WO2005116608A1

WO2005116608A1 - Method of preparing specimen for estimation of crystal property and method of estimating crystal property

Info

Publication number: WO2005116608A1
Application number: PCT/JP2005/010288
Authority: WO
Inventors: Hiroshi Kitano; Hiroaki Adachi
Original assignee: Nikon Corporation; Sasaki, Takatomo; Mori, Yusuke; Takano, Kazufumi; Inoue, Tsuyoshi; Matsumura, Hiroyoshi
Priority date: 2004-05-31
Filing date: 2005-05-30
Publication date: 2005-12-08
Also published as: JP4317919B2; JPWO2005116608A1

Abstract

Referring to view (a), organic crystal (1) together with culture solution (3) used in growing thereof is ladled up by means of looped holder (2) and retained in the holder (2) by means of the surface tension of the culture solution (3). In this form, the organic crystal (1) and the culture solution are frozen, and within the range of X-ray irradiation, the culture solution (3) and holder (2) at portions lying in front of and behind the organic crystal are cut off by ultraviolet irradiation (see view (b)). Although in the figure the culture solution (3) and holder (2) at portions lying above the organic crystal (1) are also removed, these portions are not necessarily to be removed. As a result, in the region of X-ray transmission, any influence upon X-ray of the culture solution (3) and holder (2) lying in front of and behind the organic crystal can be avoided, so that noise reduction in X-ray diffractometry can be attained.

Description

Method for preparing crystal characteristic evaluation sample and characteristic evaluation method

Technical field

The present invention relates to a method for preparing a sample used for evaluating characteristics of an organic crystal, particularly a polymer crystal, and a method for evaluating characteristics of a crystal using a sample prepared by this method. book

Background art

In recent years, attention has been paid to research on organic materials having higher functions and higher performances than the properties of inorganic materials, and next-generation organic devices are being developed. To this end, the development of new organic materials is indispensable.In the fields of applied chemistry, the newly synthesized materials are crystallized, and the molecular structure is determined by X-ray crystal structure analysis to determine their functions. Infer. In addition, when converting a crystal into a device, it is necessary to accurately evaluate various physical characteristics such as crystal quality and the state of the crystal surface. On the other hand, research on the postgenome called the proteome is gaining momentum. Of particular interest is research that seeks to elucidate the three-dimensional structure of proteins, which is called structural genomics. Analysis of protein structure and function is an important research field in the life sciences, and is expected to lead directly to the treatment of diseases and drug discovery, and to the elucidation of life phenomena. Is required. Thus, crystals of various organic materials are widely used to evaluate the properties of those materials.

For many organic materials, crystallization conditions and growth conditions for obtaining high-quality single crystals have not been established. Always difficult. For this reason, it is difficult to grow only an ideal single crystal, and in some cases, it may be polycrystalline or a crystal with defects may be obtained. ing. In addition, similar to the growth of inorganic crystals and organic low-molecular crystals, crystals having a shape that reflects the molecular structure and growth conditions are grown, and thus the crystal shape may be unsuitable for measurement.

For example, when performing X-ray crystal structure analysis, which is one of the evaluations of crystal properties, usually, a crystal in a growth vessel is taken out, held by a crystal holder, and placed on a stage of an X-ray diffraction apparatus. X-ray irradiation is performed. At that time, it is difficult to operate and maintain the crystal quality of the organic crystal.Therefore, the crystal held in the crystal holder is not an ideal single crystal suitable for measurement. It may be measured in the state. Specifically, there are cases where a plurality of single crystals are included in a crystal holder, when a crystal in a crystal holder has a defect, and when measurement is performed while spraying liquefied nitrogen or the like. If measurement is performed in such an environment, X-rays will penetrate through substances other than the object to be measured. The problem is that reflections are included in the measurement data.

Disclosure of the invention

The present invention has been made in order to solve such a problem, and in a sample used for evaluating the characteristics of an organic crystal, a substance other than the crystal whose characteristics are to be evaluated, which causes a decrease in measurement accuracy, is removed. An object of the present invention is to provide a method for preparing a characteristic evaluation sample so as not to affect the measurement, and a method for evaluating a crystal characteristic using the sample. A first invention for achieving the above object is a method for producing a sample used for evaluating characteristics of an organic crystal, wherein the sample is held near an organic crystal to be evaluated after the organic crystal is held by holding means. It is characterized by removing, modifying, or miniaturizing at least a part of the substances that are not evaluated by irradiating it with ultraviolet light.

In the present invention, of the sample including the crystal and the holding means, a substance other than the object to be evaluated which is present in the vicinity of the object to be evaluated and which is a factor of deteriorating the accuracy in the property evaluation is removed by irradiating ultraviolet light. , Reforming, or miniaturization. In the present specification and claims, the term "modified" means that the influence on the property evaluation is reduced by, for example, making the crystal amorphous. The term “substances not to be evaluated” refers to all substances including non-evaluable organic crystals, holding means, and deposits near the organic crystal to be evaluated. However, the surrounding gas does not belong to this category. The term “miniaturization” refers to miniaturization of the crystal to reduce its influence on the property evaluation. Here, “characteristic evaluation” is a concept including so-called well-known physical, chemical, and mechanical characteristic evaluation in general. As described above, when growing an organic crystal, it is difficult to grow an ideal single crystal.Therefore, when a single crystal is included in the crystal holder, or when the crystal in the crystal holder is In some cases, it contains defects. When the crystals are kept frozen by containing a crystal growth solution such as water such as proteins or organic salts, the frozen solution around the crystals is unnecessary for measurement. According to the present invention, the influence of substances other than the evaluation target on the crystal characteristic evaluation result among the samples including the crystal and the holding means is reduced, so that accurate characteristic evaluation can be performed. It is. Also, by exposing the crystal surface, the surface or surface layer can be accurately evaluated. In the present invention, ultraviolet light is used because, by using ultraviolet light having a large photon energy, a chemical bond can be easily broken, so that removal and modification can be easily performed. is there. In addition, shattering occurs when light irradiation causes abrasion, and the material around the light-irradiated part is mechanically broken by the impact. In this case, too, ultraviolet light having a large photon energy is used. Is preferable. A second invention for achieving the object is the first invention, wherein the characteristic evaluation of the organic crystal is a characteristic evaluation of the organic crystal using a diffraction phenomenon of radiation, and the ultraviolet light is At least a part of an irradiation area is a part where the radiation passes through the sample. In the present invention, since the influence of substances other than the above-described substances present in the portion through which the radiation used for the characteristic evaluation passes on the measurement results is reduced, the absorption of the irradiated radiation is suppressed, and Noise is reduced and accurate characteristic evaluation is possible. The radiation referred to in the present specification and claims includes X-rays, electron beams, neutron beams, and the like. Characteristic evaluations using radiation diffraction phenomena include crystal structure analysis and X-ray topography.

A third invention for achieving the above object is the second invention, wherein the characteristic evaluation of the organic crystal is a crystal structure analysis.

In the crystal structure analysis, substances other than the organic crystal to be evaluated, which are present in the portion through which the synchrotron light passes, basically cause a decrease in measurement accuracy. Structural analysis can be performed at. In addition, structural analysis may not be possible if the sample contains multiple organic crystals, but unnecessary by irradiating unnecessary crystals other than the crystal to be evaluated with ultraviolet light. The influence of a simple crystal It is possible to perform a desired structural analysis by excluding it.

A fourth invention for achieving the above object is the first invention, which is a characteristic evaluation of a surface or an internal state of the organic crystal using a probe that comes into contact with or comes close to the organic crystal, By irradiating light, the surface of the organic crystal is exposed. In the evaluation of characteristics of organic crystals, when a probe is brought into contact with or in close proximity to an object to be evaluated, accurate characteristic evaluation may not be performed unless the organic crystal to be evaluated is exposed. In the present invention, a substance other than the evaluation target existing in the vicinity of the evaluation target organic crystal is removed by irradiating ultraviolet light, so that a desired evaluation can be performed. Examples of such characteristics evaluation using a probe include a scanning probe microscope such as a scanning tunneling microscope and an atomic force microscope. The reason for using ultraviolet light is the same as the reason described in the description of the first invention.

A fifth invention for achieving the above object is the first invention, wherein the characteristic evaluation of the organic crystal is a characteristic evaluation of the organic crystal using electromagnetic wave absorption, interference, or scattering. At least a part of a region to be irradiated with ultraviolet light is a portion where the electromagnetic wave passes through the sample.

In the present invention, substances other than the evaluation target existing in the portion through which the electromagnetic waves used for characteristic evaluation pass are removed, modified, or miniaturized by irradiating with ultraviolet light, so that these substances are measured. The influence on the result can be reduced, and the characteristics of the crystal can be evaluated with high accuracy. Examples of such characteristics evaluation using electromagnetic waves include absorption spectrum measurement and photoluminescence measurement. The reason for using ultraviolet light is the same as the reason described in the description of the first invention. A sixth invention for achieving the above object is any one of the first invention to the ^fifth invention, wherein the ultraviolet light is an ultraviolet laser light.

By using an ultraviolet laser beam, high irradiation illuminance can be obtained, and removal, modification, or miniaturization can be easily performed. In addition, since the irradiation position can be controlled with high accuracy, it is possible to give an effect only to a target place. ,

A seventh invention for achieving the above object is the sixth invention, wherein the ultraviolet laser light is a short pulse ultraviolet laser light having a pulse width of 100 ns or less.

When removing a substance, as described above, the force S that removes by breaking the chemical bond using ultraviolet light, and in that case, when heat is generated, the organic crystal to be evaluated is damaged. May cause If a short pulse ultraviolet laser beam with a pulse width of 100 ns or less is used, heat generation can be suppressed, and substances can be removed without causing significant damage to surrounding parts. When the material is ground, the material around the irradiated part is mechanically pulverized by the impact generated by the abrasion. Therefore, it is desirable to use light that can locally give a large impact, and it is preferable to use ultraviolet light having a short pulse width and a high energy density.

An eighth invention for achieving the above object is any one of the first invention to the seventh invention, wherein the organic crystal comprises a resin, a protein, a saccharide, a lipid, an organic supramolecular complex and a nucleic acid. Among them, it is characterized by being at least one crystal.

The first to seventh inventions are particularly suitable for evaluating the physical properties of polymer crystals in which it is particularly difficult to grow a high-quality single crystal and the crystal is broken. This is a suitable method.

A ninth invention for achieving the above object is characterized in that, using the characteristic evaluation sample manufactured by the method for producing a crystal characteristic evaluation sample according to any one of the first to eighth inventions, This is a method for evaluating the characteristics of a crystal, which comprises evaluating the characteristics of the crystal.

In the present invention, the characteristics of the organic crystal can be evaluated while suppressing the influence of substances other than the evaluation object, and as a result, more accurate evaluation results can be obtained. Brief Description of Drawings

FIG. 1 is a diagram schematically showing an example of the first embodiment of the present invention. FIG. 2 is a diagram schematically showing an example of the second embodiment of the present invention. FIG. 3 is a diagram showing a result and a photograph of the first example of the present invention.

FIG. 4 is a diagram showing a result and a photograph of the second example of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a diagram schematically showing an example of the first embodiment of the present invention. As shown in (a), when performing the characteristic evaluation, the organic crystal 1 is scooped up by the loop-shaped holder 2 together with the culture solution 3 used for its growth, and the organic crystal 1 is held in the holder 2 by the surface tension of the culture solution 3. Is held. In this state, the organic crystal 1 and the culture solution are frozen, and the holder 2 and the culture solution 3 before and after the organic crystal are removed by irradiating ultraviolet light (b). In the figure, the holder 2 and the culture solution 3 above the organic crystal 1 are also removed, but this part does not necessarily need to be removed.

By doing this, the holder 2 before and after the organic crystal can be Since the influence of the liquid 3 is eliminated, adverse effects in the characteristic evaluation are reduced.

The method of cutting and removing is performed by irradiating with ultraviolet light and scanning along the line to be cut and cutting by ablation, or irradiating all the parts to be removed with ultraviolet light and removing by abrasion. U. At that time, as described above, it is preferable to use an ultraviolet laser beam as the ultraviolet light, and it is preferable to use a short pulse ultraviolet laser beam having a pulse width of 100 ns or less. In the embodiments and examples of the present invention, the culture solution 3 and the holder 2 correspond to the “holding means” in the claims and the disclosure of the invention.

FIG. 2 is a diagram schematically showing an example of the second embodiment of the present invention. As shown in (a), the organic crystal 1 is scooped by the loop-shaped holder 2 together with the culture solution 3 used for growing it, and is held in the holder 2 by the surface tension of the culture solution 3. You. At that time, the organic crystal 4 other than the object to be measured is simultaneously scooped and is in the evaluation area. Even if the characteristic evaluation is performed in such a state, the obtained result may be obtained by accumulating information from the organic crystal 1 and the organic crystal 4, so that accurate evaluation may not be performed. This is the same regardless of whether the organic crystal 4 is the same organic crystal as the organic crystal 1 to be measured, a different organic crystal, or a foreign substance.

In this state, the organic crystals 1 and 4 and the culture solution are frozen, and the organic crystals 4 other than the measurement target are removed by irradiating ultraviolet light (b). In this case, ultraviolet light is irradiated along the vertical cutting line, and the holder 2 and the culture solution 3 on the rear side of the organic crystal 1 are removed together with the organic crystal 4. On the other hand, the holder 2 and the culture solution 3 in front of the organic crystal 1 are left without being removed. In that sense, the first embodiment can provide more accurate evaluation results, but if the effects of the holder 2 and the culture solution 3 on the measurement are small, this is sufficient. It is.

In the second embodiment, the organic crystal 4 other than the object to be measured is removed by cutting the holder 2 and the culture solution 3, but by irradiating the organic crystal 4 with ultraviolet light, The organic crystal 4 may be modified or miniaturized so as not to affect the characteristic evaluation.

In particular, when the organic crystal 1 is a biopolymer crystal, a liquid containing glycerol, ethylene glycol, polyethylene glycolone, methylinopentane, paraffin oil, paraton oil, etc. These culture solutions can be removed by irradiating appropriate ultraviolet light. The culture solution 3 is not limited to the above substances, and the technique described in the present specification is applicable as long as it is a substance that absorbs ultraviolet light to be irradiated.

(Example 1)

The X-ray crystal structure analysis of a protein crystal, which is one of the organic crystals, was performed by applying the method for preparing a sample for property evaluation in the present invention. The grown protein crystals (egg white lysozyme crystals) are scooped together with a culture solution consisting of paraton oil in a holder, frozen at 150 ° C or less, and placed around the egg white lysozyme crystals. The material in the X-ray irradiation area was removed by UV laser irradiation, and the X-ray diffraction results before and after the removal were compared. As the light source of the irradiation device for removal, a short-pulse ultraviolet laser generated by the eighth harmonic of a fiber light source with a wavelength of 1547 mn was used. The output light had a wavelength of 193 nm and a pulse width of Ins. After passing through the mechanical shutter, ultraviolet light was reflected by two optical galvanometer mirrors, and the sample was focused and irradiated by a synthetic quartz lens. The light density on the irradiation surface was 50 mJ / cm ² , the spot diameter was 20 μιη, and the laser irradiation position was adjusted by rotating the galvanomirror. Changed.

The results of the X-ray crystal structure analysis are shown in Fig. 3 along with the photographs of the crystals. In FIG. ³ , what is described as "before" in the photograph indicates a state in which egg white lysozyme crystals are scooped and frozen by a holder. “After (l)” shows the state of removal around the egg white lysozyme crystal by ultraviolet laser irradiation. The one with “Aftei '(2)” is the one in which the culture solution around the egg white lysozyme crystal is almost removed and the crystal is processed into a spherical shape, and the one with “After (3)” is In addition, additional processing was performed on the crystal to reduce the remaining crystal parts.

Fig. 3 shows the results of X-ray diffraction performed in these "before", "After (2)", and "After (3)" states. In Fig. 3, the X-ray generator, detector, temperature, distance to the detector, aperture angle, and irradiation time are shown on the right side of the photograph.

X-ray diffraction results are shown at the bottom of the photo. The “before” state, the “After (2)” state, the “Space Group” in “After (3)”, Cell dimensions (lattice woman), Resolution limit (resolution), Total of observations (number of all reflections measured), No. of unique observation (number of independent reflections), Completeness (data integrity), Rmerge (Data accuracy), Estimated mosaicity (crystal mosaicity) and Iσ (diffraction intensity).

The fact that the lattice constant is not changed by the laser processing means that the crystal structure of the object to be measured is not changed by the processing, which is a desirable result. The smaller the resolution is, the better, but the fact that this value hardly changes by processing means that the crystal structure of the object to be measured does not change by processing, which is a desirable result. Similarly, the number of all reflections measured, the number of independent reflections, and the integrity of the data can be reduced by processing. No change means that the crystal structure of the object to be measured has not been changed by the processing, which is a desirable result.

On the other hand, the data accuracy and the mosaic value of the crystal are smaller in the “After (2)” state and “After (3)” than in the “before” state. These values mean that the smaller the value, the higher the reliability of the analysis result.It is clear that the removal of substances around the measurement object by processing resulted in highly reliable data. Represents.

In addition, the intensity of diffraction is reduced by laser processing, but this is because the size of the crystal to be analyzed is reduced by processing, which is a reasonable result.

(Example 2)

The X-ray crystal structure analysis of protein crystals was performed by applying the method for preparing a sample for property evaluation in the present invention. When the egg white lysozyme crystal, which is the object to be measured, is scooped by a holder and two egg white lysozyme crystals are brought into close contact with each other and are in the X-ray transmission region, the measurement object is irradiated with ultraviolet light. The measurement was carried out by removing the lysozyme crystals which were not used. The result is shown in Fig. 4 together with a photograph of the crystal.

The photograph in the upper center of FIG. 4 shows the state of the crystals scooped by the holder, and the upper left side schematically shows the state. Using the same ultraviolet light irradiation device as in Example 1, the entire holder was cut vertically to remove egg white lysozyme crystals not to be measured. The upper right photograph shows the state after removal, the lower left photograph shows the crystal state before removal, and the lower right photograph shows the crystal state after removal. As can be seen from the lower photo, some of the egg white lysozyme crystals to be measured were also removed during processing.

The data of the used X-ray generator etc. are shown on the right side of the photo, Indicates X-ray diffraction data. Twin is the data when two organic crystals exist before processing, and Detwin is the data when the organic crystal that is not the object to be measured is removed by processing.

In the case of Twin, diffraction analysis from two crystals resulted in a pattern that was accumulated, and structural analysis could not be performed.In contrast, in the case of Detwin, an X-ray diffraction pattern from one single crystal was obtained. Data analysis was carried out without any problems.

Claims

The scope of the claims

1. A method for preparing a sample used for evaluating the characteristics of an organic crystal, comprising: holding at least the organic crystal by a holding unit; and at least a part of a substance other than the evaluation target existing near the organic crystal to be evaluated. A method for producing a sample for evaluating the characteristics of a crystal, characterized in that the sample is removed, modified, or refined by irradiating it with ultraviolet light.

2. The characteristic evaluation of the organic crystal is the characteristic evaluation of the organic crystal using a diffraction phenomenon of radiation, and at least a part of the region irradiated with the ultraviolet light is a part where the radiation passes through the sample. The method for producing a crystal characteristic evaluation sample according to claim 1, wherein the method is characterized in that:

3. The method according to claim 2, wherein the evaluation of the characteristics of the organic crystal is a crystal structure analysis.

4. The characteristic evaluation of the organic crystal is a characteristic evaluation of a surface or an internal state of the organic crystal using a probe that is brought into contact with or in proximity to the organic crystal, and by irradiating the ultraviolet light, 2. The method according to claim 1, wherein the surface is exposed.

5. The characteristic evaluation of the organic crystal is the characteristic evaluation of the organic crystal using absorption, interference, or scattering of electromagnetic waves, and at least a part of the region to be irradiated with the ultraviolet light is such that the electromagnetic waves irradiate the sample. 2. The method for producing a crystal characteristic evaluation sample according to claim 1, wherein the sample is a portion that passes.

6. The method for producing a crystal characteristic evaluation sample according to claim 1, wherein the ultraviolet light is an ultraviolet laser light.

7. The crystal characteristic evaluation according to claim 6, wherein the ultraviolet laser light is a short pulse ultraviolet laser light having a pulse width of 100 ns or less. Method of preparing sample for use.

8. The characteristic of the crystal according to claim 1, wherein the organic crystal is at least one crystal of a resin, a protein, a saccharide, a lipid, an organic supramolecular complex, and a nucleic acid. Method for preparing evaluation sample.

9. A method for evaluating characteristics of an organic crystal, wherein the characteristics of a crystal are evaluated using a sample manufactured by the method for manufacturing a sample for evaluating characteristics of a crystal according to claim 1.