JP2003344774A - Method and apparatus for adjusting optical axis of light source of microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and apparatus for adjusting the optical axes of light sources of a microscope, which method can easily, quantitatively and exactly adjust the optical axes of the light sources of the microscope. <P>SOLUTION: A sample 9 for adjustment uniformly dispersed with fluorescent molecules is used and while detecting the difference between the fluorescence intensity obtained by irradiating the sample 9 for adjustment with first light of the intensity distribution having the maximum at the center of the optical axis and the second light of the intensity distribution having the maximum and the fluorescence intensity obtained by irradiating the sample with the first light of the intensity distribution having the maximum and the second light of the intensity distribution having the minimum, the optical axes of the first light source 1 emitting the first light and the second light source 2 emitting the second light are so set as to maximize the above difference. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope, in particular, a new optical microscope of high performance and high function, which obtains a high spatial resolution by illuminating a stained sample with light of a plurality of wavelengths from a highly functional laser light source. The present invention relates to a method and an apparatus for adjusting an optical axis of a light source in the air.

[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 will be described with reference to FIGS. FIG. 6 shows the electronic structure of the valence orbits of the molecules constituting the sample. First, the electrons of the valence orbits of the molecules in the ground state (S0 state) shown in FIG.
1 is excited by the light of the first state (S1) shown in FIG.
State). Next, it is similarly excited by the light of another wavelength λ2 to be 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. 7 and the emission of fluorescence and phosphorescence of FIG. In this microscopic method, first, the molecules constituting the sample are excited to the S1 state by light having a resonance wavelength λ1 by laser light or the like, and at this time, the number of molecules in the S1 state within a unit volume is , Increases as the intensity of the applied light increases.

Here, 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 λ and λ2 and a transmission image of wavelength λ2 is taken, the contrast of the transmission image can be completely controlled by the light of wavelength λ1.

Further, when the deexcitation process by fluorescence or phosphorescence in the excited state of FIG. 8 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. 6 has an energy level unique to each molecule, the wavelength λ1 is different depending on the molecule, and at the same time, the wavelength λ2 is also unique 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. 2001-1
In Japanese Laid-Open Patent Publication No. 00102, a fluorescence microscope having a high spatial resolution exceeding the diffraction limit by using a double resonance absorption process is also proposed.

FIG. 10 is a conceptual diagram of a double resonance absorption process in a molecule. A molecule in the ground state S0 is excited by the light of wavelength λ1 to S1 which is the first excitation state, and further by the light of wavelength λ2 the second state. The state of being excited by S2 which is an excited state is shown. Note that FIG. 10 shows that the fluorescence from S2 of a certain molecule is extremely weak.

In the case of a molecule having optical properties as shown in FIG. 10, a very interesting phenomenon occurs. FIG. 11 shows
Similar to FIG. 10, 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. 11, 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 field of application of microscopes. 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.

In the case of FIG. 11, however, two kinds of light of wavelength λ1 and wavelength λ2 are spatially superposed well, and the fluorescent region is suppressed by irradiation of light of wavelength λ2, for example, wavelength λ1. 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 the 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 labeler molecule that stains various molecules that is a target is chemically bound to a biomolecule that has been subjected to biochemical staining technology 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 itself 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 a 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, when 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 a sample can be observed with high spatial resolution, but also by adjusting the side chain chemical group 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, the above-mentioned JP-A-11-951 is used.
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, a pump (especially laser light) having a wavelength λ1 for exciting a fluorescent labeler molecule from the S0 state to the S1 state is pumped. As shown in FIG. 12, pump light is emitted from the light source 81 and erase light is emitted from the light source 82, and the pump light is the dichroic mirror 83, as shown in FIG. After being reflected on the sample 95 by the annular optical system 84, the erase light is converted into a hollow beam by the phase plate 86, and then the dichroic mirror 83.
Has been proposed in which the light is transmitted through and is spatially superposed on the pump light to be condensed on the sample 95 by the annular optical system 84.

According to this microscope, fluorescence other than in the vicinity of 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 the fluorescent labeler molecules existing in the numerical aperture of the ring zone optical system 84) are observed, and as a result, super-resolution is exhibited. The phase plate 86 for hollowing the erase light is, for example, as shown in FIG. 13, configured to give a phase difference π at a position point-symmetric with respect to the optical axis, or a liquid crystal surface. A liquid crystal spatial modulator can be used.

[0027]

However, according to various experiments conducted by the present inventors, although the observation principle in the conventionally proposed super-resolution microscope is certainly excellent, it should be solved in practical use. It turned out to be a challenge.

That is, in the above-mentioned super-resolution microscope, since it is necessary to align the laser beams of two colors having different wavelengths under special conditions, it becomes difficult to make optical adjustments in the system, and especially the yield in the manufacturing line is increased. The problem of lowering occurs. Specifically, since the concentrated beam intensity distribution of the erase light of the two colors of light has an annular shape, the other pump light having the Gaussian intensity distribution is erased on the observation sample surface. When the light is focused in the center of the band, the super-resolution is most strongly expressed, but this is based on the assumption that the focused pump light and erase light are imaged according to the wave optics theory.

If the pump light and the erase light are imaged according to the wave optics theory in this way, the condensing intensity pattern of the pump light is a Gaussian type with perfect optical axis symmetry, while the erase light is also completely optical axis symmetrical. Because of the ring shape, the region where the fluorescence signal can be obtained is limited to the vicinity of the optical axis where the condensing intensity pattern of the pump light has a maximum value and the condensing intensity pattern of the erase light has a minimum value.

However, in reality, the condensing intensity patterns of the pump light and the erase light are not completely symmetrical with respect to the optical axis, and are often formed with some deviation in intensity. For example, the condensing intensity pattern when the erase light is made into a hollow beam by using a phase plate or a liquid crystal spatial modulator is, for example, as shown in FIG. become.

Therefore, when the pump light and the erase light are overlapped on the sample under the condition that the intensity minimum of the erase light and the intensity maximum of the pump light are coincident with each other, for example, as shown in FIG. The light intensity of the pump light is concentrated on one side of the erase light, and the shape of the fluorescence intensity distribution is disturbed. As a result, the fluorescence signal is lowered and the resolution cannot be improved.

As a method for solving this problem, it is possible to directly observe the sample and adjust the optical axes of the pump light and / or the erase light so that the resolution is maximized.

However, although the super-resolution microscope is a kind of fluorescence microscope, there is currently no resolution chart capable of quantitatively providing spatial resolution. Therefore, the adjustment may take time and cost may increase, and the adjustment is empirical by the adjuster, which makes it difficult to perform accurate adjustment.

Therefore, an object of the present invention made in view of the above points is to provide a method and an apparatus for adjusting a light source optical axis of a microscope, which can easily and quantitatively and accurately adjust the light source optical axis of the microscope. .

[0035]

In order to achieve the above object, the invention according to claim 1 is the first excitation from the ground state of a molecule stained with a molecule having three electronic states including at least the ground state. A first light source that generates a first light that causes a transition to a state, and the molecule from the first excited state,
A second light source that generates a second light that makes a transition to a second excited state having a higher energy level and an irradiation region of the first light and the second light are at least partially overlapped with each other, and In adjusting the optical axis of the light source of a microscope having an irradiation optical system and a detection means for detecting the light emission from the sample, the adjustment is performed by using an adjustment sample in which fluorescent molecules are uniformly dispersed as the sample. The sample for irradiation is irradiated with the first light having an intensity distribution having a maximum in the center of the optical axis and the second light having an intensity distribution having a maximum through the above optical system, and fluorescence from the sample for adjustment is emitted. A first step of detecting the intensity by the detecting means, and a first light having an intensity distribution having a maximum and a second light having an intensity distribution having a minimum on the adjustment sample via the optical system. Irradiation, and the fluorescence intensity from the adjustment sample is detected by the detection means. A second step of detecting, a third step of detecting the difference between the and the fluorescence intensity detected in the first step, the fluorescence intensity detected by the second step, the first
Difference between the fluorescence intensities obtained in the third step by repeating the first step to the third step while relatively moving the optical axis of the light source and the optical axis of the second light source. And a fourth step of setting the optical axes of the first light source and the second light source at a position at which is maximum.

According to a second aspect of the present invention, first light is generated which causes the molecules of a sample stained with molecules having at least three electronic states including at least the ground state to transition from the ground state to the first excited state. The first light source and the molecule are the first
Second light source for generating a second light that causes a transition from the excited state to the second excited state having a higher energy level, and at least a part of the irradiation regions of the first light and the second light overlap. In adjusting the optical axis of the light source of the microscope having an optical system for irradiating the sample together, and a detecting means for detecting the light emission from the sample, an adjusting sample in which fluorescent molecules are uniformly dispersed as the sample is used. The adjustment sample is irradiated with the first light having an intensity distribution having a maximum in the center of the optical axis and the second light having an intensity distribution having a maximum in the center of the optical axis via the optical system. Then, the first step of detecting the fluorescence intensity from the adjustment sample by the detection means, the adjustment sample with the first light having an intensity distribution having a maximum in the center of the optical axis, The second light of the intensity distribution having a minimum near the center and the above optical system And irradiate, and the fluorescence intensity from the adjustment sample is detected by the detection means, the fluorescence intensity detected in the first step, and the fluorescence intensity detected in the second step. A third step of detecting the difference from the intensity,
Fluorescence obtained in the third step by repeating the first to third steps while relatively moving the optical axis of the first light source and the optical axis of the second light source. The first light source and the second light source are provided at positions where the difference in intensity is maximum.
And a fourth step of setting the optical axis of the light source.

The invention according to claim 3 is the invention according to claim 1 or 2.
In the method of adjusting the optical axis of the light source of the microscope according to the item 1,
The step (1) is characterized by further including a step of adjusting the intensity of the first light and the intensity of the second light so that the fluorescence intensity from the observation sample is minimized. .

The invention according to claim 4 is the method for adjusting the optical axis of a light source for a microscope according to claim 1, 2 or 3, wherein the second light in the second step is the light from the second light source. Is obtained by intensity-modulating the liquid crystal with a liquid crystal spatial modulator.

The invention according to claim 5 relates to claims 1, 2, 3
Alternatively, in the method of adjusting the light source optical axis of the microscope according to 4,
In the fourth step, the optical axis of the second light source is fixed,
It is characterized in that the optical axis of the first light source is moved.

The invention according to claim 6 generates the first light that causes the molecule of the sample stained with the molecule having at least three electronic states including the ground state to transit from the ground state to the first excited state. The first light source and the molecule are the first
Second light source for generating a second light that causes a transition from the excited state to the second excited state having a higher energy level, and at least a part of the irradiation regions of the first light and the second light overlap. A light source optical axis adjusting device for a microscope, comprising: an optical system for irradiating the sample together; and a detecting means for detecting the light emitted from the sample, wherein a first intensity distribution having a maximum at the center of the optical axis. The first fluorescence intensity detected by the detection means when the sample is irradiated with the light and the second light having the maximum intensity distribution through the optical system, and the first fluorescence intensity having the maximum intensity distribution. Intensity comparison means for detecting a difference between the light and the second light having an intensity distribution having a local minimum, which is detected by the detection means when the sample is irradiated with light through the optical system; Based on the fluorescence intensity difference detected by the intensity comparison means Is characterized in that it has a, an optical axis moving means for relatively moving the optical axis of the serial first light source and the second light source.

According to a seventh aspect of the present invention, the first light is generated which causes the molecule of the sample stained with the molecule having at least three electronic states including the ground state to transit from the ground state to the first excited state. The first light source and the molecule are the first
Second light source for generating a second light that causes a transition from the excited state to the second excited state having a higher energy level, and at least a part of the irradiation regions of the first light and the second light overlap. A light source optical axis adjusting device for a microscope, comprising: an optical system for irradiating the sample together; and a detecting means for detecting the light emitted from the sample, wherein a first intensity distribution having a maximum at the center of the optical axis. The first fluorescence intensity detected by the detecting means when the light and the second light having the intensity distribution having the maximum in the center of the optical axis are irradiated to the sample through the optical system, and the center of the optical axis When the sample is irradiated with the first light of the intensity distribution having the maximum intensity and the second light of the intensity distribution having the minimum intensity in the vicinity of the center of the optical axis through the optical system, the sample is detected by the detecting means. Intensity comparing means for detecting a difference from the second fluorescence intensity, and the intensity Optical axis moving means for relatively moving the optical axis of the first light source and the optical axis of the second light source based on the fluorescence intensity difference detected by the comparing means. Is.

The invention according to claim 8 is claim 6 or 7
In the light source optical axis adjusting device for a microscope described in (1), the optical axis moving means and the second optical axis of the first light source are located at a position where the fluorescence intensity difference detected by the intensity comparing means becomes maximum.
It is characterized in that the optical axis of the light source is moved relatively.

According to a ninth aspect of the present invention, in the light source optical axis adjusting device for a microscope according to the sixth, seventh or eighth aspect, the first light and / or the first light intensity is adjusted so that the first fluorescence intensity is minimized.
Alternatively, it is characterized by further comprising intensity adjusting means for adjusting the intensity of the second light.

The invention according to claim 10 is the invention according to claim 6, 7,
In the light source optical axis adjusting device for a microscope described in 8 or 9, the light from the second light source is intensity-modulated by a liquid crystal spatial modulator, and a second intensity distribution having a local minimum near the center of the optical axis. It is characterized in that it is configured to obtain light.

The invention according to claim 11 is the invention according to claim 6, 7,
In the light source optical axis adjusting device for a microscope according to 8, 9, or 10, the optical axis moving means fixes the optical axis of the second light source and moves the optical axis of the first light source. It is a feature.

The principle of the present invention will be described below with reference to FIGS. 1 and 2.

FIG. 1 shows a state in which pump light and erase light that has not been beam-shaped are condensed on an evaluation sample having a fluorescent layer in which fluorescent molecules are dispersed at a uniform concentration.
FIG. 2 shows a state in which pump light and erase light shaped like a Bessel primary beam shaped into a hollow beam are condensed on an evaluation sample having a fluorescent layer in which fluorescent molecules are dispersed at a uniform concentration. Shows.

In the state of FIG. 1, since the pump light and the erase light spatially completely overlap each other, the fluorescence intensity from the evaluation sample should be suppressed to almost zero.
From this state, if the erase light is converted into a hollow beam as shown in FIG. 2, the intensity of the erase light is strong near the central portion where the erase light is weak, that is, the peripheral portion of the condensed beam of the pump light is erased. Since the fluorescence signal is detected from the part, the spatial resolution can be improved.

Here, the super-resolution effect is most strongly manifested when the intensity of the pump light and the erase light is constant, the fluorescence signal intensity is as weak as possible in the state of FIG. 1, and conversely the state of FIG. Then, the fluorescence signal intensity is as strong as possible. That is, in the state of FIG. 1, the condensed pump light and the erase light completely overlap on the evaluation sample, and in the state of FIG. 2, the strongest light below the diffraction limit from the hollow portion of the erase light. This is because the signal in the fluorescent region is taken out. Therefore, if the pump light and the erase light have ideal perfect optical axis symmetry intensity distribution,
The obtained fluorescence signal is contributed by the region having the maximum intensity on the optical axis of the pump light.

However, as described above, in reality, the pump light and the erase light deviate from the ideal condensing intensity distribution of perfect optical axis symmetry, so that the pump light and the erase light are slightly non-coaxial. , A Gaussian-type fluorescence intensity distribution is obtained, and the fluorescence signal contributing to super-resolution is maximized.

Therefore, as shown in FIGS. 1 and 2, the fluorescence intensity when the pump light and the erase light are irradiated to the evaluation sample having the fluorescent layer in which the fluorescent molecules are dispersed at a uniform concentration is measured. However, if the optical axes of the pump light and the erase light are adjusted under the condition that the difference between them becomes maximum, it becomes possible to obtain a fluorescent image with excellent super-resolution, and further, the fluorescent light in the state of FIG. When the signal is as close to zero as possible, it becomes possible to obtain a fluorescence image with a good S / N.

Moreover, since the condition shift from FIG. 1 to FIG. 2 and vice versa can be realized by, for example, inserting / removing the phase plate or turning on / off the voltage applied to the liquid crystal spatial modulator, it is extremely possible. It becomes possible to quantitatively optimize the super-resolution condition by a simple method. Further, since the evaluation sample to be used may have a fluorescent layer in which fluorescent molecules are dispersed at a uniform concentration, for example, a block in which rhodamine 6G is dispersed in PMMA (polymethylmethacrylate) is prepared, and this is prepared. Since it can be cut into a thin film, it can be easily obtained.

[0053]

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

FIG. 3 is a view showing the constitution of an example of a microscope for carrying out the light source optical axis adjusting method according to the present invention, and FIG. 4 is a perspective view showing the schematic constitution of an example of the liquid crystal spatial modulator shown in FIG.
FIG. 6 is an input / output characteristic diagram showing a phase modulation degree of read light with respect to write light intensity in the liquid crystal spatial modulator shown in FIG.

The microscope according to the present embodiment is a laser scanning type microscope in which the spatial resolution of erase data is spatially modulated by a liquid crystal spatial modulator to form a hollow beam, thereby exhibiting super-resolution. Then, as the first light source, Nd: Y
The AG laser 1 is used and Nd: YAG is used as the second light source.
Using a dye laser 2 that uses laser light from the laser 1 as excitation light, a biological sample stained with rhodamine 6G is observed. In addition, Rhodamine 6G is 530n
Since the absorption is maximum in the wavelength band of m, the Nd: YAG laser 1 uses its second harmonic (532 nm).

The second harmonic wave from the Nd: YAG laser 1 is split into two light beams by the beam splitter 3, one of which is reflected as a pump light (first light) by the mirrors 4 and 5, and then the cube half. The light is transmitted through the mirror 6, further reflected by the cube half mirror 7, and focused on the surface of the sample 9 by the microscope objective lens 8.

The other light beam split by the beam splitter 3 is made incident on the dye laser 2 as excitation light, and the dye laser 2 generates erase light (second light). The dye laser 2 uses a mixture of rhodamine B and DCM dissolved in methanol as a dye.
This dye laser 2 oscillates laser light having a wavelength of about 600 nm by being excited by a double harmonic of the Nd: YAG laser 1, and rhodamine 6G is in the first electronic excited state (S1) in the wavelength region. Therefore, there is an absorption band that can be excited to a higher electronically excited state of higher energy.

The erase light from the dye laser 2 is
After the wavefront is shaped by the spatial filter 11 so that the wavefronts are aligned, it is reflected by the mirror 12 and is incident on the diffractive surface of the liquid crystal spatial modulator 13, whereby the diffracted light is spatially modulated into a first-order Bessel beam, and the space The modulated erase light is coaxially aligned with the pump light by the cube half mirror 6, and is focused on the surface of the sample 9 through the cube half mirror 7 and the microscope objective lens 8.

In normal sample observation, the sample 9 is placed on the sample moving stage 15 and the sample moving stage 15 is two-dimensionally driven to two-dimensionally scan the sample 9 with pump light and erase light. Then, the fluorescence emitted from the sample 9 is collimated by the microscope objective lens 8 and transmitted through the cube half mirror 7, and further condensed by the condenser lens 17 to be incident on the bandpass filter 18. Here, only the fluorescence signal component is transmitted. Photomaru 19 as a detection means
The light is received by, and the output is taken into a personal computer (personal computer) 20 to form an image.

In such a structure, the liquid crystal spatial modulator 13
In the field of optical information processing represented by optical communication, optical measurement, optical calculation, etc., is actively applied to correction of phase distortion due to information propagation and control of optical phase term in Fourier plane. In particular, it is called a photo-addressable parallel alignment liquid crystal spatial modulator capable of purely controlling only the phase.

As shown in FIG. 4, the liquid crystal spatial modulator 13 has transparent electrodes 24 and 25 formed on the inner surfaces of two glass substrates 22 and 23 facing each other, and has these transparent electrodes 24 and 25. Amorphous silicon (α-Si: H) as a photoconductive layer between the glass substrates 22 and 23
It has a multilayer structure in which a layer 26, a light shielding layer 27, a dielectric mirror 28, and a liquid crystal layer 31 sandwiched between alignment films 29 and 30 are arranged. Antireflection films 32 and 33 are formed on the outer surfaces of the glass substrates 22 and 23, respectively.

In the liquid crystal spatial light modulator 13, the writing light is made incident from the glass substrate 22 side to make the α-Si: H layer 26.
Of the read light from the glass substrate 23 side, the read light is reflected by the dielectric mirror 28 via the liquid crystal layer 31, and further emitted from the glass substrate 23 via the liquid crystal layer 31. used.

As described above, when the writing light is made incident on the α-Si: H layer 26, the impedance of the α-Si: H layer 26 is lowered according to the incident intensity of the writing light. The voltage applied to the liquid crystal layer 31 via the liquid crystal layer 31 rises according to the intensity of the writing light, and the liquid crystal molecules move accordingly to modulate the reading light, that is, the refractive index for the reading light is modulated, as shown in FIG. In addition, a phase change of 2π radians or more can be applied to the reading light.

Therefore, according to the phase change characteristic curve of the reading light with respect to the writing light intensity shown in FIG. 5, the writing light intensity is modulated to modulate the refractive index of each region in the two-dimensional plane of the liquid crystal layer 31 to a desired value. For example, since a desired phase change can be given to the read light, the erase light can be shaped into an ideal hollow beam by using the liquid crystal spatial modulator 13 in the microscope as described above.

Although it is desirable to arbitrarily select the wavelength of the light to be modulated, in the case of the liquid crystal spatial modulator 13 as shown in FIG. 4, the read light with respect to the unique write light intensity is used for each used wavelength. Since the phase change characteristic curve is given and stored in the database, the user can easily form any beam shape at any wavelength.

Further, in recent years, a liquid crystal spatial modulator and a liquid crystal display are coupled via an optical image transmission element, and a pattern image corresponding to a desired phase modulation pattern of read light is directly input from a personal computer and displayed on the liquid crystal display. Then
A liquid crystal spatial modulator capable of inputting a video signal in which the displayed pattern image is directly projected on the liquid crystal spatial modulator to modulate the readout light with a desired phase pattern is also commercially available. If such a liquid crystal spatial modulator is used, beam formation can be performed from a personal computer for system control, so that it is extremely compatible with a laser scanning microscope, which is premised on computer imaging.

In the present embodiment, when adjusting the optical axis of the light source of the above-mentioned microscope, Rhodamine 6 is used as the sample 9.
Using a sample for adjustment made of a PMMA thin film containing G, the voltage applied to the liquid crystal layer 31 of the liquid crystal spatial modulator 13 is controlled to be on / off to selectively spatially modulate the erase light,
Sample 9 for adjusting the pump light and the spatially modulated erase light
On the basis of the difference between the fluorescence intensity emitted from the adjustment sample 9 when it is focused on and the fluorescence intensity emitted from the adjustment sample 9 when the pump light and the erase light that is not spatially modulated are focused on the adjustment sample 9. By displacing the mirrors 4 and 5, the optical axis of the pump light is moved with respect to the erase light, and the optical axis of the light source is adjusted.

Therefore, first, the adjustment sample 9 is excited only by the pump light, the fluorescence intensity at that time is measured by the photomultiplier 19, and the measured value (I ₀ ) is loaded into the personal computer 20. Next, without applying a voltage to the liquid crystal layer 31 of the liquid crystal spatial modulator 13, that is, without spatially modulating the erase light, the erase light and the pump light are condensed so as to overlap the adjustment sample 9, The fluorescence intensity at that time is measured by the photomultiplier 19 and the measured value (I ₁ ) is loaded into the personal computer 20. The pump light and the erase light have their intensities adjusted by intensity adjusting means or an automatic position adjusting mechanism such as a galvanometer mirror system so that the measured value I ₁ is minimized. Here, if the pump light and the erase light are completely on the same axis, the fluorescence is remarkably suppressed. Therefore, the measured value I ₁ of the photomultiplier 19 is comparable to the measured value I ₀ of the pump light alone. The amount becomes smaller.

After that, the liquid crystal layer 3 of the liquid crystal spatial modulator 13 is changed from the irradiation state of the pump light and the non-space modulated erase light.
A voltage is applied to 1 to spatially modulate the erase light so as to form a hollow beam, the fluorescence intensity at that time is measured by the photomultiplier 19, and the measured value (I ₂ ) is taken into the personal computer 20. Here, if the pump light and the erase light are completely coaxial, the measured value I ₂ should be larger than the measured value I ₁ . This is because the erase light is made into a hollow beam even on the light collecting surface, and the erase light is reduced to 1 in the light collecting area of the pump light.
This is because there is a partial spatial non-overlapping location, where strong fluorescence is generated without being suppressed.

In principle, if the erase light and the pump light have the intensity distribution with perfect optical axis symmetry, the measured value I ₂ is a fluorescence signal generated only near the intensity center narrower than the condensed size of the pump light. , Which is exactly the signal component that contributes to the super-resolution effect. Then, at this time, | I ₁ −I ₂ | becomes maximum.

However, in reality, as described above, the erase light and the pump light do not have a perfect optical axis symmetric intensity distribution, and have a slight intensity bias,
The super-resolution effect is maximized under the condition that the pump light and the erase are not focused on the same axis. That is, | I ₁ −I ₂ | does not become maximum in the state where the pump light and the erase are coaxially condensed.

Therefore, in the present embodiment, | I ₁ -I ₂
While measuring |, the angles of the mirrors 4 and 5 are adjusted so that | I ₁ −I ₂ | is maximized. Specifically, by calculating | I ₁ −I ₂ | by the personal computer 20 and displacing the mirrors 4 and 5 by controlling the automatic position adjusting mechanism 21 as the optical axis moving means based on the calculation result. , | I ₁ −I ₂ |, the angles of the mirrors 4 and 5 are adjusted.

Generation of pump light from the Nd: YAG laser 1, generation of erase light from the dye laser 2,
The personal computer 20 controls the modulation / non-modulation of erase light by the liquid crystal spatial modulator 13.

As described above, according to this embodiment, the erase light is selectively spatially modulated by the liquid crystal spatial modulator 13, and the pump light and the spatially modulated erase light are condensed on the adjustment sample 9. While detecting the difference between the fluorescence intensity at this time and the fluorescence intensity when the pump light and the erase light that is not spatially modulated are condensed on the adjustment sample 9, the mirrors 4 and 5 are displaced so as to maximize the difference. Since the optical axis of the light source is adjusted by doing so, the optical axis of the light source can be adjusted easily and quantitatively and accurately, and the spatial resolution of the microscope can be maximized.

Moreover, since the optical axis of the light source can be adjusted automatically in a short time at the initial stage of the microscope production line, the number of manufacturing steps can be reduced and the cost of the microscope can be reduced.

The present invention is not limited to the above-described embodiment, but various modifications and changes can be made. For example, although the optical axis of the pump light is adjusted with respect to the erase light in the above-described embodiment, conversely, the optical axis of the erase light is adjusted with respect to the pump light, or | I ₁
-I 2 _| may be to adjust the optical axis of both to maximize. Further, in the above embodiment, the personal computer 20 used for the microscope is provided with the intensity comparison calculation function and the optical axis adjustment control function to perform the optical axis adjustment. However, | I ₁ -I It is also possible to adjust the optical axis while calculating ₂ |. Furthermore, the present invention is not limited to the two-dimensional scanning by moving the sample by the sample moving stage, and may be applied to a microscope in which the sample is fixed and two-dimensional scanning is performed by moving the pump light and the erase light by, for example, a galvanometer mirror. It can be effectively applied.

[0077]

As described above, according to the present invention, an adjusting sample in which fluorescent molecules are uniformly dispersed is used, and the adjusting sample has a first intensity distribution having a maximum at the center of the optical axis. When the fluorescence intensity obtained when the light and the second light of the intensity distribution having the maximum are irradiated, and the first light of the intensity distribution having the maximum and the second light of the intensity distribution having the minimum are irradiated. While detecting the difference between the obtained fluorescence intensity and the optical intensity of the first light source that emits the first light and the second light source that emits the second light so as to maximize the difference. Therefore, the optical axis of the light source can be adjusted easily and quantitatively and accurately, and the spatial resolution of the microscope can be maximized.

[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 a diagram showing a configuration of an example of a microscope for carrying out a light source optical axis adjusting method according to the present invention.

FIG. 4 is a perspective view showing a schematic configuration of an example of the liquid crystal spatial modulator shown in FIG.

5 is an input / output characteristic diagram showing the degree of phase modulation of read light with respect to the write light intensity in the liquid crystal spatial light modulator shown in FIG.

FIG. 6 is a conceptual diagram showing an electronic structure of a valence electron orbit of a molecule constituting a sample.

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

FIG. 8 is likewise a conceptual diagram showing a second excited state.

FIG. 9 is a conceptual diagram similarly showing a state in which the second excited state returns to the ground state.

FIG. 10 is a conceptual diagram for explaining a double resonance absorption process in a molecule.

FIG. 11 is also a conceptual diagram for explaining a double resonance absorption process.

FIG. 12 is a diagram showing a configuration of an example of a conventional super-resolution microscope.

FIG. 13 is a plan view showing the configuration of the phase plate shown in FIG.

FIG. 14 is a diagram showing an example of a condensing intensity pattern of erase light that is formed into a hollow beam.

FIG. 15 is a diagram for explaining a conventional problem.

[Explanation of symbols]

1 Nd: YAG laser 2 dye laser 3 beam splitter 4,5 mirror 6,7 Cube half mirror 8 Microscope objective lens 9 samples (samples for adjustment) 11 Spatial filters 12 mirror 13 Liquid crystal spatial modulator 15 Sample moving stage 17 Condensing lens 18 band pass filter 19 Photomaru 20 personal computers 21 Automatic position adjustment mechanism 22,23 glass substrate 24,25 transparent electrodes 26 Amorphous Silicon (α-Si: H) Layer 27 Light-shielding layer 28 Dielectric mirror 29,30 Alignment film 31 Liquid crystal layer 32, 33 Antireflection film

Continued front page    (72) Inventor Keiki Ikedaki             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. (72) Inventor Masaaki Fujii             2-3-1, Ryumi Minami 2-3, Okazaki-shi, Aichi             No. 303 (72) Inventor Takeshi Watanabe             2-72 Koromachi, Okazaki City, Aichi Prefecture Manno Ohno             No. II 202 (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) 2G043 AA03 DA01 EA01 FA01 FA02                       FA06 GA02 GB01 GB19 HA01                       HA02 HA06 HA09 KA02 KA05                       KA09 LA02                 2H043 AD08 AD11 AD21                 2H052 AA07 AA09 AC04 AC14 AC27                       AC30 AC34 AF02

Claims (11)

[Claims] 1. A first light source for generating a first light that causes the molecule of a sample stained with a molecule having at least three electronic states including a ground state to transit from a ground state to a first excited state. A second light source that generates a second light that causes the molecule to transit from the first excited state to a second excited state having a higher energy level; and a second light source that emits the second light. In adjusting the optical axis of the light source of the microscope having an optical system for irradiating the sample with at least a part of the irradiation region overlapping and irradiating the sample, a fluorescent molecule is uniformly dispersed as the sample. Using the prepared adjustment sample, the adjustment sample is provided with first light having an intensity distribution having a maximum in the center of the optical axis and second light having an intensity distribution having a maximum through the optical system. Irradiate the firefly from the sample for adjustment. A first step of detecting the intensity by the detecting means, and a first light having an intensity distribution having a maximum and a second light having an intensity distribution having a minimum are applied to the adjustment sample via the optical system. A second step of irradiating and detecting the fluorescence intensity from the adjustment sample by the detection means, the fluorescence intensity detected in the first step, and the fluorescence intensity detected in the second step. The third step of detecting the difference between the first step and the second step, while relatively moving the optical axis of the first light source and the optical axis of the second light source. Then, the first light source and the second light source are placed at the position where the difference in fluorescence intensity obtained in the third step is maximum.
And a fourth step of setting the optical axis of the light source of the light source. 2. A first light source for generating a first light for transitioning the molecule of the sample stained with a molecule having three electronic states including at least the ground state from the ground state to the first excited state. A second light source that generates a second light that causes the molecule to transit from the first excited state to a second excited state having a higher energy level; and a second light source that emits the second light. In adjusting the optical axis of the light source of the microscope having an optical system for irradiating the sample with at least a part of the irradiation region overlapped and irradiating the sample, a fluorescent molecule is uniformly dispersed as the sample. Using the prepared adjustment sample, the adjustment sample is provided with the first light having an intensity distribution having a maximum in the center of the optical axis and the second light having an intensity distribution having a maximum in the center of the optical axis. For adjustment by irradiating through an optical system First detecting fluorescence intensity from fee by the detection means
And a second light having an intensity distribution having a maximum in the center of the optical axis and a second light having an intensity distribution having a minimum in the vicinity of the center of the optical axis through the optical system. Second step of irradiating the sample for adjustment and detecting the fluorescence intensity from the adjustment sample by the detection means, the fluorescence intensity detected in the first step, and the fluorescence intensity detected in the second step. And a third step of detecting the difference between the first step and the second step, while relatively moving the optical axis of the first light source and the optical axis of the second light source. Repeatedly, the first light source and the second light source are arranged at the position where the difference in fluorescence intensity obtained in the third step is the maximum.
And a fourth step of setting the optical axis of the light source of the light source. 3. The first step further includes the step of adjusting the intensity of the first light and the intensity of the second light so that the fluorescence intensity from the observation sample is minimized. The light source optical axis adjusting method for a microscope according to claim 1 or 2, wherein. 4. The second light in the second step is obtained by intensity-modulating the light from the second light source with a liquid crystal spatial modulator. Method of adjusting the optical axis of the light source of the microscope. 5. The fourth step is characterized in that the optical axis of the second light source is fixed and the optical axis of the first light source is moved. The method of adjusting the optical axis of the light source of the microscope according to. 6. A first light source for generating a first light that causes the molecule of the sample stained with a molecule having at least three electronic states including a ground state to transition from the ground state to a first excited state. A second light source that generates a second light that causes the molecule to transit from the first excited state to a second excited state having a higher energy level; and a second light source that emits the second light. A light source optical axis adjusting device for a microscope, comprising: an optical system for irradiating the sample with at least a part of an irradiation region overlapped thereon; It has a maximum intensity and a first fluorescence intensity detected by the detection means when the sample is irradiated with the first light of the intensity distribution and the second light of the intensity distribution having the maximum intensity. Has a first light and a minimum of intensity distribution Intensity comparison means for detecting a difference between the second fluorescence intensity distribution and the second fluorescence intensity detected by the detection means when the sample is irradiated with the second light through the optical system; and the intensity comparison means. A light source light of a microscope, comprising: an optical axis moving unit that relatively moves the optical axis of the first light source and the optical axis of the second light source based on the detected fluorescence intensity difference. Axis adjustment device. 7. A first light source for generating a first light for transitioning the molecule of the sample stained with a molecule having three electronic states including at least a ground state from the ground state to a first excited state, A second light source that generates a second light that causes the molecule to transit from the first excited state to a second excited state having a higher energy level; and a second light source that emits the second light. A light source optical axis adjusting device for a microscope, comprising: an optical system for irradiating the sample with at least a part of an irradiation region overlapped thereon; The first fluorescence intensity detected by the detection means when the sample is irradiated with the first light of the intensity distribution and the second light of the intensity distribution having a maximum in the center of the optical axis through the optical system. And the intensity distribution with a maximum in the center of the optical axis The first light and the second light having an intensity distribution having a local minimum in the vicinity of the center of the optical axis, and the second fluorescence intensity detected by the detection means when the sample is irradiated through the optical system. Intensity comparison means for detecting a difference, and optical axis movement for relatively moving the optical axis of the first light source and the optical axis of the second light source based on the fluorescence intensity difference detected by the intensity comparison means. A light source optical axis adjusting device for a microscope, comprising: 8. The optical axis moving means is arranged so that the first difference is at a position where the fluorescence intensity difference detected by the intensity comparing means becomes maximum.
8. The light source optical axis adjusting device for a microscope according to claim 6, wherein the optical axis of the light source of 1 is moved relative to the optical axis of the second light source. 9. The method according to claim 6, further comprising intensity adjusting means for adjusting the intensity of the first light and / or the second light so that the first fluorescence intensity is minimized. Or the light source optical axis adjusting device for a microscope according to the item 8. 10. The light from the second light source is intensity-modulated by a liquid crystal spatial modulator to obtain second light having an intensity distribution having a local minimum near the center of the optical axis. The light source optical axis adjusting device for a microscope according to claim 6, 7, 8 or 9. 11. The optical axis moving means fixes the optical axis of the second light source and moves the optical axis of the first light source. Or the light source optical axis adjusting device for a microscope according to the item 10.