is placed around the human brain 96, emits fluorescence due to the collision of the electron beam, and is used to localize the relative position in each section arranged on the film surface. Be useful. The diamond thin film 105 may be removed and the whole may be put into a vacuum. Every time the upper surface of the sample mass 95 is scraped by about 1 μm with the device shown in FIG.
7, the pressing plate 98, the film 102 with a section, etc. are moved forward, the fluorescence identification type reagent is applied on the exposed surface of the sample mass, and the apparatus of FIGS. 11-12, the sample lump 95 is lifted by about 1 μm, and the knife 97 and the pressing plate 98 are lifted.
The surface may be scraped with and the device shown in FIGS.

A laser light source for generating ultraviolet rays or visible light is placed instead of the electron gun 108, and the parallel laser beam is focused by a projection lens with a diameter of about 10 μm to open ten small holes with a diameter of about 0.5 μm. Also, each hole is made to function as a point light source by hitting a small photomask having a shape like a plate 106, and a half mirror inclined at an angle of 45 ° which rises toward the front is provided in the space between the coils 109 and 110. Approximately half the amount is directed upward, and an optical lens placed instead of the electron lens 111 is used to measure a diameter of 0.5 μ in the sample piece.
Approximately 10 m focal points are formed, and the fluorescence and reflected light from the sample piece are magnified by the optical microscope 112 through the optical lens placed at the position of the electron lens 111 and the above-mentioned half mirror, and the light receiving element 113 is used. It may be photoelectrically converted and sent to a computer.

In this case, a ring-shaped piezoelectric body was inserted between the container 104 and the periphery of a photomask placed in place of the plate 106, and a sawtooth wave voltage of about 1 MHz was applied to the ring-shaped piezoelectric body to move the photomask back and forth. You can move the light focus up and down. (Piezoelectric element inserted between the lower surface of the upper wall of the container 104 and the optical lens or between the half mirrors to vertically drive the lens or mirror, or a piezoelectric element inserted between the light receiving element 113 and the lower wall of the container 104. Then, the light receiving element may be driven up and down to perform Z-axis scanning.) Three perforated plates such as plate 106 are arranged in the vertical direction and emitted from three laser light sources of red, green and blue. Light may be applied to the rear surface of each perforated plate through three thick optical fibers, and the image may be focused on a sample piece through a dichroic mirror and a lens, for a total of 30 focal points of 3 colors. (30 optical fibers may be used.) 60 optical fiber terminals are arranged horizontally above the sample surface, 30 of which are connected to laser light sources of three colors, and the other 30 are photomultiplier tubes. The light emitted from the optical fiber connected to the light source may be reduced through the lens, hit the sample surface, and the reflected light may enter the terminal of the light receiving optical fiber through the same lens. (An ultraviolet laser may be used to guide the fluorescence generated from the imaged portion on the sample surface to the optical fiber for light reception by the same lens.) With the above-mentioned various systems, DNA of many healthy people and clinical patients As a result of analyzing the base sequence of RNA, the protein molecular structure, the structure of the brain, etc., a huge amount of data is generated, and a large-capacity recording device is required to record the data.

Japanese Patent Application No. 1-119161 "Recording / analyzing device for wiring structure of nerve fibers in brain" (applicant / inventor Akihiro Fujimura) applies metal plating on a synthetic resin disk, and a photoresist on it. Applying a fine electron beam squeezed with an electron lens or a fine electric current emitted from the tip of an electrode needle to the photoresist intermittently to cure it, take out the disc and wash away the photoresist in the non-cured part, then soak it in a corrosive solution, It describes how to remove metal in the non-hardened part and record a large amount of data on the wiring structure of nerve fibers in the brain,
Since the removal of the photoresist and the chemical treatment of immersing in the corrosive liquid are required, the following may be carried out.

FIG. 13 is a front view of an information recording device for recording a large amount of analysis data and the like. 114 is a fixed base. Reference numeral 115 is a left-right direction driving device mounted on it. 116 is a motor mounted on it. Reference numeral 117 denotes a semi-transparent recording disk made of glass or synthetic resin containing a large amount of fluorescent substance fine particles with a diameter of about 20 cm attached to its shaft. Reference numeral 118 is a recording film having a thickness of about 10 nm, which is attached to the upper surface of the recording film by vapor deposition or another method, and evaporates due to heat generation by a collision of electrons or a chemical reaction,
One or two layers of substances that cause any detectable change such as sublimation, deformation, change in conductivity, change in secondary electron emission ability, fluorescence generation ability, disappearance, etc. Stacked products, for example, single layer aluminum / tin / lead / other metals, compounds of metals and iodine / bromine / sulfur / inorganic acids / organic acids / others, synthetic resins containing carbon / metal / other fine particles , Iron oxide / manganese peroxide / copper nitrate / other solid oxidizer layer, aluminum / magnesium / polysilicon / other oxidant layer, aluminum on inorganic / organic fluorescent film It is a layered product, etc.

Numeral 119 is a column standing on a fixed base. Reference numeral 120 denotes an electron gun with an electron lens attached thereto, which has an electron lens for coupling the electron beams emitted from 30 small electron guns arranged in the radial direction of the disk 117 onto the recording film 118.
Reference numeral 121 is a light receiving element with a lens mounted on a fixed base. These are stored in a vacuum container. Motor 116
Rotates the disk 117 at a speed of about 1 revolution per second, and the drive unit 115 moves them to the right at a speed of about 20 nm per second.

30 of the light receiving element 113 relating to the sample mass 95
The serial digital output of the channel is added to the control grids of the 30 electron guns in the electron gun 120 to control the density of each electron beam. On the recording film 118, 30 electron beams with a diameter of 10 nm are focused at a distance of 2 μm within a range of 60 μm in the radial direction. Part is heated, the material of the recording film 118 is evaporated, sublimated, or melted to form a donut-shaped hole,
If the compound decomposes and becomes transparent, or conversely it is colored, loses its fluorescence, becomes transparent due to the oxidizing action of the oxidizing agent,
A change, such as an increase in the secondary electron emission ability, occurs in a dot shape, and a pit occurs. (The film 118 may be oxidized by irradiating it with an electron beam of an electron gun 120 in a dilute oxygen atmosphere.) Thirty spiral tracks are formed at the same time, but 10 discs are formed.
When it is rotated 0 times, the driving device 115 rapidly moves to the right by 60 μm, and thereafter moves at a speed of 10 nm per second to draw the next spiral track.
Further, it goes right by 60 μm rapidly.

In this way, recording is performed for about 100,000 seconds even on a portion of the disk 116 which is in a fairly small range. This storage capacity is 7 × 10 13 bits (8750 gigabytes) if one pit is written in a square of 20 nm and the total recording area is 280 cm 2. (If the diameter of the pit is further reduced, the recording capacity is further increased.) When reading this, the electron gun 120 continuously emits 30 electron beams accelerated at low density or low voltage,
If the disk 117 is driven in the same manner as when writing while the surface thereof is being applied to the recording film 118, when the electron beam hits the pit created by the disappearance of the recording film, the electron beam hits the disk 117 and generates fluorescence. Then, 30 images are generated on the light receiving element 121, and respective outputs are generated.

When the transparency of the recording film is changed or the color is changed, the electron beam passes through the recording film and hits the disk,
The amount of reflection of the emitted fluorescence changes and is detected by the light receiving element.
Even when the fluorescence of the recording film changes, the amount of light emitted from the recording film also changes and is detected. When the conductivity or the secondary electron emission capacity is changed, it passes through the shaft of the motor 116 connected to the recording film,
The value of the current flowing through the high-voltage power supply circuit changes and is detected.

Reading and writing may be performed by using an electrode needle (probe / probe) of STM or AFM. A metal film is provided on the disk 117, a photoresist is applied on the disk 117, exposed by electron beam irradiation, the cured photoresist is removed, and the metal plating layer is etched with a corrosive solution.
The same recording can be performed, but it cannot be read immediately as in the above case.

Although the recording capacity per unit area is inferior to that of the apparatus shown in FIG. 13, the following large capacity recording apparatus using magnetic field may be used. FIG. 14 is a front view of parts of a magnetic head for a large capacity hard disk. FIG. 15 is a rear view of other components. FIG. 16 is a side view when they are assembled. Reference numeral 122 denotes a head substrate formed by plating a metal electrode layer on the front and rear surfaces of a PZT or other piezoelectric plate having a thickness of about 10 μm and attaching an insulating coating on the front and rear surfaces. Reference numerals 123 and 124 denote coils made of copper, aluminum or the like, which are formed on the front surface by a method such as lithography. The magnetic cores 125 and 126 are made of an insulating material such as ferrite and have a width of 1 μm and a thickness of 0.1 μm formed in the coil. Cover with an insulating film. The core information may be thickened.) 127 is a head substrate made of the same material as 122, but with a small vertical width. 128,
129 is a coil built on the right side of the front surface. 1
30 and 131 are magnetic cores contained in them. 132
Is a stepped cover plate made of the same material as the core. 133 is a copper-coated lead wire built on the surface. 134,
135 is an adhesive layer.

Substrates 122, 12 with these coils and the like
As an example of the manufacturing process of No. 7, first, a copper or aluminum film having a thickness of about 0.1 μm is plated on the entire surface by vapor deposition / sputtering / other methods, and a photoresist is applied on the film to coat the front surface of the core. The front shape of the coil, excluding the corresponding coil components, is exposed using a photomask, the photoresist is cured, and the uncured portion is washed away.
Remove the metal in the exposed part with a corrosive liquid, put a metal mask with a window corresponding to the core open, sputter the material of the core, form it on the coil part, remove the metal mask, and metal plating the entire surface Then, a photoresist is applied on it, a photoresist is applied to expose it, the photoresist on the portion other than the shape of the front of the coil is washed away, and the exposed portion is removed by a corrosive solution.

An adhesive layer 134 is attached to the rear surface of the substrate 122, the lower end is aligned and attached to the front surface of the substrate 127, and the rear surface of the cover plate 132 is adhered to a rear surface of the coating plate 132 containing carbon particles having a uniform particle size of about 0.1 μm in a diluted manner. Adhesive layer 1 composed of an agent
35 is attached and pressed onto the front surfaces of the substrates 122 and 127 to be adhered, and the ends of the lead wires 133 overlap the front surfaces of the upper ends of the respective coils, and are electrically connected by carbon particles. (Because carbon particles are sparse between lead wires and the like, a short circuit does not occur.) Each end of the lead wire 133 is connected to an input / output circuit of a computer. A cover cover made of the same material as the cover 123 and having an upper end protruding forward and reaching the cover 132 is attached to the rear surface of the substrate 127.

The magnetic head block thus formed is arranged so that the left-right direction coincides with the radial direction of the hard disk, and is appropriately moved in the left-right direction by a driving device to write or read on the magnetic surface of the rotating disk. Or A space of 3 μm is provided between the cores 125 and 126 and between the cores 130 and 131 to form a coil.
In case of using only one substrate, a gap of 3 .mu.m is generated between tracks formed on the disk, and in order to fill it, it is necessary to perform scanning so that the head block is arranged between them.

However, when two substrates are used, the cores of both substrates fill the gaps with each other, so that fine scanning in the radial direction becomes unnecessary. In addition, actually, 15 or more cores (including clock pulse cores) are provided on the substrates 122 and 127 at a pitch of 4 μm, and 30 cores at a pitch of 2 μm are provided.
The above-mentioned image information is written on the track of the book all at once.

The upper end of each core is relatively close to the upper ends of the covering covers 132 and the like, and the magnetic lines of force from the upper ends of the cores pass through the covering covers at the front and rear and extend downward from the lower ends thereof with a large radius. Entering the lower end of each core, each core will perform perpendicular magnetic recording on the disk surface.
It is also possible to provide an extra 64 cores and use them to input / output parallel data of a 64-bit personal computer at once.

If such a head block is used to write on a hard disk having a diameter of 10 cm, information of about 12 gigabytes (1.5 gigabytes) can be recorded, and a capacity comparable to that of a magneto-optical disk can be obtained. . It may be recorded on a magnetic tape to obtain a larger amount of information. For example, one board (L including some information processing circuits
The silicon substrate of SI may be used), and the thing which built 1500 cores with a coil at 4 μm pitch is 2
It is also possible to stack the sheets into 3000 head blocks and record information of 30 channels from a head group of 100 heads each.

In this way, the relative moving speed of the head can theoretically be 1/3000 as compared with the conventional helical scan VTR or the like. In this case, if the tape width is reduced by 1/6000 due to long-term use or the like, even if one end of the head row is aligned with the track at the time of writing, the head at the other end is displaced by 1 μm and contacts the tracks. become.

As a countermeasure against such misalignment, head blocks are attached on the fixed base through the vertically moving piezoelectric bodies in the direction in which the head rows are arranged vertically, and the head block is attached to the top, middle and bottom ends of the head block. Using three heads as monitor heads, write a clock pulse on the tape from three persons at the time of writing, and at the time of reading, apply a voltage to the piezoelectric body for vertical movement with negative feedback so that the output of the monitor head at the lower end becomes maximum. The substrate 12 is controlled so that the output of the monitor heads at the middle and upper ends are also the same.
The vertical length of the substrate may be adjusted by controlling the voltage applied to 2, 127.

As another measure, a tape having a synthetic resin layer containing magnetic particles attached to the surface of a glass tape having a thickness of about 5 μm and a synthetic resin layer attached to the back surface may be used.
A metal tape may be used to prevent the tape width from changing. Alternatively, in addition to an input / output head block with a head pitch of 2 μm, several head blocks with a slightly narrower pitch are installed side by side, and the one that produces the most appropriate output among them should be used during playback. You may

The core and coil attached to the substrate 127 may be attached to the back surface of the substrate 122, and the substrate 127 may be omitted.
Widen the width of the end of the magnetic core attached to the front surface of the substrate 122,
Other coils and cores may be omitted. As described above,
A method for rapidly and inexpensively producing a glass film having a thickness of about 5 μm such as a glass film 102 to which a sample piece is attached and a glass tape for magnetic tape will be described below.

FIG. 17 is a side view of a glass film manufacturing apparatus having a width of 20 cm. 136 is a crucible containing molten glass of a thousand and a hundred degrees. Reference numeral 137 denotes a nozzle attached to the lower surface of the nozzle. The lower end of the opening has a front-rear width of 20 μm and a left-right width of 0.1.
It is about mm. 138 is a molten glass hanging from the nozzle. Reference numerals 139 and 140 denote drawing rollers having a diameter of 2 cm, which are made of metal or ceramics to which glass is hard to adhere, and are cooled to a temperature below the softening point of the glass by air blown from below (not shown). It is pushed to the left by a hard spring, and the distance between the rollers does not fall below 5 μm, but it is possible to open it further. 141 and 142 are lead films attached to the upper and inner surfaces of both rollers, and the lower end thereof is attached to the winding roller 143. The front and rear width is 20 cm, and the thickness is 1
It is made of a film of carbon, silicon dioxide, metal or the like, which has a thickness of about μm and is produced by vapor deposition or another method.

The molten glass in the crucible 136 is pressed downward by a piston (not shown), and the molten glass 138 hangs down from the nozzle 137 to form a film having a lateral width of 0.1 mm.
Eventually, when the fact that the lead film 141, 142 is reached is detected by an infrared sensor or the like, the rollers 139, 140 start to rotate and the molten glass has a lateral width of 3 μm.
m, then sandwiched between lead films, roller 1
It is wound up by 43.

When the ends of the lead film passed through the rollers 139 and 140 without winding, the molten glass 13 was passed thereafter.
8 directly contacts the roller surface and is molded to a thickness of 5 μm,
At the same time, it is cooled and solidified, and then sent downward. The upper end of the molten glass 138 has a lateral width of 0.1 mm, but since the rollers 139 and 140 rotate at a circumferential speed 20 times the hanging speed, the molten glass is stretched to have a thickness of 5 μm, and the roller 139, It reaches 140.

The surface tension acts on the molten glass 138 and the front-back width tends to be narrowed. However, the lower end of the nozzle 137,
Since the distance to the rollers 139, 140 is small, the rollers reach the rollers 139, 140 with almost no narrowing in the front-rear width. (Because the molten glass 138 is thin, the temperature drops in a short time.) When starting without using the lead films 141 and 142, first, the drooping speed of the molten glass 138 is slowed to a cooling state, and then slowly rotated. Rollers 139, 1
40, and passed between the rollers downward. Soon after, the rotational speeds of the rollers 139 and 140 and the drooping speed ratio of the molten glass 138 were both maintained at 20, while increasing both speeds, and a glass film having a thickness of 5 μm was formed. When it begins to occur, it is cut at the height of the lower end of the roller 143 with a cutter (not shown), the wind is blown from the right side to wind the cut end of the film around the roller 143, and the same winding as described above is performed.

The nozzle 137 is made of conductive ceramics or the like and is energized to raise the temperature to improve the fluidity of the molten glass passing through the inside, or the nozzle 137 and the roller 1
Between 39 and 140, blow cold air to the molten glass 138,
You may cool appropriately. In order to prevent the glass film wound around the roller 143 from being unwound due to surface charging or the like, water, ionized air, or the like may be blown onto the glass film during winding.

A crucible containing glass having a refractive index smaller than that of the glass in the crucible 136 is provided on the left and right of the crucible 136, both nozzles are provided on the left and right of the nozzle 137, and a laminated body of molten glass discharged from the three nozzles is provided. By passing between the rollers 139 and 140, stretching and allowing parallel light to enter from one end, a light transmitting film that propagates without leaking out may be produced and used for various purposes.

Next, various points will be described in relation to the application of the above-mentioned apparatus. Conventionally, when analyzing the base sequence of DNA, heat or alkali is applied to the extracted DNA to deform it, unpair it, cleave it with a restriction enzyme, add urea during electrophoresis, etc. Therefore, even if the strong DNA base sequence is preserved, secondary structure, tertiary structure,
Mutual bonds in the molecule, such as hydrogen bonds, and bonds of external molecules are all destroyed.

Therefore, a molecular reagent is attached to the frozen fracture surface while keeping the DNA in the cell nucleus, and
It is necessary to perform structural analysis. It is due to cell differentiation and various reversible or irreversible structural changes associated with functional expression,
It is considered that the state where the binding of various proteins, RNA and other substances occurs will be captured.

As a structural change, a double strand may be partially opened (there may be another molecule at both open ends to prevent re-pairing), or one of the paired strands may be one. It is cut off at one place and has a branch shape, or it is cut off at two places and is in a removed state. On the contrary, there may be a case where a function is regulated by expressing a function in the meantime or using a restriction enzyme created by itself, a DNA polymerase or the like to remove a part of the base sequence. As a material change (the basis of structural change is a material change and it is difficult to separate the two), when the base sequence changes, one strand is linked and RNA / protein / other substances are inserted. If
When the function changes due to binding between adjacent bases of an open DNA strand, directly or through another substance, an open DN
It is conceivable that the A chain may be bound to another substance to change its function.

These changes are reversible (when necessary substances are produced in a required amount, when substances are accompanied by cell division, when certain conditions are met, and when they return), and when they are irreversible. Can be. DNA that controls cell differentiation
It is irreversible in change, and when it receives a physical information (ligand) from an unchanged or irreversible part and expresses a function, it often causes reversible change.

If an analogy is sought for electronic circuits, irreversible functional changes may be soldered to replace wiring,
It is equivalent to cutting a part of the prepared wiring, a functional change that is hard to return to the original corresponds to the on / off of the snap switch, and a very temporary change corresponds to pressing the button switch to energize the pulse. After the button switch is pressed once, the change is retained as long as the same state is maintained, which is equivalent to setting / resetting the magnet switch or the flip-flop. (When making a copy of these hard models or soft models, there may be those that are easily reset and those that are not easily reset.) Known information such as the nucleotide sequence and gene sequence of DNA, and the known information related to its function, Input it into the software of the computer, and replace the latter function with the function of the switch, analog amplifier, negative / positive feedback circuit, various analog / digital logic circuits, and other electronic circuits as described above, and the unknown part with various It is also possible to create a research field in which an assumed model is automatically created, replaced and fitted, functional simulation is performed, and a more consistent overall model is built.

Ontogeny is the process in which one fertilized egg repeats division and differentiates into a large number of differentiated cells. Exists. However, in the process of differentiation, each DNA should undergo some kind of change as described above.
It should also be copied during cell division after maturation.

Therefore, there should be a switch that is reset when copying and a switch that is copied in the switched state. If they were all reset, all of the epithelial cells and connective tissue cells would return to their original fertilized egg state during division.
The change caused by decomposition is not only the change in DNA but also the R around it.
It may have to be copied, including changes in NA, protein, nucleus, cytoplasm, etc. However, there seems to be a distinction between essential and non-essential conditions.

It is necessary to capture changes in DNA and its peripheral substances (intracellular DNA environment) associated with such differentiation as they are. The evolutionary mechanism by neoderwinism does not affect the environment that irreversible changes (mutations) in germ cell DNA affect somatic cells (successfully growing one rice while acclimatizing to a cold environment). Even if you do, the adaptive ability will not be inherited to the offspring),
Mutations caused by the effects of radiation and mutagens that directly act on the germ cell DNA itself, and mutations occur almost randomly in the germ cell DNA itself, regardless of external factors,
It is said that the strains that are inherited by the offspring, are naturally selected by the offspring, and have mutations suitable for the environment will survive and prosper.

However, it is possible that there is a mechanism for inheriting the acquired trait obtained as a result of adapting to the environment within the range of the DNA (gene) that is naturally born.
The question arises as to whether there is nothing at all. The following possibilities are theoretically possible. First, when environmental factors act on somatic cells as well as on phagocytic cells to cause mutations, and within the range of the functions originally possessed by DNA (not mutations), changes in DNA due to the aforementioned differentiation. That is when (gene expression) occurs, it is copied and inherited. (May be the DNA changes associated with differentiation are preserved in the copy associated with cell division.) Second, environmental factors cause somatic cells around germ cells or at distant locations. This is the case when a physiological or non-physiological substance reaches the germ cell, changes its DNA, and inherits it.

Third, environmental factors cause some changes in the DNA of somatic cells, some of the changed DNA fragments are lost, and copies of DNA and RNA are prevented from being decomposed by nucleases in blood. Of the protein of the above, it is integrated into the plasmid of the invading bacterium, or it is integrated into the gene of the virus, reaches the germ cell, and is integrated into its DNA.
When the DNA switch in germ cells is switched. (It is also possible that a physiologically or pathologically normal or mutated DNA fragment exits the cell and metastasizes to adjacent cells or distant cells after development or maturation. [Although it is found in unicellular organisms.]) The mechanism seems to be unlikely, but in the history of biology, it is a series of discovering things that human beings do not expect in living organisms, and in human heads It's possible that something imaginable is actually being done.

In 1971, I told a professor of biology at a university that a creature might kill itself, but it didn't matter at all. Since there are annual and perennial plants in closely related plants,
He conceived that the one-year-old objects that have little structural difference do not die with aging, but rather kill with their own functions. Then, in 1972, J, R, Ke
The concept of "apoptosis" has been proposed by rr et al.

The chromatin in the nucleus is wound around the histone octamer protein in a state where the double strand of DNA is twisted like a rope, which is further wound into a solenoid coil and further into a coil or a coil. , And is said to form a folded state. When expressing a part of its function or making a copy during proliferation, in order to make frequent contact with surrounding environmental substances, it is partially linearized and one of the chains is cut at one place. , It is said that the pairing will open.

At this time, if the intermediate portion of the coil is stretched,
A twist occurs in the DNA strand, and in order to remove the twist,
It is said that a part of the DNA that idles occurs at one place. However, I think that even if there is no idling mechanism for removing the twist, a configuration that does not cause the twist when the coil is stretched is possible.

For example, a length of 10 m corresponding to DNA,
Two red and black vinyl cords are lined up in parallel, both ends are fixed with a space of 1 m, and they are wound around a magnet rod with a diameter of 3 cm and a length of 1 m, which is equivalent to histone, about 100 times and wound in a single layer coil shape. , Suppose that the shape is fixed. (One twist is generated for each winding.) This magnet bar is a collection of many small magnets corresponding to small molecules of histone protein, and can be separated arbitrarily. The winding becomes straight, but the twist disappears.

If the separated magnet rods are rejoined while the straightened portion of the cord is wound around the magnet rod again, the original state is restored. D related to the differentiation of these cells, adaptation to the environment, mutation and evolution, cell division, substance metabolism, etc.
The NA and its surrounding structure are captured by the above device. When the invention described in each claim of the claims is not similar in other fields, it can be slightly modified and used for other purposes.

[0135]

EFFECTS OF THE INVENTION By carrying out the present invention, it becomes possible to analyze the base sequence of a nucleic acid, the molecular structure of a protein, etc., much more rapidly than conventional methods and devices, and to supplement and enhance its function, It is possible to obtain a computer system, an electron microscope system, a sample preparation device, etc. for making it easier to use and expanding the range of applications.

[Brief description of drawings]

FIG. 1 is a plan view of a sample preparation device with a DNA single-stranded reagent to be examined using the present invention.

FIG. 2 is a vertical sectional view thereof.

FIG. 3 is a plan view of a DNA nucleotide sequence analysis device embodying the present invention.

FIG. 4 is a vertical cross-sectional front view of the electron microscope unit covered with a vacuum container.

FIG. 5 is a vertical left side view of the computer or the like.

FIG. 6 is an enlarged vertical cross-sectional view of a handy image scanner 31, a stereoscopic image input device, and the like.

FIG. 7 is a vertical sectional view of a closed vacuum vessel type electron microscope.

FIG. 8 is a longitudinal sectional view of an embryo differentiation induction test device.

FIG. 9 is a plan view of the manufacturing process of the rigid thin needle.

FIG. 10 is a vertical sectional front view of an apparatus for serially cutting a whole human brain.

FIG. 11 is a plan view of an apparatus for reading data of a sample piece attached to the glass film 102 at high speed.

FIG. 12 is a vertical sectional front view thereof.

FIG. 13 is a front view of an information recording device that records a large amount of analysis data and the like.

FIG. 14 is a front view of components of a magnetic head for a large capacity hard disk.

FIG. 15 is a rear view of another component.

FIG. 16 is a side view when they are assembled.

FIG. 17 is a side view of a glass film manufacturing apparatus having a width of 20 cm.

[Explanation of symbols]

 1 UV irradiation device. 2 water layers. 3 Metal plate electrode. 4 Nitrocellulose membrane. 5 water supply pipe. 6 Drain cock. 7 glass tubes. 8 1 chromosome. 9 Glass sample plate. 10 Transparent electrode. 11 Fixed base. 12 Sample plate driving device. 13 Sample DNA with fluorescent recognition type molecular reagent. 14 props. 15 Scanning electron microscope. 16 TV camera with optical microscope. 17 Light source. 18 color photon counter. 19 lenses. 20-21 Dichroic mirror. 22-24 Red, green, and blue photomultiplier tubes for receiving light. 25 Vacuum container. 26 cables. 27 computers. 28 Main switch. 29 LCD display. 30 Transparent plate with groove. 31 Handy image scanner. 32 click switch. 33 cables. 34-35 Transparent rotating shaft. 36 Transparent endless belt. 37 TV camera. 38 Transparent disk. 39 Encoder. 40 Stereoscopic image input device. 41 TV camera. 42-43 Light source. 44 Transparent plate. 45 Dilute black ink. 46 White rubber film. 47 Fixed base. 48 columns. 49 Vacuum container for ordinary electron microscope. 50 electronic lens. 51 Fluorescent plate. 52 Video camera. 53 Metal disk. 54 Air barrier membrane. 55 Sample film. 56 jacks. 57 Vacuum container for scanning electron microscope. 58 electron gun. 59-60 X-axis deflection electrode. 61 Y-axis deflection electrode. 62 Metal disk. 63 Air barrier membrane. 64 water. 65 color photon counter. 66 fixed base. 67 Petri dish. 68 Embryos in the developing process. 69 capsules. 70 Micropores. 71 Drug particles. 72 Jack. 73 containers. 74 Synthetic resin tube. 75 Aqueous drug solution. 76 Heat-resistant metal wire. 77-79 Metal-coated mineral fibers. 80-81 Metal coated anti-fiber. 82 Diamond Dice. 83 Gas burner nozzle. 84-85 rollers. 86 Gas burner nozzle. 87 Fixed base. 88 sample container. 89 Jack. 90-91 Piezoelectric body. 92 Movable plate. 93-94 Distance meter. 95 sample mass. 96 Human brain in sample mass. 97 knife. 98 Sample pressing plate. 99-100 grooves. 101 Connecting plate. 102 glass film. 103 Fixed base. 104 Vacuum container. 105 Diamond thin film. 106 Accelerating electrode plate. 107 Electron beam scattering plate. 108 electron gun. 109-110 Deflection coil. 111 electronic lens. 112 Optical microscope. 113 Light receiving element. 114 fixed base. 115 Left-right direction drive device. 116 motor. 117 Recording disk. 118 recording film. 119 props. 120 electron gun. 121 Light receiving element. 122 head substrate. 123-124 coils. 125-126 Magnetic core. 127 head substrate. 128-129 coils. 130-131 magnetic core. 132 Cover plate. 133 leads. 134-135 adhesive layer. 136 Crucible. 137 nozzles. 138 Molten glass film. 139-140 Stretching roller. 141-142 Lead film. 143 Rolling roller.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/58 A G06T 7/00 // C12N 15/00

Claims (11)

[Claims] 1. Four kinds of bases (atomic groups) in a nucleic acid molecule
Fluorescence discrimination type that emits fluorescence of different colors to label what the reagent molecule is, such as a nucleobase or the like, which can specifically bind to a specific atomic group in a protein molecule A labeled molecule, or a morphologically discriminating labeled molecule consisting of molecules having different shapes and sizes which can be distinguished by an electron microscope etc. and which are markedly different,
A large number of molecular standard reagents are bound to one nucleic acid molecule or protein molecule, and the shapes of sample molecules and reagent molecules are visualized by scanning electron microscope, transmission electron microscope, atomic force microscope, etc. What is the atomic group in the sample molecule to which the reagent molecule is bound, by distinguishing the color of fluorescence generated each time the scanning electron microscope's fine electron beam collides with the fluorescent reagent molecule by the photoelectric conversion device and the computer? The shape or size of the shape-identifying reagent molecule with a computer from the images of electron microscope or atomic force microscope, and what is the atomic group in the sample molecule to which the reagent molecule is bound? A method for analyzing the nucleotide sequence in one nucleic acid molecule, the molecular structure of a protein, etc. to identify 2. An apparatus for binding a fluorescence discrimination type molecular reagent or a morphology discrimination type molecular reagent to a sample molecule, and an ordinary scanning electron microscope, transmission electron microscope, tunnel scanning electron microscope, etc. for visualizing the morphology of the sample molecule. With an intermolecular force microscope,
Fluorescence discrimination type molecule bound to sample molecule One molecule of reagent is captured by the fine electron beam of scanning electron microscope and the generated fluorescence is captured to discriminate the kind of reagent System consisting of photoelectric conversion device and computer, or fluorescence discrimination An apparatus for analyzing a nucleotide sequence of a nucleic acid, a molecular structure of a protein, and the like, which is provided with an image recognition computer for identifying the type of a single molecule reagent of a type molecular reagent. 3. A number of parallel grooves are arranged at regular intervals,
Place the transparent plate on the surface over the image display device such as a paper surface or a computer display, attach a transparent endless belt or a flat plate having a groove that meshes with the groove on the bottom surface of the hand image scanner, and output terminal of the scanner Input device that connects the computer to the input terminal of the computer, and illuminates the uneven surface at an angle to generate shadows and light and shade due to the unevenness created in the diluted ink layer, which is input to the computer as a video signal with a video camera. An apparatus for analyzing a nucleotide sequence of a nucleic acid, a molecular structure of a protein and the like according to claim 2, which is provided with a device. 4. A vacuum container of an electron microscope is divided into an upper container and a lower container with a sample as a boundary, and a conical trapezoidal electron beam whose diameter becomes smaller toward the sample is passed through the walls of both the containers facing the sample. A thin film, which is made of carbon, alumina, and others, that is impermeable to air but impervious to electron beams but is permeable to electron beams, is attached to the surface of the walls facing the sample that is provided with light, and the holes are closed to drive the upper or lower container up and down. The apparatus for analyzing a nucleotide sequence of a nucleic acid or a molecular structure of a protein according to claim 2, which is provided with a driving device for allowing the sample to be taken in and out. 5. Acetylcholine / noradrenaline /
Generation process such as a capsule with micro-release holes that enclose other arbitrary chemical substances, small pellets containing those substances, a container containing a solution of these substances, and a solution administration device consisting of a fine solution transport tube connected to the container, etc. 3. An apparatus for gradually administering a chemical substance to a minute site in an embryo, and a nucleic acid sequence of the nucleic acid according to claim 2 for analyzing a base sequence in a nucleic acid extracted from an embryo whose development is controlled by the administration of the chemical substance. A nucleic acid base sequence analysis system comprising a base sequence analyzer. 6. A bundle of a large number of carbon fibers, silicon carbide fibers, and other hard long fibers whose surfaces are metal-plated, which is heated to a temperature at which the coating metal melts and softens, and which has a small exit from the entrance. A base sequence of a nucleic acid, comprising a device for internal tissue collection using a hard needle or a hard tube integrally formed by fusion-bonding and pressure-welding coated metals through a die having holes, and the device according to claim 2. System for analysis of molecular structure of proteins and proteins. 7. An upper open box-shaped sample block container is provided,
On the bottom of the container is provided an actuator plate that drives the sample lump up and down, and along the bottom surface of the container, there is a knife that drives horizontally, and on the bottom surface of the horizontal plate attached to the leg that contacts the top edge of the container. , The proximity edge is near the blade edge of the knife, and directly or through the piezoelectric body, a sample block pressing plate that is connected to the lower surface of the horizontal plate is provided, and the lower surface of the pressing plate is at the same height as the blade edge of the knife. Or a base sequence of a nucleic acid in a brain or other tissue, a molecular structure of a protein in a brain or other tissue according to claim 2, further comprising a sample piece preparation device that is driven horizontally while maintaining a slightly higher position. Other fine structure analysis system. 8. A plurality of substantially parallel laser beams or electron beams are scatteredly arranged such that the mutual distance between the focal points connecting to the sample surface is larger than the wavelength of the reflected light or fluorescence generated from the focal portion. Through a half mirror or a magnetic field inclined at 45 °, changing the direction by 90 °,
Multiple simultaneous detection of fluorescent reagents, etc., in which an image is formed on the sample surface through an optical lens or an electronic lens, and reflected light or fluorescence generated from the sample surface is imaged on a light receiving element having multiple light receiving surfaces using the optical lens. Using the device,
An apparatus for analyzing a nucleotide sequence of a nucleic acid or a molecular structure of a protein according to claim 2. 9. A disk-shaped or tape-shaped recording medium driven by a motor is subjected to melting, evaporation, sublimation, decomposition, oxidation, etc. by electron beam irradiation or energization, resulting in deformation, volume increase / decrease, alteration, or change in conductivity. A thin layer of a substance that causes, for example, a computer-controlled high-density, high-energy, minute electron beam squeezed by an electron lens, and the tunnel current flowing from the tip of the electrode needle for a scanning tunnel electron microscope are intermittent An information recording device that is provided with a digital information writing device that is applied to the recording medium and a reading device that can weakly and finely continuously apply an electron beam or tunnel current to the recording medium and immediately read the written information from the change in the current. An apparatus for analyzing a nucleotide sequence of a nucleic acid or a molecular structure of a protein according to claim 2, which uses a computer having a reproducing apparatus. 10. When a voltage is applied, a large number of magnetic heads arranged vertically are formed on the left and right surfaces of a piezoelectric head substrate that expands and contracts in the vertical direction by a method such as lithography, and the voltage is applied to the lower end of the substrate. When mounted on a fixed base via a vertically moving piezoelectric body that expands and contracts vertically, when writing on a magnetic tape that drives horizontally while contacting each magnetic head, the upper and lower ends of the head row and the monitor head in the middle part The clock pulse is recorded from, and at the time of reading, the output of each monitor head is maximized and even,
Using a large-capacity magnetic data recorder, which is provided with a circuit for applying a voltage to the vertically moving piezoelectric body and the head substrate,
An apparatus for analyzing a nucleotide sequence of a nucleic acid or a molecular structure of a protein according to claim 2. 11. A nozzle for drawing out a molten glass film and a roller pair for stretching the film are provided, and in order to prevent the film width from narrowing due to surface tension, the distance from the tip of the nozzle to the roller is set equal to or less than the film width, A sample piece attached to a glass film made by a glass film manufacturing apparatus having a thickness of about 5 μm, which is provided with a device for blowing cold air to the film between the tip of the nozzle and the roller, or a magnetic substance is used for the lath film. An apparatus for analyzing a DNA base sequence or a protein molecular structure according to claim 2, which uses a layered data recording magnetic tape.