JP2003066338A - Microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high-function microscope which is capable of detecting the chemical base characterized by a specific vibration level by measuring a photoresponse at a super-high resolution by using light of two wavelengths; a IR ray or far IR ray corresponding to the resonance wavelength excitable to the specific vibration level in the molecules or crystals existing within a sample without using a far UV light source and a ray corresponding to the wavelength excitable to an electron excitation state of a high level from the specific vibration level. SOLUTION: This microscope has the first light source 1 which generates the first light for causing the transition of the molecules in a sample 15 from a base state to the high vibration level of a lowest electron state, the second light sources (1 and 3) which generate the second light for causing the transition of the molecules described above from the high vibration level of the lowest electron state to a quantum state of a higher energy level, optical systems (5, 7 and 9) which partly superpose the irradiation regions of the first light and the second light on each other over the sample 15, and a photodetector 21 which detects the emitted light from the regions irradiated with the first light and the second light on the sample 15.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a microscope, and more particularly, it uses two wavelengths of light, at least one of which is infrared or near infrared, and which is specific to molecules or crystals present in a sample. Corresponding to the resonance wavelength that can be excited to the vibrational level, the other light corresponds to the wavelength that can be excited from the vibrational level to the higher electronic excited state, and these two wavelengths of light partially overlap with each other spatially. And irradiating the sample, the photoresponse (eg, fluorescence) induced from a region narrower than the diffraction limit of light is measured, and the chemical group characterized by a particular vibrational level is detected with high sensitivity. It concerns a functional microscope.

[0002]

2. Description of the Related Art The technique of an optical microscope is old and various types of microscopes have been developed. In recent years, further advanced microscope systems have been developed due to advances in peripheral technologies such as laser technology and electronic image technology.

Under such a background, for example, Japanese Patent Laid-Open No. 8-1
In Japanese Patent No. 84552, there is proposed a high-performance microscope capable of not only controlling the contrast of an obtained image but also performing chemical analysis by using a double resonance absorption process generated by illuminating a sample with light of a plurality of wavelengths. There is.

This microscope uses double resonance absorption to select a specific molecule and observes absorption and fluorescence due to a specific optical transition. This principle is shown in FIGS.
Will be described with reference to. FIG. 12 shows the electronic structure of the valence orbits of the molecules that make up the sample. First, the electrons of the valence orbit of the molecules in the ground state (S0 state) shown in FIG. 12 are excited by light of wavelength λ1. To the first excited state (S1 state) shown in FIG. Next, it is similarly excited with light of another wavelength λ2 to obtain the second excited state (S2 state) shown in FIG. In this excited state, the molecule emits fluorescence or phosphorescence and returns to the ground state as shown in FIG.

In microscopy using the double resonance absorption process,
The absorption image and the emission image are observed using the absorption process of FIG. 13 and the emission of fluorescence and phosphorescence of FIG. In this microscopic method, first, a laser beam or the like is used to generate light having a resonance wavelength λ1.
As described above, the molecules constituting the sample are excited to the S1 state. At this time, the number of molecules in the S1 state within the unit volume increases as the intensity of the irradiation light increases.

Since the linear absorption coefficient is given by the product of the absorption cross section per molecule and the number of molecules per unit volume, in the excitation process as shown in FIG. The linear absorption coefficient with respect to will depend on the intensity of the light having the wavelength λ1 initially irradiated. That is, the linear absorption coefficient for the wavelength λ2 can be controlled by the intensity of the light of the wavelength λ1. This means that if the sample is irradiated with light of two wavelengths of wavelength λ and wavelength λ2 and a transmission image with wavelength λ2 is taken, the contrast of the transmission image is wavelength λ1.
It shows that it can be completely controlled by the light.

Further, when the deexcitation process by fluorescence or phosphorescence in the excited state of FIG. 14 is possible, the emission intensity thereof is proportional to the number of molecules in the S1 state. Therefore, when used as a fluorescence microscope, the image contrast can be controlled.

Further, the microscopy using the double resonance absorption process enables not only the above-mentioned control of image contrast but also chemical analysis. That is, since the outermost shell valence orbit shown in FIG. 12 has an energy level unique to each molecule, the wavelength λ1 is different depending on the molecule,
At the same time, the wavelength λ2 becomes peculiar to the molecule.

Here, it is possible to observe an absorption image or a fluorescence image of a specific molecule to some extent even when illuminating with a conventional single wavelength, but generally, the wavelength range of the absorption band of some molecules is Due to the overlap, accurate identification of the chemical composition of the sample is not possible.

On the other hand, in the microscopic method using the double resonance absorption process, since the molecules that absorb or emit light are limited by the two wavelengths of wavelength λ1 and wavelength λ2, the chemical composition of the sample can be identified more accurately than the conventional method. Is possible. Further, when exciting a valence electron, only light having a specific electric field vector with respect to the molecular axis is strongly absorbed. Therefore, if the polarization direction of wavelength λ1 and wavelength λ2 is determined and an absorption or fluorescence image is taken, the same result is obtained. Even molecules can be identified in the orientation direction.

Recently, for example, Japanese Patent Laid-Open No. 10-14
As disclosed in Japanese Patent No. 2151, a fluorescence microscope having a high spatial resolution exceeding the diffraction limit by using a double resonance absorption process has also been proposed. FIG. 16 is a conceptual diagram of a double resonance absorption process in a molecule. A molecule in the ground state S0 is excited to S1 which is a first excited state by light having a wavelength λ1, and further in a second excited state by light having a wavelength λ2. It shows a state of being excited by a certain S2. Note that FIG. 16 shows S of a certain molecule.
It shows that the fluorescence from 2 is extremely weak.

An extremely interesting phenomenon occurs in the case of a molecule having optical properties as shown in FIG. Figure 17
Similar to FIG. 16, it is a conceptual diagram of the double resonance absorption process.
The axis represents the spread of the spatial distance, and shows the spatial area A1 irradiated with the light of wavelength λ2 and the spatial area A0 not irradiated with the light of wavelength λ2.

In FIG. 17, in the spatial area A0, the wavelength λ
A large number of molecules in the S1 state are generated by the excitation of light of No. 1, and at that time, fluorescence emitted at the wavelength λ3 is seen from the spatial region A0. However, in the space region A1, since the light having the wavelength λ2 is irradiated, most of the molecules in the S1 state are immediately excited to the higher S2 state, and the molecules in the S1 state do not exist. This phenomenon has been confirmed by some molecules. As a result, the fluorescence of wavelength λ3 completely disappears in the spatial area A1, and since the fluorescence from the S2 state does not exist originally, the fluorescence itself is completely suppressed in the spatial area A1 and the fluorescence is emitted only from the spatial area A0. Become.

This has a very important meaning when considered from the application field of the microscope. That is, in a conventional scanning laser microscope or the like, laser light is condensed into a microbeam by a condenser lens to scan an observation sample, and the size of the microbeam at that time is the numerical aperture and wavelength of the condenser lens. The diffraction limit is determined by and, and in principle, a higher spatial resolution cannot be expected.

However, in the case of FIG. 17, two kinds of light of the wavelength λ1 and the wavelength λ2 are spatially superposed well, and the fluorescent region is suppressed by the irradiation of the light of the wavelength λ2. Focusing on the light irradiation area, the fluorescent area can be made narrower than the diffraction limit determined by the numerical aperture and wavelength of the condenser lens, and the spatial resolution can be substantially improved. Therefore, by using this principle,
It becomes possible to realize a super-resolution microscope, for example, a fluorescence microscope, which uses a double resonance absorption process exceeding the diffraction limit.

Further, in order to enhance the super resolution of the microscope,
For example, Japanese Patent Application Laid-Open No. 11-95120 discloses a fluorescent labeler molecule for fully utilizing the function of the super-resolution microscope, the irradiation timing of two light beams of wavelength λ1 and wavelength λ2 to be used, and the like. . In this prior art, there are three quantum states including at least the ground state, and the transition at the time of deexcitation from a higher energy state excluding the first excited state to the ground state is dominated by the thermal relaxation process rather than the relaxation process by fluorescence. A sample in which a fluorescent probe molecule that stains various target molecules is chemically bound to a biomolecule that has undergone a biochemical staining technique is excited to the S1 state with light having a wavelength λ1 that excites the molecule to be stained, Then, the fluorescence from the S1 state is suppressed by immediately exciting it to a higher quantum level with light of wavelength λ2. In this way, the spatial resolution can be improved by artificially suppressing the spatial fluorescent region by utilizing the optical properties of the molecule.

The optical properties of such molecules can be explained from the viewpoint of quantum chemistry. That is, in general, in a molecule, each atom constituting the molecule is connected by a σ or π bond. In other words, the molecular orbital of a molecule is
It has a σ molecular orbital or a π molecular orbital, and the electrons existing in these molecular orbitals play an important role in binding each atom. Among them, the electrons in the σ molecular orbital bond each atom strongly and determine the interatomic distance within the molecule, which is the skeleton of the molecule. On the other hand, the electrons in the π molecular orbital make little contribution to the bond of each atom, and are rather bound to the whole molecule by an extremely weak force.

In many cases, when an electron in the σ molecular orbital is excited by light, the atomic spacing of the molecule is greatly changed, and a large structural change including dissociation of the molecule occurs. As a result, most of the energy given to the molecule by light changes into thermal energy due to the kinetic energy and structural change of atoms. Therefore, the excitation energy is not consumed in the form of light called fluorescence. Further, since the structural change of the molecule occurs at an extremely high speed (shorter than picosecond), even if fluorescence occurs in the process, its lifetime is extremely short.

On the other hand, the electron in the π molecular orbital hardly changes the structure of the molecule even when excited, stays in the high quantum discrete level for a long time, and emits fluorescence in the order of nanosecond. It has the property of de-excitation.

According to quantum chemistry, it is equivalent that a molecule has a π molecular orbital and that it has a double bond, and a molecule having abundant double bonds should be selected as the fluorescent labeler molecule to be used. Is a necessary condition. This indicates that even in a molecule having a double bond, the fluorescence from the S2 excited state is extremely weak in a 6-membered ring molecule such as benzene or pyrazine (for example, M. Fujii et.al. Chem. Phys). .Lett.1
71 (1990) 341).

Therefore, if a molecule containing a 6-membered ring molecule such as benzene or pyrazine is selected as the fluorescent labeler molecule, the fluorescence lifetime from the S1 state is long, and moreover, the molecule is obtained by exciting from the S1 state to the S2 state by photoexcitation. Since the fluorescence from the can be easily suppressed, the super resolution can be effectively used. That is, if observation is performed by staining with these fluorescent labeler molecules, not only a fluorescent image of the sample can be observed with high spatial resolution, but by adjusting the side chain chemical groups of the molecule, Since only specific chemical tissues can be selectively stained, even detailed chemical composition of a sample can be analyzed.

In general, the double resonance absorption process occurs only when the wavelength of two lights, the polarization state, etc. satisfy a specific condition. Therefore, by using this, the structure of the molecule can be known in great detail. it can. That is, there is a strong correlation between the polarization direction of light and the orientation direction of molecules, and when the polarization direction of light of two wavelengths and the orientation direction of molecules form a specific angle, the double resonance absorption process occurs. It happens strongly. Therefore, 2
By simultaneously irradiating the sample with light of two wavelengths and rotating the polarization direction of each light, the degree of fluorescence disappearance changes, so it is also possible to obtain information on the spatial orientation of the tissue to be observed from that state. You can This is also possible by adjusting the wavelengths of the two lights.

As described above, Japanese Patent Laid-Open No. 11-951 mentioned above.
According to the technique described in Japanese Patent Publication No. 20, it is understood that it has a high analysis capability in addition to the super resolution. Furthermore, the wavelength λ
By devising the irradiation timing of two lights of 1 and wavelength λ2, S / N can be improved, fluorescence can be suppressed effectively, and super-resolution can be more effectively expressed. It will be possible.

As a specific example of such super-resolution microscopy, for example, in Japanese Patent Laid-Open No. 2001-100102, pumping light of wavelength λ1 (especially laser light) that excites fluorescent labeler molecules from the S0 state to the S1 state. As shown in FIG. 18, pump light is emitted from the light source 51 and erase light is emitted from the light source 52, and the pump light is the dichroic mirror 53. After being reflected by, the light is condensed on the sample 55 by the annular optical system 54, the erase light is made into a hollow beam by the phase plate 56, and then the dichroic mirror 53.
Has been proposed in which the light is transmitted and is spatially superposed on the pump light and is condensed on the sample 55 by the annular optical system 54.

According to this microscope, fluorescence other than near the optical axis where the intensity of the erase light is zero is suppressed, and as a result, a region (Δ <0.61 · λ) narrower than the spread of the pump light is suppressed.
For 1 / NA and NA, only fluorescent labeler molecules existing in the numerical aperture of the ring zone optical system 54) are observed, and as a result, super-resolution is exhibited. The phase plate 56 for converting the erase light into a hollow beam, as shown in FIG.
The phase difference is provided at a position symmetrical with respect to the optical axis.

[0026]

However, according to various experiments conducted by the present inventors, the observation principle in the super-resolution microscope proposed hitherto is certainly excellent in terms of spatial resolution. It was found that there is a point to be improved in the amount of information that can be obtained, that is, in the structure classification ability. That is, in a super-resolution microscope, pump light and erase light are collected on a sample stained with a fluorescent labeled molecule to detect the fluorescence from the fluorescent labeled molecule. Excited to a so-called high vibronic level where the electronic state and the vibrational state are combined by light,
After that, it is thermally relaxed to the lowest vibrational level of the first electronic excited state, and then emits fluorescence to the ground state to be deexcited (Kash
law of a). In this case, the only information obtained is "which spatial position in the sample the fluorescent labeled molecule is stained with".

However, in the field of bioscience,
Since the sample is a living body, there is a strong demand for real-time and high-resolution observation of cell vital activities.
There is also a need for detailed information on "where in the cell the fluorescent labeling molecule is bound and in what state".
Moreover, there is also a high demand to see life activity in its natural state while maintaining high spatial resolution without staining the sample if possible.

In general, one of the reasons for staining a biological sample with a fluorescently labeled molecule is that most biomolecules are first to ground state.
The energy for transitioning to the electronically excited state is large, and a far-ultraviolet light source is required as an excitation light source, but at this stage, there is no stable commercial laser light source in the far-ultraviolet region, and a laser microscope system should be configured in this region. This is because it cannot be done. Note that there are xenon lamps and mercury lamps as far-ultraviolet light sources, but these light sources are not practical because they have low brightness and are considerably poor in quality in comparison with laser light sources in terms of light divergence and polarization.

Therefore, an object of the present invention made in view of the above point is that an infrared ray corresponding to a resonance wavelength capable of being excited to a specific vibrational level in a molecule or a crystal existing in a sample without using a far-ultraviolet light source. Alternatively, by using light of two wavelengths, that is, a far infrared ray and a ray corresponding to a wavelength capable of being excited to a higher electronic excited state from a particular vibrational level, an optical response induced from a region narrower than the diffraction limit of light is used. An object is to provide a highly functional microscope capable of measuring and detecting a chemical group characterized by a specific vibrational level with high sensitivity.

[0030]

In order to achieve the above object, the invention of a microscope according to claim 1 generates a first light which causes a molecule in a sample to transit from a ground state to a high vibrational level of a lowest electronic state. A first light source, a second light source for generating a second light that causes the molecule to transit from a high vibration level of the lowest electronic state to a quantum state of a higher energy level, and the first light source.
Of the above-mentioned light and the above-mentioned second light, which partially overlaps the irradiation area on the above-mentioned sample, and light for detecting the light emission from the above-mentioned area where the above-mentioned first light and the above-mentioned second light are irradiated. And a detector.

The invention according to claim 2 is the microscope according to claim 1, characterized in that the first light source emits infrared light of 700 nm or more or light in the near infrared region. is there.

The invention according to claim 3 is the invention according to claim 1 or 2.
The microscope according to claim 1, wherein the first light or the second light is used.
Is characterized by having a spatial filter for spatially intensity-modulating at least one of the light.

The invention according to claim 4 is the microscope according to claim 3, characterized in that at least one of the first light source and the second light source is a pulse light source.

The invention according to claim 5 is the microscope according to claim 4, characterized in that the first light source and the second light source are coherent light sources.

The invention according to claim 6 is the microscope according to claim 5, wherein at least one of the first light source and the second light source is a variable wavelength light source.

The invention according to claim 7 is the microscope according to any one of claims 1 to 6, wherein the wavelength of the first light is -CH, -NH, -OH, C = C, C. = O, -C
It is characterized in that it excites to the fundamental tone of any of the chemical groups such as H ₂ , —CHOH, and —CN, or a high overtone state thereof.

In a preferred embodiment of the microscope according to the invention, first and second light of different wavelengths are used, at least one of which is infrared or near infrared and these lights are spatially partly overlapped. Then, by irradiating the sample, a photoresponse signal from a region narrower than the diffraction limit of light is detected. At that time, the infrared or near-infrared wavelength corresponds to the resonance wavelength that can excite a specific vibrational level in the molecule or crystal present in the sample, and the other light changes from that vibrational level to a higher electronic excited state. Corresponding to the excitable wavelength, the chemical group characterized by the vibrational level is detected with high sensitivity by measuring the optical response (for example, fluorescence) induced by the irradiation of two wavelengths of light.

The principle will be described below.

That is, the microscope according to the preferred embodiment of the present invention is based on the double resonance process using infrared light or near infrared light as pump light. Here, the interaction between infrared light or near-infrared light and a substance is very characteristic, and it is caused by a vibrational motion state caused by a bond state between atoms constituting a substance and a structure in a molecule. Strongly reflects the rotational movement state. These oscillating motion state and rotational motion state change the interaction between infrared light or near infrared light and a substance very sensitively, and as a result, not only the detection of the elements constituting the substance but also the chemical group It is also possible to know the chemical bond state including and the environment of the substance in the sample.

The background will be described spectroscopically with reference to FIG. FIG. 1 shows a quantum structure of a general molecule, and a plurality of complicated vibrational and rotational levels belong to a specific electronic state. In other words, an electronically excited state that occurs when a valence electron of a molecule or the like changes the electron orbit, for example, a vibrationally excited state in which the distance between adjacent atoms changes due to thermal excitation, and a rotation that occurs when a molecule or the like makes a rotational motion. A material has various quantum states by complexly coupling three excited states.

In general, a substance, particularly a molecule, is excited (so-called electronic excitation) from the ground state (S0) having the lowest energy to the first electronic excited state (S1) that is bound to a specific vibrational / rotational level. In order to do so, light in the visible / ultraviolet wavelength range must be irradiated. In other words, the energy between these states is large, and when the wavelength of these lights is converted into photon energy, it becomes 2 eV or more. Currently, the majority of biomolecules, which are important research subjects in the field of life science, have excitation wavelengths of very short wavelengths from ultraviolet to far ultraviolet, and when converted to photon energy, they are 4 eV.
The energy is extremely high.

On the other hand, in the transition between vibrational / rotational states belonging to the same electronic state (vibrational / rotational excitation), the energy difference between the states is smaller than that in electronic excitation, so the photon energy of light required for excitation is It is small and the wavelength band is in the infrared / near infrared region.

In this regard, for example, “near infrared spectroscopy”,
The Spectroscopic Society of Japan, Measurement Method Series 32, Toshihiro Ozaki, Kawada
Satoshi, Academic Society Publishing Center 1996, the absorption spectra of various substances in the infrared / near-infrared region are shown (infrared / near-infrared spectroscopy).・ An absorption line due to rotational excitation has been confirmed. These each line, -OH in substance, _-CH 3, -C
This is due to vibrational / rotational transition of chemical groups such as H and —NH ₃ , and its spectral shape is peculiar to a substance, and detailed data commonly known as “molecular fingerprint” has been completed.

In general, the material composition of a substance is clarified by measuring the optical response including the absorption of the substance in the infrared / near infrared region. Not only that, -OH, _-CH 3, -
Chemical groups such as CH and —NH ₃ sensitively and largely change when another substance (particularly a molecule) is present in the surrounding environment, for example, in a space adjacent to the chemical group or when the phase is changed. That is, the wavelength of the absorption line changes.

As a specific example, in fluorescent microscope observation, when a living tissue is dyed with a fluorescent dye molecule, the dye molecule physically or chemically adsorbs to a specific chemical group or site of the tissue, and at that time, it is adsorbed. The electronegativity of the dye molecule changes, and the absorption spectrum in the infrared / near infrared region changes subtly due to the difference in pH. This change provides information on the environment in which the dye molecule exists in the tissue.

In this way, when infrared / near infrared light is used,
Not only the substance composition but also the state in which the substance exists in the sample is clarified. By combining this extremely excellent infrared / near-infrared light analysis capability with the double resonance absorption process (infrared double resonance absorption process), an outstanding microscopic spectroscopic method can be realized in terms of spatial resolution. .

FIG. 2 is a conceptual diagram of the infrared double resonance absorption process which is the basis of the present invention. In FIG. 2, a molecule in the ground state (S0) is excited to a specific vibrational / rotational level at a higher level by the infrared or near-infrared light (pump light) having the first wavelength, and the state (S0 (V1) ) Another second
The first electronic excited state (S
It is excited to any vibration / rotation state belonging to 1) (vibration / rotation resonance absorption). Here, the probe light is generally light in the visible or ultraviolet region.

FIG. 3 shows the deexcitation process after the infrared double resonance absorption process. As shown in FIG. 3, in some cases, any one of the vibration / rotation states of S1 is thermally relaxed to the lowest vibration / rotation state of S1, and then the fluorescence of the third wavelength is emitted from the vibration / rotation state of S0, so that S0 Is de-excited to any of the vibration / rotation states belonging to. After that, the heat is relaxed again to return to the ground state. Further, in some cases, photochemical change occurs in S1 to generate other chemical substances or secondary products such as ions and electrons.

Thus, in the infrared double resonance absorption process,
At least as a requirement, infrared or near-infrared light will
Since it is necessary to perform rotational resonance absorption, various optical responses detected in the infrared double resonance absorption process have the same contents as the absorption spectra of various substances in the infrared / near infrared region.

Therefore, a characteristic of the infrared double resonance absorption process is that the detection sensitivity is extremely high. For example, infrared / near-infrared spectroscopy basically takes an absorption spectrum of infrared or near-infrared light, but if absorption is weak due to intensity fluctuation of the light source, etc., infrared / near-infrared spectroscopy It becomes difficult to capture the change in intensity. On the other hand, in the infrared double resonance absorption process, since the secondary product is not generated unless the sample is irradiated with the pump light and the probe light, it is easy to detect the presence or absence of the phenomenon. This is called zero level detection and is a technique used in fluorescence microscopes, photoelectron microscopes, fluorescent X-ray analysis and the like, and its high detection sensitivity is well-established.

Now, the infrared double resonance absorption process can be expected to be a very interesting phenomenon in terms of imaging theory. FIG. 4 (a),
FIG. 4B shows a state of detecting a molecule to be analyzed in the observation sample by the infrared double resonance absorption process.
FIG. 4A shows a state in which the pump light, which is infrared or near-infrared light, and the probe light, which is visible or ultraviolet light, are spatially partially overlapped and condensed on the sample, and FIG.
(B) shows the degree of overlap between the pump light and the probe light at the condensing part.

In the infrared double resonance absorption process, an optical response peculiar to the infrared double resonance absorption process is observed only when the pump light and the probe light are simultaneously irradiated. Therefore, FIG.
As shown in FIG. 3, when the pump light and the probe light are spatially partially overlapped and collected on the sample, the observation target molecule existing only in the overlap region generates a photoresponse signal. . This meaning is extremely important from the viewpoint of spatial measurement.

For example, a laser scanning microscope as shown in FIG. 5 is known as a conventional microscope. In this laser scanning microscope, a laser beam from a laser light source 61 is passed through a reflection mirror 62, a beam splitter 63 and an objective lens 64 to be focused on an observation sample 65 placed on a stage, and the focused laser is focused. The sample 65 is scanned by the laser beam by moving the beam and the sample 65 relative to each other, and the sample 6 is irradiated with the laser beam.
5, the optical response such as fluorescence is separated from the outward path by the beam splitter 63 via the objective lens 64, and then the filter 66,
A photodetector 69 such as a photomultiplier tube receives the light through the relay lens 67 and the pinhole 68, and an image is formed on a computer.

In general, the spatial resolution of this type of microscope is
It depends on the size of the focused beam. In detail, it is determined by the diffraction limit defined by the focusing optical system and the wavelength of the laser beam, and in principle, a spatial resolution higher than that cannot be expected.

However, if the phenomenon shown in FIG. 4 is used, since the optical response is a part of the focused laser beam, a super-resolution microscope method capable of observing a target substance with a high spatial resolution exceeding the diffraction limit is realized. . The epoch-making feature of this super-resolution microscopy is the high spatial resolution as well as the high analytical capability peculiar to the infrared double resonance absorption process. Have. in addition,
In the infrared double resonance absorption process, excitation between states corresponding to the sum of photon energies of pump light and probe light becomes possible. For example, tyrosine, which is a normal biogenic amino acid molecule,
For tryptophan, nucleic acid, etc., the photon energy for exciting S0 to S1 state needs to be at least 4 eV,
An ultraviolet light source having a wavelength of 300 nm or less is required.

However, at present, there is no inexpensive and stable commercial laser light source that emits light of a wavelength shorter than 300 nm, and the condensing optical system is expensive because the glass material that can be used in the ultraviolet band is expensive. Also, the manufacturing cost will increase. In addition, considering living cells as a sample, ultraviolet light is highly damaged, and is not suitable for observing living organisms that bioresearchers long for. However, if the vibrational level existing near 900 nm (1.4 eV in terms of photon energy) of -CH disclosed in the above-mentioned "near infrared spectroscopy" is used by the infrared double resonance absorption process. , By setting the wavelength of the probe light to 475 nm (2.6 eV in terms of photon energy), we observe the fluorescence emitted between some vibration and rotation levels of S1 and S0 after the infrared double resonance absorption process. can do.

In this case, the wavelength of the light source becomes long, and the damage to the sample can be reduced, and there are various commercially available laser light sources corresponding to the wavelengths of the pump light and the probe light. For example, there is an argon laser (476.5 nm) that corresponds to 475 nm of probe light, and an inexpensive and highly reliable AlGaAs-based commercial semiconductor laser can be used as one that corresponds to 900 nm of pump light.

In the super-resolution microscopy using the infrared double resonance absorption process, the method of spatially superimposing the pump light and the probe light on each other for enhancing the spatial resolution is as shown in FIG. In addition to the method of simply shifting the positions of the light and the probe light to collect the light, various methods such as spatially intensity-modulating at least one of the lights and overlapping them are conceivable. For example, as shown in FIGS.
It is also possible to give modes to the three regions of the beam surfaces of both the pump light and the probe light, and to overlap some of them.

In FIG. 6A, the pump light and the probe light are divided into three regions in the beam plane, and the intensity is modulated in the adjacent regions with the phase shifted by π. When such a beam is focused to the diffraction limit, the formed beam pattern has three spatial modes, although the outer size is defined by the diffraction limit, as shown in FIG. 6B. . That is, the phase of light is reversed in the adjacent regions, and the electric field strength is canceled at the boundary surface.
Therefore, if only one part of the pump light and the probe light having three spatial modes is overlapped as shown in FIG. 6C, the spatial region that responds to the infrared double resonance absorption process is 3
Limited to one part. That is, the spatial resolution exceeds the optical diffraction limit and is improved three times.

There are various patterns in the method of overlapping the pump light and the probe light using this spatial modulation method. For example, FIG.
As shown in (a) to (c), modes are provided in two regions of the beam surfaces of the pump light and the probe light,
There is also a method of overlapping a part on the sample surface. In this case, the spatial resolution in the horizontal direction is doubled. Also,
There is also a method of overlapping as shown in FIGS. 8A to 8C to improve the spatial resolution in both the vertical and horizontal directions. As in the case of FIG. 7, this method has a mode in two regions of the beam surfaces of the pump light and the probe light so that the light beam is partially overlapped on the sample surface and condensed.
At this time, by shifting one part in the vertical direction, the region where the pump light and the probe light overlap in the vertical direction is limited.
For example, if the pitch is shifted by half a pitch in the vertical direction, the spatial resolution is improved about twice in that direction.

[0061]

BEST MODE FOR CARRYING OUT THE INVENTION An embodiment of a microscope according to the present invention will be described below with reference to FIGS.

FIG. 9 is a diagram showing a schematic configuration of a super-resolution microscope using an infrared double resonance absorption process as one embodiment of the present invention. This super-resolution microscope is a laser scanning microscope with spatial resolution improved by spatially modulating both probe light and pump light, and the fluorescence spectrum emitted from the observation sample for each laser shot. By observing and analyzing, it is possible to analyze its chemical structure and composition. This embodiment will be described below with reference to FIG.

In this embodiment, one Ti sapphire laser 1 which is a pulsed light source is used as the light source, and the T
Pump light (first light) and probe light (second light) are generated from the pulsed laser beam from the i sapphire laser 1 to observe the -CH ₂ methylene group bonded to the benzene ring. Here, the absorption by vibrational excitation of the —CH ₂ group is 8% as shown in the above “near infrared spectroscopy”.
Its existence has been confirmed in the wavelength band of 85 nm to 895 nm. This is conjugated to the tuning wavelength range of 780 nm to 920 nm of Ti sapphire laser 1 (Spectra Physics, Inc .: Products Guide).

The absorption line due to vibrational excitation of the —CH ₂ group changes to some extent depending on the environment in the sample, but the resonance absorption condition can be realized by adjusting the oscillation wavelength of the Ti sapphire laser 1. Further, when the Ti sapphire laser 1 is subjected to wavelength conversion with a non-linear optical crystal such as BBO, it is possible to generate light having a wavelength of 390 nm to 460 nm that is a harmonic. If this is converted into photon energy, 2.7 eV ~
It is 3.2 eV, and can be excited from the high vibrational excitation level belonging to the S0 state to any vibrational level belonging to the S1 state. Moreover, it should be confirmed that 390 nm to 460 nm
The nm wavelength alone cannot excite the benzene ring in the ground state to the S1 level, and no photoresponse is detected by light irradiation. Therefore, in this embodiment, the fundamental wave of the Ti sapphire laser 1 is used as pump light and the harmonic wave is used as probe light.

In FIG. 9, Ti sapphire laser 1
A laser beam (fundamental wave) emitted in pulse form is split into two optical paths by a beam splitter 2, and one of the laser beams is used as a probe light by passing through a KDP crystal 3 and being converted into a double wave. The probe light is transmitted through the phase plate 4 as a spatial filter, spatially intensity-modulated, and incident on the dichroic mirror 5. The other laser beam split into two by the beam splitter 2 is used as it is as pump light, and the pump light is used as a mirror 6 and a parallel-movable prism 7a.
After being made incident on the phase plate 8 as a spatial filter through the delay optical system 7 having the above and spatially intensity-modulated, the dynamic mirror 5 is passed through the position adjusting lens 9 and the mirror 10.
And is combined with the probe light by the dichroic mirror 5 to be emitted.

As described above, in this embodiment, the Ti sapphire laser 1 is used to form the first light source, and
The Ti sapphire laser 1 and the KDP crystal 3 are used to form a second light source. The dynamic mirror 5
The position of the pump light combined with the probe light with respect to the probe light is finely adjusted by the position adjusting lens 9 to adjust the degree of spatial overlap between the pump light and the probe light on the sample surface.

The pump light and the probe light emitted from the dichroic mirror 5 pass through the lens 11 and are reflected by the half mirror 12, and are then collected by the objective lens 13 on the observation sample 15 set on the two-dimensional moving stage 14. Light up.

On the other hand, the fluorescence emitted from the observation sample 15 upon irradiation with the pump light and the probe light is the objective lens 13.
Further, the fluorescence reflected by the half mirror 16 through the half mirror 12 and the half mirror 16 is condensed at the center of the pinhole 18 by the lens 17, and further, the transmission diffraction grating 20 as a spectrum meter is obtained by the lens 19. Highly sensitive ICC using the photoelectric conversion principle
It is incident on the D camera 21. Here, the pinhole 18 functions as a spatial filter, and plays a role of increasing the S / N ratio of the measurement by cutting off the fluorescence emitted from other than the observation sample 15, for example, the fluorescence emitted from the optical system.

A feature of this embodiment is that the transmission diffraction grating 20 is used as a spectrum meter in the detection system. By using the transmission diffraction grating 20 in this way, not only fluorescence can be measured but also the fluorescence spectrum and the time response to laser irradiation can be measured, so that the chemical structure and composition of the observation sample 15 can also be analyzed. .

Further, the fluorescence from the observation sample 15 transmitted through the half mirror 16 is focused on the CCD camera 23 by the imaging lens 22 so that the fluorescent spot image can be directly observed, whereby the objective lens 13 is focused. And so on.

In this embodiment, in order to effectively bring out the super-resolution function, the pump light and the probe light are spatially overlapped under a predetermined condition, and those lights are temporally overlapped. Matching is important. Regarding the temporal overlap, since the Ti sapphire laser 1 is a pulse light source, the prism 7a of the delay optical system 7 is moved in parallel so that the pump light and the probe light are simultaneously pulse-irradiated on the sample surface. Adjust the optical path difference of the light. In addition,
The adjustment of the optical path difference can be performed, for example, while detecting the laser scattered light from the sample surface with a high-speed PIN photodiode and measuring the output signal with a sampling oscilloscope.

On the other hand, regarding the spatial superposition of the pump light and the probe light, the superposition method shown in FIG. 8 is used in the present embodiment. That is, the pump light and the probe light are spatially intensity-modulated so that the corresponding phase plates 8 and 4 have modes in two spatial regions of the beam surface.

Therefore, the phase plate 8 has a phase plate 8 as shown in the plan view and the side view in FIGS. 10 (a) and 10 (b).
The quartz glass substrate for forming the is divided into two regions, and one of the regions is chemically etched so that the phase of the pump light is shifted by π so that a mode pattern of the pump light as shown in FIG. 10C is generated. To configure. Similarly, as shown in the plan view and the side view in FIGS. 11A and 11B, the phase plate 4 is divided into two regions on the quartz glass substrate forming the phase plate 4, one of which is used for the probe light. 11 is chemically etched so that the phase of
It is configured to generate a mode pattern of probe light as shown in (c).

In this way, as shown in FIG. 8A, the pump light and the probe light are spatially intensity-modulated so that they have modes in two spatial regions of their respective beam surfaces, and these lights are modulated. By using the position adjusting lens 9 shown in FIG. 9, the two-dimensional spatial resolution is substantially doubled as shown in FIG.

The present invention is not limited to the above-mentioned embodiment, but various modifications and changes can be made. For example, in the above embodiment, the -CH ₂ methylene group was observed, but for example, -CH (3rd overtone: 90
0 nm to 910 nm), -NH secondary amine (3rd overtone:
It is also possible to visualize the chemical composition in the sample with high resolution by using the vibrational excitation levels of 1010 nm to 1040 nm) and free-OH alcohol (3rd harmonic: 730 nm to 745 nm). In addition, as described in "Near infrared spectroscopy" above, C = C, C = O, -CHOH, -CN
Chemical groups such as can be separated.

In the above embodiment, the Ti sapphire laser 1 is used in common, the fundamental wave thereof is used as the pump light, and the fundamental wave converted into the harmonic wave by the KDP crystal 3 is used as the probe light. A light source that emits light (first
Light source) and a light source (second light source) that emits probe light may be provided independently. As described above, when the first light source and the second light source are provided independently, at least one of the light sources is a pulse light source, and more preferably, a wavelength variable light source according to the chemical group to be observed by super-resolution. Try to obtain the desired first and second wavelengths.

[0077]

As described above, according to the present invention, the first light for transitioning the molecule in the sample from the ground state to the high vibration level of the lowest electronic state and the molecule for the high vibration of the lowest electronic state. The second light that causes a transition from a level to a quantum state with a higher energy level is partially overlapped and irradiated on the sample, and the light emission from the irradiation region is detected. Without using a light source, luminescence induced from a region narrower than the diffraction limit of light can be detected, and a chemical group characterized by a specific vibrational level can be detected with high sensitivity.

[Brief description of drawings]

FIG. 1 is a diagram for explaining the principle of the present invention.

FIG. 2 is also a diagram for explaining the principle of the present invention.

FIG. 3 is likewise a diagram for explaining the principle of the present invention.

FIG. 4 is a diagram showing an aspect of simultaneous irradiation of pump light and probe light.

FIG. 5 is a diagram showing a schematic configuration of a scanning laser microscope.

FIG. 6 is a diagram showing another mode of simultaneous irradiation of pump light and probe light.

FIG. 7 is a diagram showing still another mode.

FIG. 8 is a diagram showing still another mode.

FIG. 9 is a diagram showing a schematic configuration of a super-resolution microscope using an infrared double resonance absorption process as one embodiment of the present invention.

FIG. 10 is a diagram showing a configuration of a phase plate for pump light shown in FIG. 9 and a spatial mode pattern of pump light.

FIG. 11 is also a diagram showing a configuration of a phase plate for probe light and a spatial mode pattern of probe light.

FIG. 12 is a conceptual diagram showing an electronic structure of a valence orbit of a molecule constituting a sample.

13 is a conceptual diagram showing a first excited state of the molecule of FIG.

FIG. 14 is likewise a conceptual diagram showing a second excited state.

FIG. 15 is a conceptual diagram similarly showing a state in which the second excited state returns to the ground state.

FIG. 16 is a conceptual diagram for explaining a double resonance absorption process in a molecule.

FIG. 17 is likewise a conceptual diagram for explaining a double resonance absorption process.

FIG. 18 is a diagram showing a configuration of an example of a conventional super-resolution microscope.

FIG. 19 is a plan view showing the structure of the phase plate shown in FIG. 18.

[Explanation of symbols]

1 Ti sapphire laser 2 beam splitter 3 KDP crystal 4,8 Phase plate 5 dichroic mirror 6,10 mirror 7 Delay optical system 7a prism 9 Position adjustment lens 11, 17, 19 lenses 12,16 Half mirror 13 Objective lens 14 Two-dimensional movement stage 15 observation samples 18 pinholes 20 Transmission type diffraction grating 21 ICCD camera 22 Imaging lens 23 CCD camera

Continued front page    (72) Inventor Keiki Ikedaki             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. (72) Inventor Masaaki Fujii             2-3-3 Ryumiminami 2-3-16, Okazaki-shi, Aichi (72) Inventor Takashige Omatsu             5-10-9 Hirado, Totsuka-ku, Yokohama-shi, Kanagawa (72) Inventor Koju Yamamoto             9-2-304 Denenchofu, Ota-ku, Tokyo (72) Inventor Tomoo Suzuki             Makuhari Tech 1-3, Nakase, Mihama-ku, Chiba City, Chiba Prefecture             Nogarden D-10E Nippon Ropa Co., Ltd.             -In F term (reference) 2H052 AA08 AA09 AC13 AC30 AC34                       AF07 AF14

Claims (7)

[Claims] 1. A first light source for generating a first light for transitioning a molecule in a sample from a ground state to a high vibrational level of a lowest electronic state, and the molecule from the high vibrational level of the lowest electronic state. A second light source for generating a second light that transits to a quantum state having a higher energy level, and an optical system for partially overlapping the irradiation regions of the first light and the second light on the sample. And a photodetector for detecting light emission from a region of the sample irradiated with the first light and the second light. 2. The microscope according to claim 1, wherein the first light source emits infrared light having a wavelength of 700 nm or more or light in the near infrared region. 3. The microscope according to claim 1, further comprising a spatial filter spatially intensity-modulating at least one of the first light and the second light. 4. The microscope according to claim 3, wherein at least one of the first light source and the second light source is a pulse light source. 5. The microscope according to claim 4, wherein the first light source and the second light source are coherent light sources. 6. The microscope according to claim 5, wherein at least one of the first light source and the second light source is a variable wavelength light source. 7. The wavelength of the first light is --CH, --N.
_{H, -OH, C = C,} C = O, -CH 2, -CHOH,
The microscope according to any one of claims 1 to 6, wherein the microscope excites to a fundamental tone of one of the chemical groups such as -CN or a higher harmonic state thereof.