JP2013066404A - Device for observing crystallization plate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device for observing a crystallization plate, without requiring to take out the crystallization plate from a ferromagnetic field, when observing the degree of formation of a protein formed in a crystallization well installed in the crystallization plate, without disarranging the crystal growth even when the crystal is not formed yet and also without changing the magnification of an image even on adjusting the focal point of an optical system.SOLUTION: With the device for observing the crystallization plate, the crystal formed at a crystal formation part is observed, in the state in which the crystallization plate consisting of a ring body equipped with a reservoir, the crystal forming part and a pillar-shaped space is arranged in a super ferromagnetic field; and the light from the crystal formation part is reflected by a reflecting means installed at the pillar-shaped space of the ring body and directed toward a light-receiving element. The device has an objective lens-moving means capable of moving the objective lens while fixing parts except for the objective lens among the observation means to adjust the focal point. The reflecting means is positioned at the pillar-shaped space, and in the pillar-shaped space formed at the inner circumference of the ring body, the objective lens in an imaging optical system is set as an infinity corrected optical system.

Description

  The present invention relates to an apparatus for observing a crystallization plate that crystallizes proteins or the like in a pseudo microgravity state.

  Conventionally, it has been very difficult to crystallize many important proteins, and even if crystals are obtained, there are many cases where X-ray diffraction data with poor quality and resolution capable of structural analysis cannot be obtained. The stage of crystallization has become a bottleneck for X-ray crystal structure analysis of proteins.

  Here, protein crystal production by the vapor diffusion method will be described.

  In the airtight container, the water in the crystallization drop (mixture of protein solution and precipitant solution) evaporates and moves to the external liquid (reservoir) with a high concentration of precipitant, so the protein solution in the crystallization drop The concentration and the precipitating agent concentration increase, and microcrystals that form the nucleus of crystal growth are formed in the supersaturated protein solution. If the precipitant concentration is in an appropriate range, crystals are formed.

  By the way, under gravity, a protein solution concentration gradient is generated in protein crystallization, and natural convection occurs in the protein solution. That is, when moisture evaporates from the droplet surface of the protein solution, a region with a high solute concentration is formed on the droplet surface, and this high concentration region falls due to gravity, thereby generating natural convection. The convection of the protein solution reduces the quality of the crystals.

  Under gravity, protein and impurities are frequently supplied by natural convection, so that crystal growth is fast but impurities are easily taken up. In addition, collisions between proteins are likely to occur due to natural convection, many crystal nuclei are generated, and the generated crystal nuclei are likely to be small crystals. Under gravity, as described above, the protein is supplied quickly, so that the protein attached to the crystal surface in the wrong orientation is easily taken into the crystal, and the molecular arrangement of the protein is disturbed.

Crystallization using the microgravity environment of the universe was performed as a means to avoid disturbance of protein molecular arrangement under gravity, and it was proved that it is effective for protein crystal generation that gives high-resolution X-ray diffraction images. . However, space experiments have limited experimental opportunities and are difficult to comprehensively apply to structural analysis of a wide variety of proteins. Thus, it is known to create a pseudo microgravity environment by levitation using a superconducting magnet on the ground (see, for example, Patent Document 1), and a protein crystal is generated in the pseudo microgravity environment. An approach has been made.

Japanese Patent No. 3278865

  However, in the above conventional example, when observing the degree of protein production in the crystallization well provided in the crystallization plate, it is necessary to take out the crystallization plate from a strong magnetic field, and is the crystal formed? If it is not taken out in an unclear state, the state cannot be confirmed, and if no crystal is formed, there is a problem that crystal growth is disturbed.

  In other words, in order to take out the crystallization plate, the magnetic force acting on the solution changes spatially, and the work of taking out and returning the crystallization plate causes a flow in the solution, disturbing the crystal growth. There is a problem that.

Further, in the above conventional example, even if the generation degree of the protein generated in the crystallization well can be observed with the optical system without taking out the crystallization plate from the strong magnetic field, the arrangement position of the crystallization well is not limited. When the focus of the optical system is adjusted, the magnification of the image of the crystallization well changes, which makes it difficult to observe.The present invention has the problem that the protein produced in the crystallization well provided in the crystallization plate When observing the degree of formation, it is not necessary to take out the crystallization plate from a strong magnetic field, and even when crystals are not formed, crystal growth is not disturbed, that is, the process of protein crystallization can be observed in situ. In addition, an object of the present invention is to provide a crystallizing plate observation apparatus in which the magnification of an image does not change even when the focus of an optical system is adjusted.

  The observation device for a crystallization plate of the present invention contains a ring body having a columnar space, a precipitant required for protein crystallization, a reservoir provided in the ring body, and protein crystals are generated. The crystallization plate, which is a portion that is provided with the crystal generation unit provided in the ring body, is placed in a super strong magnetic field, and the crystal generated in the crystal generation unit is observed. A light-receiving element that converts an image of light from the crystal generation unit into an electrical signal, and a reflection unit that reflects light from the crystal generation unit toward the light-receiving element. Reflecting means provided in a columnar space formed on the inner periphery, and objective lens moving means for adjusting the focus by moving the objective lens while fixing other than the objective lens among the observation means, and the reflecting means Located in the columnar space, columnar space formed in the inner circumference of the ring body, an observation device of the crystallization plate, characterized in that the objective lens of the imaging optical system infinity optical system.

  According to the present invention, when observing the degree of protein production in the crystallization well provided in the crystallization plate, it is not necessary to take out the crystallization plate from a strong magnetic field and the crystal is not formed. However, the crystal growth is not disturbed, that is, the process of crystallizing the protein can be observed in situ, and the magnification of the image does not change even if the focus of the optical system is adjusted.

It is a figure which shows crystallization plate PL1 which is Example 1 of this invention, and observation apparatus OB1 which observes crystallization plate PL1. It is a figure which shows the plane of crystallization plate PL1, and the cross section of crystallization plate PL1. It is a block diagram which shows crystallization plate PL2 which is Example 2 of this invention. It is a block diagram which shows crystallization plate PL3 which is Example 3 of this invention. It is a block diagram which shows crystallization plate PL4 which is Example 4 of this invention. It is a figure which shows the fitting structure in crystallization plate PL2. It is a block diagram which shows crystallization plate PL5 which is Example 6 of this invention. It is a figure which shows the observation system 50 in the observation apparatus of the crystallization plate which is Example 7 of this invention. In the observation system 50, when an observation point differs in a Y-axis direction, it is a figure which shows the example which positions a Y-axis. In the observation system 50, it is a figure which shows the example which adjusts a focus by fixing the position of the inclination mirror 51 and moving the objective lens 52, when observation points differ in a Z-axis direction.

  The modes for carrying out the invention are the following examples.

  FIG. 1 is a diagram showing a crystallization plate PL1 that is Embodiment 1 of the present invention and an observation apparatus OB1 that observes the crystallization plate PL1.

  FIG. 2 is a diagram showing a plane of the crystallization plate PL1 and a cross section of the crystallization plate PL1.

  That is, FIG. 2 (1) is a diagram showing a plane of only the crystallization plate PL1, and FIG. 2 (2) is a sectional view taken along line II-II in FIG. FIG.

  The crystallization plate observation device OB1 is provided with a vertical hole in FIG. 1 at the center of the superconducting magnet Mg1, and the vertical hole can be moved up and down (movable in the axial direction), and can rotate about the axis. An observation system 50 (which can be rotated in the circumferential direction) is provided. The light source 55 provided in the observation system 50 can also move up and down in the vertical hole of the superconducting magnet Mg1, and can rotate about the axis.

  The observation system 50 includes an inclined mirror 51, an objective lens 52, a tube lens 53 (also referred to as an imaging lens), a CCD camera 54, a light source 55, a rotating unit, and a moving unit. The tilt mirror 51 reflects the crystal image generated by the crystal generation unit 40, and the objective lens 52 temporarily converts the light reflected by the tilt mirror 51 into parallel light and forms an image on the CCD camera 54 by the tube lens 53. . The CCD camera 54 has a light receiving element such as a CCD, and converts the crystal image generated by the crystal generation unit 40 into an electrical signal. Note that other observation means may be provided instead of the CCD camera.

  The rotating means is means for rotating the reflecting means, which is the tilted mirror 51, in the circumferential direction of the ring body 10 in the columnar space 20 formed on the inner periphery of the ring body 10. The moving means is means for moving the reflecting means in the axial direction of the ring body 10 in the columnar space 20, and means for adjusting the focus by moving only the objective lens 52 with the respective parts fixed.

  The light source 55 is provided in the columnar space 20 of the crystallization plate PL1 below the inclined mirror 51 in FIGS. 1 and 2 (2), and from the side (side surface) in FIGS. 1 and 2 (2). The crystal generation unit 40 is irradiated with light.

  The crystallization plate PL1 includes a ring body 10, a reservoir 30, a crystal generation unit 40, and a reflection surface M1. If total reflection is used in the reflective surface M1, the same effect as that of the mirror surface can be obtained even if it is not a mirror surface.

  The ring body 10 is formed of a transparent material such as synthetic resin or glass, and as shown in FIG. 2, a columnar space 20 and 12 chambers are provided, and one reservoir 30 and 2 are provided in one chamber. One crystal generation unit 40 is provided. In the above embodiment, the number of chambers other than 12 may be provided.

  A crystal generation unit 40 is provided inside the ring body 10. The reason why the crystal generation unit 40 is provided inside the ring body 10 is that the closer to the center of the ring body 10, the better the uniformity of the magnetic force field, that is, the crystal generation unit 40 is arranged inside the ring body 10. By providing, a more uniform magnetic force field can be utilized. Further, when the reservoir 30 and the crystal generation unit 40 are combined, the ring body 10 is provided with a plurality of the above sets. Note that one reservoir 30 and one crystal generation unit 40 may be provided in one chamber.

  The reservoir 30 is a part that stores an external liquid having a high concentration of a precipitant such as polyethylene glycol, ammonium sulfate, or NaCl. Further, steps 31 and 32 are provided in the upper portion of the reservoir 30, and a gap 33 is provided between the reservoir 30 and the crystal generation unit 40.

  The crystal generation unit 40 is also called a crystal well or a drop well, and is a part where a protein placed in a strong magnetic field is crystallized. As shown in FIG. 1, a superconducting magnet Mg1 for generating a high magnetic force field is provided on the outer periphery of the crystallization plate PL1. Due to the high magnetic force field generated by the superconducting magnet Mg1, a pseudo microgravity environment is created in the central cavity of the superconducting magnet Mg1.

  The crystal generation unit 40 is a part on which a crystallization drop that is a mixed solution of a protein solution and a precipitant solution is placed, and is a part where crystallization of the protein proceeds. It is provided in the vicinity. That is, the crystal generation unit 40 is provided between the reservoir 30 and the inner periphery 11 of the ring body 10 (between the reservoir 30 and the central axis of the ring body 10). Note that the crystal generation unit 40 and the reservoir 30 may be provided at substantially the same distance from the central axis of the ring body 10.

  The initial condition of the crystallization drop is, for example, that the precipitant concentration in the crystal generation unit 40 is half the precipitant concentration in the reservoir 30.

  That is, the crystallization plate observation apparatus OB1 includes a crystal generation unit in a crystallization plate PL1 including a ring body 10 provided with a reservoir 30 that stores a protein solution and a crystal generation unit 40 that generates protein crystals. 40 is an observation device for observing the state of crystallization at 40.

  The ring body 10 is provided with a reflective surface M1. The reflecting surface M1 is a reflecting surface provided in the lower part of the crystal generation unit 40 in FIG. 2B in the ring body 10, and the horizontal light in FIG. (2) The light is reflected vertically upward, and the crystal generation unit 40 is irradiated. The upward light passes through and scatters through the crystal generation unit 40 and reaches the inclined mirror 51. In other words, the reflecting surface M1 is a reflecting means installed so that the light from the light source 55 irradiates the crystal generating unit 40, and the light transmitted through and scattered by the crystal generating unit 40 reaches the CCD camera 54. It is a reflecting means provided in a columnar space formed on the inner periphery of the body 10. The reflection surface M1 is a total reflection means or a specular reflection means. That is, the reflection surface M1 is a total reflection means or a specular reflection means that reflects the light of the light source provided in the columnar space 20 of the ring body 10 of the crystallization plate PL1 so as to reach the crystal generation unit 40.

  Furthermore, in FIG. 2 on the outer periphery of the ring body 10, a concave portion 71 is provided at the upper portion, and a convex portion 72 is provided at the lower portion in FIG. When two or more crystallization plates PL1 are overlapped with each other, the convex portion 72 of one crystallization plate PL1 and the concave portion 71 of the crystallization plate PL1 stacked thereon are fitted to each other. The concave portion 71 and the convex portion 72 will be described later with reference to FIG. 3, 4, 5, and 7, the concave portion 71 and the convex portion 72 are not shown.

  Next, the operation of the crystallization plate observation apparatus OB1 will be described.

  First, when a magnetic field is generated by the superconducting magnet Mg1 and a substance is placed in this magnetic force field, a magnetic force acts depending on the magnetic susceptibility of the substance. Generate gravity.

  The crystal generation unit 40 and the reservoir 30 of the crystallization plate PL1 are sealed, and water in the crystallization drop (mixed solution of protein solution and precipitant solution) provided in the crystal generation unit 40 evaporates, and the reservoir 30 It moves to the external liquid with a high concentration of precipitating agent. Accordingly, the protein solution concentration and the precipitating agent concentration in the crystallization drop provided in the crystal generation unit 40 are increased, and microcrystals serving as the nucleus of crystal growth are formed in the supersaturated protein solution. If the precipitant concentration is in an appropriate range, protein crystals are produced. In Example 1, since protein crystallization is performed in a pseudo microgravity state, even if a protein solution concentration gradient occurs during protein crystallization, natural convection does not occur in the protein solution. Crystal quality does not deteriorate.

  When observing the crystallization plate PL1, first, the crystallization plate PL1 is placed in a strong magnetic field, and the observation system 50 is installed in the columnar space 20 of the crystallization plate PL1. The light from the light source 55 is reflected by the reflecting surface M 1, transmitted and scattered by the crystal generation unit 40, reflected by the inclined mirror 51, becomes parallel light by the objective lens 52, and is reflected on the light receiving surface of the CCD camera 54 by the tube lens 53. Form an image. In this case, since the crystallization plate PL1 is placed in a strong magnetic field, the protein is crystallized under microgravity, and the crystallization process can be observed.

  That is, since the observation system (microscope optical system) 50 is disposed at the axial portion of the crystallization plate PL1 having a ring structure, in-situ observation in an ultra-strong magnetic field environment is possible. Since the illumination light from the light source 55 is totally reflected or specularly reflected in the crystallization plate PL1, the crystal generation unit (drop well) 40 is illuminated from the transmission direction or from the side, and the contrast is improved. Can do.

  Under pseudo microgravity, the movement of molecules occurs by diffusion, so crystal growth is slow, but impurities are difficult to be incorporated. Moreover, since the collision between proteins is difficult to occur under a pseudo microgravity state, the number of generated crystal nuclei is small, and the generated crystal nuclei are likely to be large. Moreover, under the pseudo microgravity state, as described above, since the supply of protein is slow, the protein attached to the crystal surface in the wrong orientation is easily reoriented, and there is little disorder in the molecular arrangement of the protein. That is, according to the first embodiment, it is possible to prevent quality deterioration due to exposure to the gravitational field.

  Protein crystals are precipitated by removing water from the supersaturated protein solution by vapor diffusion or the like. X-rays are diffracted by the regularly arranged protein molecule layers in the crystal and are observed as diffraction spots reflecting the properties of the crystal. In other words, when impurities are incorporated into the solution or the molecular arrangement is disturbed and the crystal becomes nonuniform, the X-ray diffraction ability is reduced and only an ambiguous three-dimensional structure can be obtained. Since the molecular arrangement is not disturbed, the crystal quality can be improved, and thus precise structural information of the protein can be obtained.

  According to Example 1, when a protein is crystallized in a crystallization plate in a strong magnetic field, an observation device can be inserted into the columnar space of the ring body, thereby removing the crystallization plate from the strong magnetic field. Protein crystallization can be observed, that is, the process of protein crystallization can be observed.

  Further, in the conventional example, in order to take out the crystallization plate, the magnetic force acting on the solution is spatially changed, and the work of taking out and returning the crystallization plate causes a flow in the solution. Growth is disturbed. However, according to Example 1, since it is not necessary to take out the crystallization plate in the crystallization process, there is no work of taking out or returning the crystallization plate, and the flow in the solution by moving the crystallization plate is reduced. It does not occur and crystal growth is not disturbed.

  In addition, according to the first embodiment, when the pseudo microgravity state is generated by the magnetic force, it is possible to obtain abundant experimental opportunities, that is, the experimental cycle is speeded up and can be observed when necessary. In addition, the experiment conditions can be customized, that is, the crystallization temperature, the experiment period, the crystallization method, and the shape of the crystallization plate can be arbitrarily selected. In addition, the influence of gravity can be examined.

  If a light source 56 that irradiates light from a direction different from that of the light source 55 is provided, the amount of light that the light from the light source 56 reaches the crystal generating unit 40 and its periphery, and is transmitted and scattered through the crystal generating unit 40. Will increase.

  Furthermore, according to Example 1, in the drug discovery field, the three-dimensional structure of the target protein can be determined accurately with a resolution of one base, and the usefulness thereof is dramatically improved. The three-dimensional structure of a disease-related protein can be determined with high accuracy, drug design based on the three-dimensional structure is possible, and the drug discovery process can be greatly accelerated. In addition, modification of enzyme function (stability, reaction specificity, improvement of reaction speed, etc.) based on the three-dimensional structure of useful proteins enables drug discovery of high-performance proteins, which is low cost and environmentally friendly. On the other hand, it is possible to construct an industrial process with low load, high efficiency, and space saving.

  In the first embodiment, the mirror coating applied to the ring body 10, and means for causing total reflection by the transmission member having a large refractive index and the transmission member having a small refractive index are used as the reflection surface M1. You may do it.

  In the above embodiment, if the columnar space 20 has a margin, a plurality of tilted mirrors 51 may be provided. By doing so, it is not necessary to rotate the observation system 50 in the circumferential direction.

  Further, in the first embodiment, the steps 31 and 32 are provided, and the area of the reservoir 30 is increased by the steps 31 and 32, and the increase of the aqueous solution can be suppressed by the increased area in the pseudo microgravity state. .

  FIG. 3 is a block diagram showing a crystallization plate PL2 that is Embodiment 2 of the present invention.

  FIG. 3 (1) is a plan view showing the crystallization plate PL2, and FIG. 3 (2) is a view showing a III-III cross section in FIG. 3 (1).

  The crystallization plate PL2 is an embodiment in which a crystal generation unit 41 is provided instead of the crystal generation unit 40 in the crystallization plate PL1. The crystal generation unit 41 is an example in which the step on the inner periphery 11 side of the crystal generation unit 41 (step 40s shown in FIG. 2 (2)) is eliminated.

  If there is a step 40 s (see FIG. 2 (2)) on the inner peripheral side of the crystal generation unit 40, a non-flat region is formed on the liquid crystal side surface (inner peripheral 11 side) of the crystallization solution (protein + precipitation agent). The In particular, when a crystal is formed in the vicinity of the interface with air in a droplet, there is a possibility that observation by an optical system may be hindered. Therefore, as shown in FIG. 3B, only the inner circumference 11 side is flattened in the crystal generation unit 40, that is, the step 40s is removed.

  FIG. 4 is a block diagram showing a crystallization plate PL3 that is Embodiment 3 of the present invention.

  4 (1) is a plan view showing the crystallization plate PL3, and FIG. 4 (2) is a view showing the IV-IV cross section and the observation system 50 in FIG. 4 (1).

  The crystallization plate PL3 is an embodiment in which a reflection surface M2 is provided instead of the reflection surface M1 in the crystallization plate PL2. The reflection surface M <b> 2 is a mirror that reflects the light, which is transmitted from and scattered by the crystal generation unit 41, from the light source 55 to the inclined mirror 51. Then, the two crystallization plates PL2 are overlapped, and in FIG. 4B, the light transmitted and scattered through the crystal generation part 41 of the lower crystallization plate PL2 is reflected on the upper crystallization plate PL2. The light is reflected by the surface M <b> 2 and travels toward the tilted mirror 51.

  Note that the crystallization plate PL3 shown in FIG. 4 (2) is a plate that crystallizes proteins by the sitting drop method.

  FIG. 4 (3) shows a crystallization plate PL3a, which is a plate for crystallizing proteins by the hanging drop method.

  The reflection surface M2 provided in a certain crystallization plate PL3a (in this case, the crystallization plate provided above in the drawing of the two crystallization plates PL3a shown in FIG. 4 (3)) is as described above. This is means for reflecting the light from the crystal generation part 41 of the crystallization plate PL3a set under the crystallization plate PL3a to the columnar space 20 of the certain crystallization plate PL3a.

  Since the crystallization plate PL3a can be observed from the top rather than observing from the side of the drop with an optical system, it is important that the drop well (crystal generation unit 40) has no problem even if it is circular. It is. It is also significant that the drop well has a circular shape when the crystal is taken out for an X-ray diffraction experiment.

  In addition, the point which can observe the drop in crystallization plate PL3a from the top is the same also in crystallization plate PL3. That is, the reflection surface M2 is provided in order to reflect the light from the crystal generation part 41 of the crystallization plate PL3 set below to the columnar space 20 of the crystallization plate PL3 set above. .

  In FIG. 4 (3), the observation system 50 is shown as a substitute for an eye mark.

  That is, the reflection surface M2 in the crystallization plates PL3 and PL3a is a total reflection means for allowing light from a light source provided in the columnar space of the ring body of the crystallization plate to reach the crystal generation unit and totally reflect light from the crystal generation unit. Or a mirror reflection means for specularly reflecting light from the crystal generation unit, which is a mirror that reflects light from the crystal generation unit provided in one crystallization plate to the observation system, and is another crystallization plate It is the mirror provided in.

  In order to cope with the hanging drop method by the specular reflection means by the reflection surface M2, the seal (cover) for sealing the crystallization plate PL3a may be changed. In other words, if the seal (cover) that is hermetically sealed is changed by cutting out and using a material seal suitable for the hanging drop method (commercially available seal) in a shape suitable for the crystallization plate group of the embodiment, A hanging drop method can be realized.

  The crystallization plate PL3a has a ring-shaped protrusion 42b on the adhesion surface side (the lower side in FIG. 4 (3)) of the sealing seal SL. The protrusion 42b has a rectangular structure drawn on the left and right of the crystallization drop 42a in FIG. 4 (3), and FIG. 4 (3) is a cross-sectional view, so the protrusion 42b is drawn as two protrusions. It is actually a ring shape. The protrusion 42b is installed so as to surround the crystallization drop 42a. By providing the protrusion 42b, the crystallization drop 42a can be held at a position where it can be easily observed from the columnar space 20 in the hanging drop method. FIG. 4 (4) is an enlarged view of the periphery of the protrusion 42b.

  That is, the crystallization plate PL3a has a ring-shaped protrusion 42b formed so as to surround the crystallization drop 42a provided on the adhesion surface side of the sealing seal SL in the hanging drop method.

  The ring-shaped protrusion 42b is effective not only when crystallizing protein or the like in a pseudo microgravity state but also when crystallizing protein or the like in a state other than the pseudomicrogravity state, for example, in a normal gravity state. is there.

  FIG. 5 is a block diagram showing a crystallization plate PL4 that is Embodiment 4 of the present invention.

  FIG. 5 (1) is a plan view showing the crystallization plate PL4, and FIG. 5 (2) is a view showing the VV cross section and the observation system 50 in FIG. 5 (1).

  The crystallization plate PL4 is a combination of the reservoir 30 and the crystal generation unit 40 in the crystallization plate PL1, and a plurality of such sets are provided, and between one set and the other set of the plurality of sets. This is an embodiment in which a gas flow path 60 is provided.

  When at least one crystallization plate PL4 is disposed in the magnet bore of the superconducting magnet Mg1 (the central cavity portion of the magnet Mg1), air or the like can flow through the gas channel 60 in the magnet bore. The temperature of the crystallization plate PL4 can be adjusted by adjusting the temperature of the air. That is, when the temperature is not controlled, liquid helium or the like is provided in the superconducting magnet Mg1 and the superconducting magnet Mg1 is cooled, so that the crystallization plate is slightly cooled. When the environmental temperature is lower than the set temperature, the appropriate temperature cannot be maintained. In addition, when the environmental temperature of the crystallization plate becomes a temperature other than the set temperature due to other factors, the appropriate temperature cannot be maintained.

  However, according to the crystallization plate PL4, the crystallization plate PL4 can be maintained at an appropriate temperature by controlling the temperature of the gas flowing through the gas flow path 60.

  In addition, the position of the gas flow path 60 in the crystallization plate is arbitrary, and the number of the gas flow paths 60 is also arbitrary.

  In the above embodiment, the temperature can actually be adjusted without the gas channel 60, but it is expected that the temperature can be more reliably controlled by providing the gas channel 60.

  FIG. 6 is a diagram showing a fitting structure in the crystallization plate PL2.

  6A is a diagram showing a VI-VI cross section in the crystallization plate PL2 shown in FIG. 3A, and FIG. 6B is a perspective view showing a part of the crystallization plate PL2.

  The fitting structure in the crystallization plate PL2 includes a recess 71 provided in a part of the upper surface of the crystallization plate PL2 in FIG. 6 (1) and a lower surface of the crystallization plate PL2 in FIG. 6 (1). It is comprised by the convex part 72 provided in the part. When the two crystallization plates PL2 are overlapped, the concave portion 71 of the lower crystallization plate PL2 and the convex portion 72 of the upper crystallization plate PL2 are fitted, and the upper surface of the lower crystallization plate PL2 and the upper The lower surface of the crystallization plate PL2 is in close contact. When the two crystallization plates PL2 are overlapped and the concave portion 71 of the lower crystallization plate PL2 and the convex portion 72 of the upper crystallization plate PL2 are fitted, the reservoir 30 of the lower crystallization plate PL2 and the upper portion The reservoir 30 of the crystallization plate PL2 coincides with the circumferential direction of the crystallization plate PL2, and the crystal generation unit 40 of the lower crystallization plate PL2 and the crystal generation unit 40 of the upper crystallization plate PL2 are A concave portion 71 and a convex portion 72 are provided so as to coincide with each other in the circumferential direction of the crystallization plate PL2.

  That is, the concave portion 71 and the convex portion 72 have a fitting structure provided on the upper and lower surfaces of the crystallization plate, and are positioning means provided in the circumferential direction of the crystallization plate.

  As described above, when the upper and lower surfaces of the crystallization plate have a fitting structure, when a plurality of crystallization plates are stacked, the distance between the upper and lower crystallization plates can be maintained uniformly. In addition, as described above, the top and bottom surfaces of the crystallization plate have a fitting structure (positioning means are provided in the circumferential direction of the crystallization plate), so that the position of each crystal generation unit can be matched in the circumferential direction. Thereby, the coordinate information in the circumferential direction in the crystallization plate can be stored.

  Coordinate information in the circumferential direction is completely matched between the stacked plates by providing irregularities at 11 places except for one place among the positions obtained by dividing the outer periphery 12 of the ring body 10 at equal intervals. That is, when the crystallization plates are stacked, the first chamber of the upper crystallization plate is always arranged above the first chamber of the lower crystallization plate. That is, by arranging the unevenness (fitting structure) asymmetrically in at least one of the plurality of locations on the outer periphery 12 of the crystallization plate, the coordinate information in the circumferential direction completely matches between the stacked plates.

  Contrary to the above, the convex portion 72 may be provided on the upper portion of the crystallization plate PL2, and the concave portion 71 may be provided on the lower portion of the crystallization plate PL2. In the embodiment shown in FIG. 6, the cross section of the concave portion 71 and the convex portion 72 is a quadrangle. However, other shapes such as a triangle and a semicircle may be used instead of the quadrangle.

  In other words, the fitting means includes a first fitting means provided on one predetermined surface of the one crystallization plate and a second fitting provided on the other surface of the one crystallization plate. Means for fitting the first fitting means and the second fitting means to each other.

  Furthermore, a fitting structure in the crystallization plate PL2 may be provided in the crystallization plates PL1, PL3, and PL4.

  In the first to fourth embodiments, a surface light source such as an EL may be provided on the outer periphery 12 of the ring body 10. When the surface light source is provided on the outer periphery 12 of the ring body 10, the light source 55 may not be provided in the columnar space 20.

  If the crystal generating part is arranged on the inner peripheral side of the ring body, the resolution and contrast are considerably improved as compared with the case where it is arranged on the outer peripheral side.

  FIG. 7 is a block diagram showing a crystallization plate PL5 that is Embodiment 6 of the present invention.

  The crystallization plate PL5 is an embodiment in which the arrangement of the reservoir 30 and the crystal generation unit 40 is changed in the crystallization plate PL1. The crystallization plate PL5 is provided with a surface light source 57 on the outer periphery of the ring body 10.

  When the observation system 50 is provided in the columnar space 20 of the crystallization plate PL5, light from the surface light source 57 is transmitted and scattered by the crystal generation unit 40, and this light is reflected by the inclined mirror 51 and sent to the CCD camera 54. The CCD camera 54 converts the image of the crystallization process in the crystal generation unit 40 into an electrical signal and outputs it. This makes it possible to reliably observe the process of crystallizing the crystallization plate PL5 without taking it out of the strong magnetic field.

  In Example 6, the crystal generation unit 40 is provided on the outer periphery 12. Even in this case, the formation process is observed if the lower part of the crystal generation unit 40 is higher than the liquid level of the solution in the reservoir 30. It is possible to observe the optical system.

  In each of the embodiments described above, the ring body 10 is a circular ring body having an outer diameter of a circle, but may be a square ring body having an outer shape of a ring shape such as a quadrangle, a pentagon, or a hexagon.

  That is, in each of the above embodiments, a crystallization plate such as the crystallization plate PL1 is placed in a strong magnetic field, and the observation system 50 is installed in the columnar space 20 of the crystallization plate. Light from the surface light source passes through the crystal generation unit 40, is reflected by the inclined mirror 51, and forms an image on the light receiving surface of the CCD camera 54 by the objective lens 52. In this case, since the crystallization plate is placed in a strong magnetic field, it is possible to crystallize under microgravity and observe this crystallization.

In this way, when protein is crystallized in a crystallization plate in a strong magnetic field, light such as EL is transmitted through the crystal generation unit 40 and an image illuminated by this transmitted light is seen, so the contrast of the image Therefore, it is possible to reliably observe the process of crystallization without removing the crystallization plate PL1 and the like from the strong magnetic field.

  Instead of the surface light source 57, a plurality of point light sources may be provided.

  Further, the light source 55 in the columnar space 20 and the surface light source (or a plurality of point light sources) on the outer periphery 12 of the ring body 10 may be used in combination. If the light source 55 and a surface light source on the outer periphery 12 are used in combination, the contrast can be further improved.

  Also in the crystallization plate PL5, the gas flow path 60 shown in FIG. 5, the fitting structure shown in FIG. 6, and the positioning means shown in FIG. 6 may be provided.

  FIG. 8 is a diagram showing an observation system 50 in the crystallization plate observation apparatus which is Embodiment 7 of the present invention.

  The observation system 50 includes an inclined mirror 51, an objective lens 52, a tube lens 53, and a CCD camera 54 as shown in FIG. In addition to the above, the observation system 50 is provided with a push load 56 and an outer sleeve 57.

  The push load 56 is composed of a plurality of thin threads and moves only the objective lens 52 in the vertical direction (the axial direction of the columnar space 20 of the crystallization plate), and moves only the objective lens 52 up and down. To adjust the focus. That is, a plurality of thin holes are provided in the tube lens 53, the thin thread is inserted into these holes, and the objective lens 52 is fixed to the lower end of the thin thread in FIG. 8 (1). In FIG. 8 (1), the upper end of the thin thread is connected to means (not shown) for adjusting the position of the objective lens 52.

  The outer sleeve 57 is a tube for fixing the CCD camera 54, the tube lens 53, the objective lens 52, and the tilting mirror 51, and performs positioning of the Y axis and θ axis of the observation system 50. The Y axis is the axial direction of the crystallization plate (the axial direction of the columnar space 20), and the θ axis is the circumferential direction of the crystallization plate. The Y-axis is positioned by moving the outer sleeve 57 up and down (moving in the axial direction of the crystallization plate), and the θ-axis is positioned by rotating the outer sleeve 57. In addition, between the objective lens 52 and the tube lens 53 is parallel light.

  Next, the operation of the observation system 50 will be described.

  First, control of the Y-axis coordinates will be described. When controlling the Y-axis coordinates, the CCD camera 54, the tube lens 53, the objective lens 52, and the tilt mirror 51 are all moved up and down simultaneously. To move the CCD camera 54, the tube lens 53, the objective lens 52, and the tilting mirror 51 all up and down simultaneously, the outer sleeve 57 is moved up and down. In this case, the vertical movement stroke is, for example, 108 mm, and the step movement amount is, for example, 2 μm.

  FIG. 9 is a diagram illustrating an example of positioning the Y axis when the observation point is different in the Y axis direction in the observation system 50. In this example, not all of the CCD camera 54, the tube lens 53, the objective lens 52, and the tilting mirror 51 are moved up and down at the same time, but only the objective lens 52 and the tilting mirror 51 are moved up and down. In this case, the distance between the objective lens 52 and the inclined mirror 51 is fixed.

  The magnification m of the infinity optical system is determined by m = f1 / f2, as shown in FIG. 9 (1). F1 is the focal length of the objective lens 52, and f2 is the focal length of the tube lens 53. In the infinity optical system, the light beam that has passed through the objective lens 52 from the crystal (observation point) of the crystal generation unit 41 does not form an image on the objective lens 52 but forms a tube lens 53 (image formation) as an infinite parallel light beam. Lens) and an image is formed by the tube lens 53. That is, in the infinity optical system, the distance between the objective lens 52 and the tube lens 53 can be freely set.

  FIG. 9 (2) is a diagram illustrating a case where the position of the observation point on the Y axis is higher in FIG. 1 than in the case illustrated in FIG. 9 (1). FIG. 9 (3) is a diagram showing a case where the Y-axis position of the observation point is lower in FIG. 1 than in the case shown in FIG. 9 (1).

  Since the focal lengths f1 and f2 are the same in both the case shown in FIG. 9A and the cases shown in FIGS. 9B and 9C, the magnification m = f1 / f2 of the infinite optical system is as shown in FIG. The cases (1), (2), and (3) are the same as each other. That is, when the Y-axis positioning is performed by simultaneously moving the objective lens 52 and the tilt mirror 51, the magnification m is constant regardless of the position of the observation point on the Y-axis.

  Next, θ control will be described. For θ control, the CCD camera 54, the tube lens 53, the objective lens 52, and the tilting mirror 51 are all rotated simultaneously to determine the θ coordinate of the observation point. To rotate all of the CCD camera 54, the tube lens 53, the objective lens 52, and the tilting mirror 51 simultaneously, the outer sleeve 57 is rotated. In this case, the rotation amount is, for example, ± 185 degrees, and the step rotation amount is, for example, 0.045 degrees.

  Next, focus adjustment will be described. In order to adjust the focus, the Z-axis coordinate of the observation point is determined by moving only the objective lens 52 up and down without moving the CCD camera 54, the tube lens 53, and the tilting mirror 51. In order to move only the objective lens 52 up and down, the push load 56 is moved up and down. The Z-axis coordinate is a coordinate in the radial direction from the center of the columnar space 20 of the crystallization plate. When adjusting the focus, the movement stroke of the objective lens 52 is, for example, 14 mm, and the step movement amount is, for example, 2 μm.

  FIG. 10 is a diagram illustrating an example of adjusting the focus by moving the objective lens 52 while fixing the position of the tilting mirror 51 when the observation point is different in the Z-axis direction in the observation system 50.

  The magnification m of the infinity optical system is determined by m = f1 / f2, as shown in FIG.

  FIG. 10 (2) is a diagram showing a case where the observation point is closer than the case shown in FIG. 10 (1) (when the observation point is close to the central axis of the crystallization plate). FIG. 10 (3) is a diagram showing a case where the observation point is farther than the case shown in FIG. 10 (1) (when the observation point is far from the central axis of the crystallization plate).

  Since the focal lengths f1 and f2 are the same in both the case shown in FIG. 10A and the cases shown in FIGS. 10B and 10C, the magnification m = f1 / f2 of the infinity optical system is The cases (1), (2), and (3) are the same as each other. That is, if the position of the tilt mirror 51 is fixed and the objective lens 52 is moved, the focus can be adjusted (Z-axis positioning). In this case, the magnification m is constant regardless of the position of the observation point on the Z-axis. is there. That is, when adjusting the focus, only the objective lens 52 is moved up and down, so that the distance between the objective lens 52 and the tube lens 53 changes, but the space between the objective lens 52 and the tube lens 53 is a space where parallel light exists. The distance in this space is a distance that does not affect the magnification m = f1 / f2 of the optical system. Therefore, even if the position of the tilting mirror 51 is fixed and only the objective lens 52 is moved up and down, the magnification m of the optical system does not change.

  That is, the light in the space between the objective lens 52 and the tube lens 53 is parallel light, and the magnification m does not change.

  The reflecting means may be located in the columnar space, and the objective lens of the imaging optical system may be a finite distance optical system in the columnar space formed on the inner periphery of the ring body. The finite optical system is an optical system in which a light beam that has passed through the objective lens 52 from the crystal (observation point) of the crystal generation unit 41 forms an image with the objective lens 52 alone.

  By producing protein crystals according to each of the above examples, it is expected that crystal structures such as proteins useful for food industry use, disease-related proteins / membrane proteins, and proteins useful for environmental industry use can be obtained quickly. The Alternatively, it is expected that the time spent for crystallization, which is a bottleneck for X-ray crystal structure analysis, can be reduced by generating protein crystals according to each of the above embodiments. Furthermore, the above embodiments are expected to contribute to the general field of protein crystallization and crystal structure analysis.

  Each of the above embodiments is a protein crystallization plate, but the object to be crystallized is not limited to protein. In other words, organic substances other than proteins may be crystallized using the above examples, and inorganic substances may be crystallized using the above examples.

OB1 ... Crystallization plate observation device,
PL1, PL2, PL3, PL3a, PL4, PL5 ... crystallization plate,
10 ... Ring body,
20 ... columnar space,
30 ... reservoir,
40 ... crystal generation part,
50 ... Observation system,
51 ... Reflector,
55. Light source,
M1, M2 ... reflective surfaces.

Claims (2)

A ring body having a columnar space, a preserving agent necessary for protein crystallization, a reservoir provided in the ring body, and a site where protein crystals are generated, which is provided in the ring body. A crystallization plate observation device for observing a crystal generated in the crystal generation unit by arranging a crystallization plate having a crystal generation unit in an ultra-strong magnetic field,
A light receiving element for converting an image of light from the crystal generation unit into an electrical signal;
A reflecting means for reflecting light from the crystal generating portion toward the light receiving element, the reflecting means provided in a columnar space formed on the inner periphery of the ring body;
Objective lens moving means for adjusting the focus by moving the objective lens while fixing other than the objective lens among the observation means;
And the reflecting means is located in the columnar space, and the objective lens of the imaging optical system is an infinite optical system in the columnar space formed on the inner periphery of the ring body. Plate observation device. A ring body having a columnar space, a preserving agent necessary for protein crystallization, a reservoir provided in the ring body, and a site where protein crystals are generated, which is provided in the ring body. A crystallization plate observation device for observing a crystal generated in the crystal generation unit by arranging a crystallization plate having a crystal generation unit in an ultra-strong magnetic field,
A light receiving element for converting an image of light from the crystal generation unit into an electrical signal;
A reflecting means for reflecting light from the crystal generating portion toward the light receiving element, the reflecting means provided in a columnar space formed on the inner periphery of the ring body;
Objective lens moving means for adjusting the focus by moving the objective lens while fixing other than the objective lens among the observation means;
And the reflecting means is located in the columnar space, and the objective lens of the imaging optical system is a finite distance optical system in the columnar space formed on the inner periphery of the ring body. Plate observation device.

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