JP3001817B2 - X-ray excitation type fluorescence microscope - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an X-ray excitation type fluorescence microscope. More specifically, the present invention relates to a high-performance, new X-ray-excited fluorescence microscope capable of improving contrast and detecting a specific chemical group or the like of an observation sample or a trace amount of a chemical group. Things.

[0002]

2. Description of the Related Art In recent years, X-ray microscopes have been actively developed as an applied technology with the technical progress of X-ray light sources and X-ray optical systems. This X-ray microscope is characterized in that X-rays, which are its light source, cause little damage to a biological sample, and that the biological sample can be observed with high resolution and no staining while the biological sample remains wet. In particular, in a wavelength range of 42.7A to 23.6A called a “water window”, X-rays have the highest absorption by carbon or nitrogen and have a high transmittance to water molecules composed of oxygen and hydrogen. Therefore, a transmission image of a protein whose main constituent atom is carbon can be observed with good contrast even in water. "Water window" limit wavelengths 42.7A and 23.6A
Correspond to the K absorption edge of carbon and the K absorption edge of oxygen, respectively. The principle of capturing an X-ray absorption image is based on the inner shell ionization process of 1S electrons of carbon and nitrogen.

Conventionally, as an X-ray microscope using such an inner shell ionization process, for example, a grazing incidence optical system represented by a Walter type, a Fresnel zone plate using a diffraction effect, and two spherical mirrors are used. Microscope system using an imaging element such as a direct-incidence type Schwarzschild optical system with a vapor-deposited X-ray multilayer mirror, or direct recording of a sample image directly on an X-ray film or photoresist without using an imaging element 2. Description of the Related Art A microscope system using a close-up photographing method is known.

On the other hand, apart from this inner-shell ionization process, an X-ray microscope using an inner-shell excitation process to the molecular orbital of a biomolecule has been developed. FIGS. 1 (a) and 1 (b) are diagrams illustrating the concepts of an inner-shell ionization process and an inner-shell excitation process, respectively. As shown in FIG. 1 (a), the inner shell ionization process uses the X-rays in the wavelength range of 42.7A to 23.6A of the “water window” to generate 1S electrons of carbon or nitrogen in the cells of the biological sample. It ionizes.

[0005] In the inner-shell excitation process, as shown in Fig. 1 (b), 1S electrons of carbon or nitrogen in cells of a biological sample are converted into electrons by X-rays in a wavelength region of a "water window". Excitation to unoccupied molecular orbitals. The molecular orbital that plays an important role in this inner-shell excitation process is π *
Orbital anti-bonding molecular orbitals, usually empty orbitals. This π * orbit is often seen when the 2p orbit of carbon constituting a biomolecule is double-bonded to the orbit of another atom. For example, a benzene ring or a nitrogen base has a strong absorption line when the X-ray wavelength is about 43.5 A (light energy: 285 eV). The energy position of these absorption lines is on the longer wavelength side by about 1 A than 42.7 A, which is the longer wavelength limit of the “water window” region (5e in terms of photon energy).
V-low energy side). Such an inner-shell excitation process
This is a kind of resonance absorption process, and strong X-ray absorption can be expected. Furthermore, since the molecular orbital has an energy level inherent to the molecule, an absorption image of only a specific molecule can be obtained by selecting the resonance wavelength of the X-ray.

Therefore, the X-ray excitation microscope using the inner-shell excitation process is different from the X-ray microscope using the inner-shell ionization process.
It has high sensitivity and is very excellent as an X-ray microscope for observing a biological sample. In recent years, a fluorescent microscope using labeling by a labeler has been developed in order to further improve the contrast of an image obtained by an X-ray excitation microscope using such an inner-shell excitation process.

[0007] In this fluorescence microscope, a fluorescent dye molecule, which is a molecule having a fluorophore, that is, a labeler, stains or labels a specific chemical group or element of a living cell molecule, and a specific molecular orbital of the labeled molecule. Then, the core electrons are resonantly absorbed by X-rays to analyze the structure and composition of living cells. For example, N-Succinimidyl-4-nitro
phenylacetate or 5- (4-Dimmethylaminophenyl) -2,4-pen
Tadienal emits strong ultraviolet fluorescence by binding to the amino group (N-terminal) of the protein. Also, O- (4-Nitrobenzyl) -N, N'-d
iisopropylisourea is the carbonyl group of protein (C-terminal)
And emits strong ultraviolet fluorescence. Furthermore, N, N, N ', N'-
Tetrakis (2-pyridylemethyl) ethylenediamine labels the signaling molecule Ca2 + that plays an important role in cells. Also, 4-Fluroro-7-sulfobenzofurazan, am
Monium salt binds to, for example, the SH group of cysteine, which is a kind of amino acid, and has a strong intensity of 515 n at 385 nm excitation.
m.

[0008] As described above, each labeler for labeling a specific chemical group or element has a feature of emitting strong fluorescence, and this feature is attributed to the molecular orbital structure of the labeler. That is, since a labeler has a chemical group having a double bond, π electrons exist in a molecular orbital usually called a π orbital. These π electrons are easily excited to higher unoccupied π orbitals or antibonding π * orbitals by ultraviolet or visible light, and emit strong fluorescence when deexcited from this excited state to the ground state. is there.

Therefore, the fluorescence microscope using the labeling by such a labeler can improve the contrast of the absorption image by the strong fluorescence generated as described above, and can analyze the structure and composition of living cells more. It can be performed with high accuracy. By the way, the molecular orbital of π orbital or π * orbit is formed by the linear bond of 2p orbital of atoms constituting a molecule.

[0010]

(Equation 1)

Is represented by Here, x _i (r) is a wave function of the 2p orbit of the i-th molecular constituent atom, and Ci is a normalization constant. In general, the 2p orbit of an atom is 1
Strong interaction with s orbital electrons. That is, the electrons in the 1s orbital have a high transition probability to the 2p orbital when photoexcitation is performed. FIG. 2 shows the relationship between the photon energy and the absorption cross section when the 1s electron transitions to each p orbital in the carbon atom and when the 1s electron is ionized. As is apparent from FIG. 2, the carbon atom is about 15 when the 1s electron transitions to the 2p orbital.
It has an absorption cross section of about 0 Mb (150 × 10 ⁻¹⁸ cm ² ), and has 100 times or more strong absorption as compared with the absorption cross section in the ionization of 1s electron by the excitation wavelength of the “water window”.

FIG. 3 shows the relationship between the photon energy and the absorption cross section when the 1s electron transitions to the π * orbit and when the 1s electron is ionized in a 6-membered ring molecule such as a benzene molecule. It is. As is clear from FIG. 3, when the 1s electron transits to the π * orbit, the 6-membered ring molecule has an absorption cross section of about 24 Mb or more at a photon energy of about 285 eV and excites the “water window”. It can be seen that it has an absorption cross section about 6 times larger than the absorption cross section in the ionization of 1s electron depending on the wavelength, and has very strong absorption like the carbon atom shown in FIG.

Also, molecules having a double bond other than the six-membered ring molecule, like the carbon atoms and six-membered ring molecules described above, have a region where the photon energy is smaller than the “water window”, ie, the “water window”. Has a very strong resonance absorption due to the inner-shell excitation process. Therefore, the labeler that performs labeling also functions as an absorption labeler for soft X-rays having a wavelength that causes an inner-shell excitation process because of abundant double bonds. That is, if a specific chemical group such as an amino group in a biomolecule is labeled and a transmission image of a biological sample is taken by using X-rays of a wavelength that resonates to cause an inner-shell excitation process, the specific chemical group can be obtained. A distribution image of only groups can be obtained. Furthermore, since the absorption by resonance is very strong, a trace amount of a chemical group can be detected.

Therefore, the X-ray microscope using the labeling by such a labeler can not only improve the image quality but also obtain a distribution image of only specific chemical groups. Even trace amounts of chemical groups can be detected. By the way, in order to realize an X-ray microscope using this labeling, it is necessary to satisfy the following two necessary conditions.

1) A molecule containing a double bond is used as a labeler. 2) Use X-rays having a wavelength width or a narrower photon energy width than the resonance line that causes the inner-shell excitation process of the labeler. In addition, when observing a biological sample, as additional conditions for improving its function, 3) X-rays are not absorbed by water molecules in order to enable imaging of a biological sample in a wet state. Are also mentioned.

Hereinafter, these conditions will be described. Regarding condition 1) The labeler is usually an organic molecule containing H, C, N, and O as main components and containing some S, Br, Pb, and the like. Therefore, the 1s electrons of C, N and O are converted to the labeler's π orbit or π
* It is preferable to use an absorption line when light is excited in orbit. The photon energy and wavelength of the resonance absorption line in this case are as follows, for example.

C: 1 s → π * 280 eV to 300 eV (44 A to 41 A) N: 1 s → π * 390 eV to 410 eV (32 A to 30 A) O: 1 s → π * 520 eV to 550 eV (24 A to 22 A) Absorption peaks due to various inner-shell excitation processes including 1s → π * have been observed.

In the case of other heavy elements such as S, Br, Pb, etc., not only 1s → π *, but also 2p, 2s, 3s,
3p, 3p. . . . Absorption peaks from various inner orbitals are observed at 2000 eV or less. Therefore, for example, if X-rays in the above wavelength band are used,
A high-performance X-ray microscope can be realized.

Condition 2) The strongest absorption peak associated with the 1s → π * transition has a line width of about 1 eV. Therefore, it is necessary to use an X-ray having a bandwidth of at least a photon energy width of 1 eV or less. When a transmission image is captured with a bandwidth larger than this, the contrast is significantly reduced because wavelengths not involved in absorption are mixed. Therefore, it is necessary to use an optical element having a wavelength dispersibility having a resolution equal to or greater than the above-mentioned bandwidth and a monochromatic light source.

Regarding Condition 3) In order to image a biological sample in a wet state, it is necessary that X-rays are not absorbed by water molecules. That is, it is necessary to use a soft X-ray having a wavelength longer than the K absorption edge of an oxygen atom in a water molecule, that is, a wavelength longer than 23.6A. By satisfying such conditions, a high-performance X-ray microscope can be realized.

Hereinafter, O- (4-Nitrobenzy) is used as a labeler.
l) An example of detection of tobacco mosaic virus protein by X-ray microscopy using hydroxylamine is shown and described.
The shape of the tobacco mosaic virus is a rod of 16 × 300 nm, and its protein is composed of a small population of 2130 proteins composed of 158 amino acid molecules. Since one peptide bond having a ketone group or an aldehyde group exists for one amino acid molecule, tobacco mosaic virus has 158 × 2130 ketone groups or aldehyde groups.

O- (4-Nitrobenzyl) hydroxylamine has a benzene ring and has a photon energy of around 285 eV and carbon 1
It has a strong X-ray absorption peak accompanying the s → π * transition. this
Calculating the strongest X-ray absorption accompanying 1s → π * transition when labeling the ketone group or aldehyde group of tobacco mosaic virus with O- (4-Nitrobenzyl) hydroxylamine, the absorption cross section σ per benzene molecule is calculated. Is about 25 Mb (25 × 10 ⁻¹⁸ cm ² ) and is the line given by the product of the absorption cross section σ and the density of ketone or aldehyde groups per unit volume at the resonant wavelength of the stained tobacco mosaic virus. Absorption coefficient μ is 13 / μm
Becomes This value is at least twice the linear absorption coefficient μ of the wavelength in the “water window” region of a normal protein. Further, the contrast C of the microscope is determined by the maximum transmittance Imax of the sample having various contrast areas and the transmittance Imax of the target area.
And is given by the following equation.

[0023]

(Equation 2)

The transmittance I of tobacco mosaic virus having a size of x = 16 nm is given by the following equation.

[0025]

(Equation 3)

When calculated using these equations, the contrast C of the tobacco mosaic virus is about 0.1,
Compared to an X-ray microscope using a wavelength in the “water window” region, it is about 2
The value is more than double. Therefore, it can be seen that an X-ray microscope using labeling by a labeler can detect, for example, a protein of tobacco mosaic virus with very high sensitivity.

However, such a conventional X-ray microscope is a transmission type microscope, that is, a bright field microscope, and a contrast of an absorption image is obtained by absorption of a constituent tissue of a sample to be observed. As is apparent, when the size of the tissue to be observed is reduced, the degree of absorption of X-rays is reduced, and there is a problem that the contrast is necessarily deteriorated.

The present invention has been made in view of the circumstances described above, and can detect a specific chemical group or element, and can detect a minute amount of a chemical group. It is an object of the present invention to provide a new high-performance X-ray microscope that can perform the measurement and can further improve the contrast.

[0029]

According to the present invention, there is provided an X-ray light source for emitting X-rays.
An X-ray focusing unit that focuses X-rays from the X-ray light source on the observation sample; a phosphorescent / fluorescent focusing unit that collects phosphorescence and fluorescence emitted from the X-ray focusing point by the X-ray focusing unit An X-ray excitation type fluorescence microscope equipped with a photodetector for detecting phosphorescence and fluorescence condensed by a phosphorescence / fluorescence condensing unit, wherein X-rays monochromatized at a photon energy of 2000 eV or less are used. The present invention provides an X-ray excitation type fluorescence microscope (Claim 1).

According to the present invention, in the above microscope, monochromatic X-rays having a wavelength width of 1 eV or less are used (claim 2).
0 eV to 550 eV and the wavelength is variable (Claim 3), and the X-ray light source is any of a synchrotron, a laser plasma light source, a plasma discharge type X-ray light source, or an electron beam excitation type X-ray tube. There is (claim 4)
In addition, an X-ray focusing objective lens is provided in the X-ray focusing section, and the X-ray focusing objective lens is one of a Fresnel zone plate, a Walter type, and a direct incidence type Schwarzschild optical type. (Claim 5) In addition, a pinhole for limiting the emission point of the X-ray from the X-ray light source is provided in the X-ray focusing section, and a separate pinhole is provided in front of the photodetector and in the phosphorescence / fluorescence focusing section. The preferred embodiment is that the X-ray focusing section and the phosphorescence / fluorescence focusing section form a confocal optical system (claim 6).

Further, the present invention relates to a chemical detection method using the above-mentioned microscope, wherein the observation sample is labeled with a labeler which is a molecule containing a double bond (claim 7). Also provide. Also,
According to the present invention, in the above method, the labeler contains a 5-membered ring or a 6-membered ring (claim 8), the labeler contains a benzene ring (claim 9), and the photon energy is 2
80 to 290 eV (Claim 10), C,
Use of 1s → π * absorption of a molecule containing N and O (claim 11) is also a preferred embodiment thereof.

[0032]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As described above, an X-ray excitation type fluorescence microscope according to the present invention includes an X-ray light source, an X-ray focusing section, a phosphorescence / fluorescence focusing section, and a photodetector. X-rays emitted from the X-ray light source are collected on the observation sample by the X-ray collector. The collected X-rays cause an inner-shell excitation process to be described later in the electronic structure of the observation sample, and phosphorescence and fluorescence emitted in the de-excitation process after the inner-shell excitation process are converted to phosphorescence and fluorescence condensed light. The light is collected by the unit and detected by the photodetector.

Such an X-ray excitation type fluorescence microscope of the present invention utilizes the principle of a dark field microscope. A dark field microscope obtains an image by scattered light, fluorescence, or diffracted light from an observation sample.
Parts that cause scattering, fluorescence, or diffraction appear shiny. Therefore, since the photons emitted from the sample are purely measured, the microstructure of the observation sample can be detected at a high S / N ratio by using, for example, a trace photon detection technique. As described above, the dark-field microscope has much higher detection sensitivity than the bright-field microscope.

As described above, the X-ray excitation type fluorescence microscope of the present invention, which uses the principle of the dark field microscope, condenses and irradiates the observation sample with X-rays, thereby exposing the electronic structure of the observation sample to inner-shell excitation. A process is performed to detect phosphorescence and fluorescence emitted in a de-excitation process after the inner-shell excitation process. That is, first, FIGS. 4A, 4B, and 4C exemplify the electronic structures of the carbon molecules of the benzene ring in the inner-shell excitation process, the Auger process, and the de-excitation process, respectively.

As shown in FIG. 4 (a), the benzene ring absorbs X-rays of wavelength λx, causing an inner shell excitation process, and the electrons A in the carbon orbit 1s are moved to the higher empty molecular orbital e2. Get excited. Thereafter, as shown in FIG. 4 (b), the excited benzene molecule is unstable in terms of energy, so that an Auger process occurs, and the charged electron B fills a vacancy in orbit 1s. At this time, the electron C in the trajectory e1 near the electron A is repelled and ionized. That is, in the Auger process, an excited state of monovalent benzene is formed. At this time, the electron A first excited in the higher empty molecular orbital e2 remains as it is. This electron A is usually called a spectator. This spectator,
As illustrated in FIG. 4C, for a certain period of time, for example, several n
sec, after staying in the orbit e2, the wavelength λf, for example, 2
Emitting light of 00 nm to 500 nm, the lower orbit e1
Fill the holes. That is, in this deexcitation process, the state returns to the monovalent ground state.

The molecular orbital where the spectator is present is extremely sensitive to the effects of the type of atoms and molecules or the chemical environment. Therefore, the wavelength λx and the wavelength λf are different depending on the type of atoms and molecules, the chemical environment, and the like. Therefore, in the X-ray excitation type fluorescence microscope of the present invention, the X-rays having a wavelength corresponding to the 1s → π * transition of the observed molecule are used, and the fluorescence by the spectator in the de-excitation process after the inner-shell excitation process as described above is used. By detecting, it is possible to detect a chemical group of an observation sample that reflects a slightly different chemical environment with excellent sensitivity.

Further, according to the chemical detection method using the X-ray excitation type fluorescence microscope of the present invention,
By labeling a specific chemical group or element of the observation sample, minute detection of Ca ions or the like can be performed, and an image with a very high contrast can be obtained. Further, since X-rays having a wavelength slightly shorter than the wavelength of visible light are used as probe light, extremely high spatial resolution can be obtained, and when an existing X-ray optical element is used, 20 to 30 is used.
An image with a pixel density of nm can be obtained.

Further, when a pulse light source is used as the X-ray light source, for example, the transient response of the observation sample can be observed with a time resolution. Further, when the configuration is the same as that of a scanning confocal fluorescence microscope, it is possible to have depth resolution. Thus, the present invention
A high-performance dark-field microscope that features the characteristics of a fluorescence microscope,
By combining the features of an X-ray microscope having a high spatial resolution, a high-performance X-ray excitation type fluorescence microscope can be realized.

Hereinafter, examples will be shown, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited by the following examples.

[0040]

【Example】

(Embodiment 1) FIG. 5 illustrates an X-ray excitation type fluorescence microscope according to an embodiment of the present invention. For example, as exemplified in FIG. 5, in the X-ray excitation type fluorescence microscope of the present invention, a synchrotron (1) is provided as an X-ray light source, and an X-ray focusing unit includes a diffraction grating (2); An oblique incidence condenser lens (3), a first pinhole (4), and a Fresnel zone plate (5) as an X-ray condenser objective lens, and a phosphorescence / fluorescence condenser section is an optical lens. A visible / ultraviolet lens (7) and a second pinhole (8) are provided, and a photodetector (9) is provided as a photodetector.

The X-rays radiated from the synchrotron (1) are separated by the diffraction grating (2) and then condensed on the first pinhole (4) by the grazing incidence condenser lens (3). The point image on the first pinhole (4) is reduced and formed on one point in the observation sample (6) by the Fresnel zone plate (5). In this way, an X-ray micro beam having a size of about several tens nm is formed. And this X
The fluorescence emitted from the observation sample (6) by the X-ray microbeam is collected on the second pinhole (8) by the visible / ultraviolet lens (7) in the ultraviolet or visible region,
The light is focused on the photomultiplier (9).

With such an X-ray excitation type fluorescence microscope of the present invention, it is possible to detect a specific chemical group or a trace amount of a chemical group, and to further improve the contrast of an obtained image. Further, as exemplified in FIG. 6A, the first pinhole (4) and the Fresnel zone plate (5) of the X-ray focusing unit, and the visible / ultraviolet light lens (7) of the phosphorescent / fluorescent focusing unit. The second pinhole (8) and the second pinhole (8) constitute a confocal optical system. Therefore, by three-dimensionally manipulating the observation sample (6), a three-dimensional fluorescence image from molecules excited by X-rays can be obtained. Obtainable.

Of course, the Fresnel zone plate (5) of the X-ray collector is used without using the first pinhole (4) of the X-ray collector and the second pinhole (8) of the phosphorescent / fluorescent collector. )
As shown in FIG. 6B, a confocal optical system may be constituted by the light and the visible / ultraviolet light lens (7) of the phosphorescent / fluorescent light condensing part. If so, a non-confocal optical system may be used.

As the X-ray light source, not only a synchrotron but also a laser plasma light source, a plasma discharge type X-ray light source, an electron beam excitation type X-ray tube or the like may be installed. As the lens, instead of the Fresnel zone plate (5), a grazing incidence optical system represented by a Walter type, a direct incidence type Schwarzschild optical type having a structure in which an X-ray multilayer mirror is deposited on two spherical mirrors, etc. May be installed.

As the optical lens of the light detecting section, not only the visible / ultraviolet light lens (7) but also an X-ray such as a Fresnel zone plate, a Walter type, or a direct incidence type Schwarzschild optical type having a sufficient fluorescence intensity. A line imaging optical system may be provided. (Embodiment 2) FIG. 6 illustrates an X-ray excitation type fluorescence microscope according to an embodiment of the present invention.

The X-ray excitation type fluorescence microscope of the present invention illustrated in FIG. 6 is provided with a laser system (10) for emitting a laser beam, and the laser beam from the laser system (10) is used for an observation sample ( 6) A mirror (11) for converging light on the upper surface and a laser light condensing lens (1)
2) is provided. Other configurations are the same as those of the X-ray excitation type fluorescence microscope of FIG.

The direction of the laser beam emitted from the laser system (10) is adjusted by a mirror (11), and the laser beam is condensed on the observation sample (6) by a laser beam condensing lens (12). After exciting the valence electrons of the molecule to be observed in 6), the X-ray light source (1)
Excites the inner-shell electrons of the same molecule into outer-shell valence orbitals at the resonance wavelength. Then, the fluorescence from the molecule after the Auger process is detected.

In this way, by causing double resonance absorption of, for example, ultraviolet light and X-rays, a specific trace amount of molecule can be detected with high sensitivity. The laser system (10) is preferably of a wavelength tunable type.
d: By using a YAG laser + dye laser and using the non-linear crystal in combination, light in the infrared to ultraviolet region can be emitted. Further, an optical parametric oscillator (OPO), a titanium sapphire laser, or the like may be used.

Of course, a light source system capable of causing double resonance may be used instead of the laser system shown in FIG. Optionally, by inserting a spectroscopic optical element such as a filter at the entrance of the light receiving surface of a detector such as a photodiode, a photomultiplier tube, or a microchannel plate, the detection sensitivity can be further improved.

Example 3 A biological sample is labeled with a labeler by the chemical detection method using an X-ray excitation type fluorescence microscope of the present invention, and the distribution of ketone groups or aldehyde groups in protein molecules of the biological sample is imaged. . O- (4-Nitrobenzyl) hydroxylamine is used as a labeler. This O-
(4-Nitrobenzyl) hydroxylamine has a benzene ring with a high fluorescence yield, and has a strong X-ray absorption peak associated with a carbon 1s → π * transition at a photon energy of around 285 eV.

O- (4-Nitrobenzy) having such characteristics
l) Labeling ketone groups or aldehyde groups in protein molecules of biological samples with hydroxylamine. And
X-rays with a photon energy of around 285 eV excite the carbon 1s electrons of the benzene ring in the inner shell, and then detect the fluorescence emitted in the de-excitation process, so that the distribution of ketone groups or aldehyde groups can be detected with high sensitivity and high contrast. Can be imaged.

Example 4 Here, labelers for various specific chemical groups or molecules that can be used in the present invention will be exemplified. 1) Labeler of thiol group (R-SH) A molecule having a maleimide group utilizing a specific reaction of an SH group, such as a molecule which causes an exchange reaction of disulfide and a rial halide.

For example: 4-Fluoro-7-sulfamoylbenzofurazan 2,2'-Dihydroxy-6,6'dinaphthyl disulfide N- (9-Acridinyl) maleimide 2) Labeler of carboxyl group (R-COOH) Bromethyl group A molecule used as a reactive group.

For example, 4-Bromomethyl-7-methoxycoumarin 3-Bromomethyl-6,7-dimethoxy-1-methyl-1,2-ditydro
quinoxaline-2-one 3) Labeler of amino group (R-NH, R-NH2) A molecule having an isothiocyanate group, ferrocenyl isothiocyanate group, nitroaryl halide, or acid chloride group as an active group.

For example: ・ Fluoresceinisothiothiocyanate, isomer-1 ・ 4- [4- (Dimethylamino) phenylazo] phenylisothiocyana
te ・ 4-Chloro-7-nitrobenzofurazan ・ 4-Fluroro-7-nitrobenzofurazan ・ 3-Chlorocarbonyle-6,7-dimethoxy-1-methyl-2 (1H) -q
uinoxalinone ・ N-Succinimidyl-4-nitrophenylacetate ・ Sulforrhodamine 101 acid chloride 4) Labeler of aldehyde group For example, ・ 1,2-Diamino-4,5-dimethoxybenzen, dihydrochloride ・ 2,2'-Dithiobis (1-aminonaphthalene) 5) Hydroxyl labeler acid chloride, etc.

For example: 3-Chlorocarboonyle-6,7-dimethoxy-1-methyl-2 (1H)-
quinoxalinone 6) Labeler of hydrophobic pocket of protein For example: ・ 3,6-Bis (dimethylamino) -10-dodecylacridinium brom
ide ・ 4-Benzylamino-7-nitrobenzofurazan ・ 4- (4-Methoxybenzylamino) -7-nitrobenzofurazan 7) Labeler of intracellular calcium ion For example: ・ O, O'-Bis (2-aminophenyl) ethyleneglycol-N, N, N ', N',
-tetraacetic acide, tetrapotassiumu salt, hydrate ・ N, N, N ', N'-Tetrakis (2-pyridylmethyl) ethylenediami ・ 1- [2-Amido-5- (2,7-dichloro-6-hydroxy-3-oxy -9-xan
thenyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-
N, N, N ', N',-tetraacetic acid 8) Intracellular pH labeler For example: ・ 2 ', 7'-Bis (carboxyethyl) -4 or 5-carboxyfluorescei
n ・ 3'-O-Acetyl-2 ', 7'-bis (carboxyethyl) -4 or 5-carbo
xyfluorescein, diacetoxymethyl ester 9) Labeler of cell membrane (phospholipid, biological membrane) For example: 1,3-Bis (1-pyrenyl) propane 1- (4-Trimethylammoniumphenyl) -6-phenyl-1,3,5-hex
atriene iodide 10) Labeler of nucleic acid such as DNA For example: 4 ', 6-diamino-2-phenylindole (DAPI) Also, any molecule having a functional group and at least one double bond is a labeler in the present invention. Can be used.

As described above, by selectively using various labelers, specific chemical groups, molecules, structures, and the like of living cells can be labeled, and their distribution images can be detected with high sensitivity. Of course, in addition to the above, any molecule having fluorescence can be used as a labeler. For example, various molecules exemplified in Table 1 can also be used as a labeler.

Embodiment 5 According to the present invention, O- (4-Nitro
In the case where a ketone group or an aldehyde group in a protein molecule of a biological sample is labeled using benzyl) hydroxylamine and a fluorescent image is captured, the contrast of the fluorescent image is controlled. As shown in FIG. 3, the absorption peak of the absorption cross section associated with the 1s → π * transition of the benzene ring has a bandwidth of about 1 eV. The use of X-rays that are monochromatic with a band width that is approximately one digit narrower than the band width of the resonance line and that have variable wavelengths makes it possible to easily adjust the contrast of the observed image.

For example, the beamline PF-BL2 of the Photon Factory of the High Energy Physics Research Institute of the Ministry of Education is
X-rays having a band width of about 50 meV can be taken out at a photon energy of about 285 eV, and a sample labeled with O- (4-Nitrobenzyl) hydroxylamine using such X-rays can be used to convert the sample of the benzene ring to 1s → Irradiation with the photon energy of the leading value of the resonance line peak of the π * transition maximizes the fluorescence emission. Further, when photographing is performed at a photon energy about 250 meV lower than the head value of the resonance line peak, the amount of fluorescent light emission is reduced by half because the absorption cross section of X-rays of benzene is reduced by half.

In the present invention, the amount of fluorescent light can be adjusted by selecting the photon energy or the wavelength of the X-ray, and the contrast of the obtained image can be controlled. For example, a cell having a large size and containing a large amount of protein emits light more than necessary, so that the microstructure cannot be observed as it is.

In such a case, with a conventional X-ray microscope,
Adjusting the contrast was very difficult because it was performed by adjusting the thickness of the sample. However, in the X-ray excitation type fluorescence microscope of the present invention, the fluorescence emission amount, that is, the contrast can be adjusted very easily only by arbitrarily selecting the photon energy or the wavelength of the X-ray as described above. Accuracy observations can be made.

(Example 6) Assuming that the density of a chemical group to be observed is N and the X-ray absorption cross section of a labeler is σx, the linear absorption coefficient μ is

[0063]

(Equation 4)

Is given by According to this formula 4, when the density N of the chemical group to be observed decreases, the conventional X-ray microscope shows that the absorption of X-rays is small and the amount of fluorescence decreases. However, in the method of the present invention, a fluorescent effect for obtaining a fluorescent image of a trace amount of a chemical group can be amplified by the molecular design of the labeler.

Normally, the absorption cross section when a core electron transitions to a π * orbit is proportional to the number of double bonds possessed by the labeler. Therefore, by adding the required number of side chains having double bonds to the labeler, the X-ray absorption cross section of the labeler can be increased in proportion to the number of added side chains. Assuming that the number of side chains to be added is m and the X-ray absorption cross section of the labeler before adding the side chain is σs, the X-ray absorption cross section of the labeler after adding the side chain is

[0066]

(Equation 5)

Therefore, the linear absorption coefficient μ is

[0068]

(Equation 6)

Is as follows. For example, O- (4-Nitrobenz
yl) hydroxylamine contains only one benzene ring with a double bond, but when m benzene ring side chains are added to it, the amount of emitted fluorescence becomes m times larger, and the Can be obtained.

[0070]

As described in detail above, according to the present invention, a new high-performance, high-performance device capable of detecting a specific chemical group or the like or a trace amount of a chemical group in an observation sample while improving the contrast. An X-ray excited fluorescence microscope is provided.

[Brief description of the drawings]

FIGS. 1A and 1B are conceptual diagrams illustrating an inner-shell ionization process and an inner-shell excitation process, respectively.

FIG. 2 is a diagram illustrating the relationship between photon energy and absorption cross section when a 1s electron transits to each p orbital in a carbon atom and when a 1s electron is ionized.

FIG. 3 is a diagram illustrating the relationship between photon energy and absorption cross section when a 1s electron transits to a π * orbit and when a 1s electron is ionized in a 6-membered ring molecule.

FIGS. 4 (a), (b), and (c) show respective excitation processes,
It is a conceptual diagram which illustrated the electronic structure of the carbon molecule of the benzene ring in an Auger process and a deexcitation process.

FIG. 5 is a schematic view illustrating the configuration of an X-ray excitation fluorescence microscope according to an embodiment of the present invention.

6 (a) and 6 (b) are schematic diagrams each exemplifying a configuration of a confocal optical system in the X-ray excitation type fluorescence microscope of the present invention of FIG. 5;

FIG. 7 is a schematic view illustrating the configuration of an X-ray excitation type fluorescence microscope according to an embodiment of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Synchrotron 2 Diffraction grating 3 Oblique incidence condenser lens 4 First pinhole 5 Fresnel zone plate 6 Observation sample 7 Visible / ultraviolet light lens 8 Second pinhole 9 Photomultiplier 10 Laser system 11 Mirror 12 Laser light condenser lens

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-9-89812 (JP, A) JP-A-8-248200 (JP, A) JP-A-9-105709 (JP, A) JP-A-7-108 318700 (JP, A) JP-A-7-128500 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G21K 7/00 JICST file (JOIS) WPI (DIALOG)

Claims (11)

(57) [Claims] 1. An X-ray light source that emits X-rays, an X-ray condensing unit that condenses X-rays from the X-ray light source on an observation sample, and an X-ray condensing unit that focuses X-rays What is claimed is: 1. An X-ray excitation type fluorescence microscope comprising: a phosphorescence / fluorescence condensing section for condensing emitted phosphorescence and fluorescence; and a photodetector for detecting phosphorescence and fluorescence condensed by the phosphorescence / fluorescence condensing section. , Photon energy 20
An X-ray excitation type fluorescence microscope characterized in that X-rays monochromized at 00 eV or less are used. 2. The X-ray fluorescence microscope according to claim 1, wherein monochromatic X-rays having a wavelength width of 1 eV or less are used. 3. An X-ray photon energy of 280 eV to 5
The X-ray excitation type fluorescence microscope according to claim 1, wherein the X-ray fluorescence microscope has a wavelength of 50 eV and a variable wavelength. 4. The X-ray source according to claim 1, wherein the X-ray light source is any one of a synchrotron, a laser plasma light source, a plasma discharge type X-ray light source, and an electron beam excitation type X-ray tube.
Line-excited fluorescence microscope 5. An X-ray focusing objective lens is provided in an X-ray focusing section, and the X-ray focusing objective lens is any one of a Fresnel zone plate, a Walter type, and a direct incidence type Schwarzschild optical type. Claims 1 to 4
X-ray excitation type fluorescence microscope. 6. A pinhole for limiting an emission point of X-rays from an X-ray light source is provided in an X-ray condensing section, and another pin is provided in front of a photodetector and in a phosphorescence / fluorescence condensing section. 6. A hole is provided, and the X-ray focusing section and the phosphorescence / fluorescence focusing section form a confocal optical system.
X-ray excitation type fluorescence microscope. 7. A chemical detection method using an X-ray excitation type fluorescence microscope according to claim 1, wherein an observation sample is labeled by a labeler which is a molecule containing a double bond. 8. The method according to claim 7, wherein the labeler has a 5-membered ring or a 6-membered ring. 9. The method according to claim 7, wherein the labeler contains a benzene ring. 10. A photon energy of 280-290 eV
The chemical detection method according to claim 9, wherein 11. The chemical detection method according to claim 7, wherein 1s → π * absorption of a molecule containing C, N, and O is used.