JP2004109368A - Microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope which can irradiate a sample with a first light and a second light while keeping a proper wave surface and without causing the fluctuation of a light axis and stably exhibits super resolution phenomena at all times. <P>SOLUTION: In the microscope, the first light from a first light source 11 and the second light from second light sources 12, 13 are partly superimposed by means of a superimposing optical system 16 and are converged to the sample 35 by means of a converging optical system 34 and optical response of the sample 35 is detected by a light detector 40. Further, the microscope is constituted in such a manner that the first light or the second light from at least one side of the first light source 11 or the second light sources 12, 13 is guided to the superimposing optical system 16 as a uniform planar wave through optical fiber optical systems 15, 25 having optical fibers 22, 28 and collimator lenses 23, 29. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a microscope, in particular, a new high-performance and high-performance optical microscope that illuminates a stained sample with light of a plurality of wavelengths from a highly functional laser light source to obtain high spatial resolution.
[0002]
[Prior art]
The technology of optical microscopes is old, and various types of microscopes have been developed. In recent years, further advanced microscope systems have been developed with advances in peripheral technologies such as laser technology and electronic imaging technology.
[0003]
Against this background, a highly functional microscope that enables not only control of the contrast of the obtained image but also chemical analysis using the double resonance absorption process generated by illuminating the sample with light of multiple wavelengths is proposed. (For example, see Patent Document 1).
[0004]
This microscope uses a double resonance absorption to select a specific molecule, and observes absorption and fluorescence caused by a specific optical transition. This principle will be described with reference to FIGS. FIG. 3 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. 3 are excited by light of wavelength λ1. Then, the first electronically excited state (S1 state) shown in FIG. 4 is set. Next, the light is similarly excited by light of another wavelength λ2 to obtain a second electron excited state (S2 state) shown in FIG. Due to this excited state, the molecule emits fluorescence or phosphorescence, and returns to the ground state as shown in FIG.
[0005]
In the microscopy using the double resonance absorption process, an absorption image and an emission image are observed using the absorption process in FIG. 4 and the emission of fluorescence and phosphorescence in FIG. In this microscopy method, first, the molecules constituting the sample are excited into the S1 state as shown in FIG. 4 by laser light or the like at the resonance wavelength λ1. At this time, the number of molecules in the S1 state in a unit volume is: It increases as the intensity of the irradiated light increases.
[0006]
Here, the linear absorption coefficient is given by the product of the absorption cross-sectional area per molecule and the number of molecules per unit volume. Therefore, in the excitation process as shown in FIG. The coefficient will depend on the intensity of the light of the wavelength λ1 which is first irradiated. That is, the linear absorption coefficient for the wavelength λ2 can be controlled by the intensity of the light having the wavelength λ1. This indicates that the contrast of the transmitted image can be completely controlled by the light of the wavelength λ1 if the sample is irradiated with light of two wavelengths of the wavelength λ1 and the wavelength λ2 and a transmission image with the wavelength λ2 is taken.
[0007]
When the de-excitation process by fluorescence or phosphorescence in the excited state in FIG. 5 is possible, the emission intensity is proportional to the number of molecules in the S1 state. Therefore, when used as a fluorescence microscope, it is possible to control the image contrast.
[0008]
Furthermore, microscopy using the double resonance absorption process allows not only control of the image contrast as described above, but also chemical analysis. That is, since the outermost valence orbit shown in FIG. 3 has an energy level unique to each molecule, the wavelength λ1 differs depending on the molecule, and the wavelength λ2 also becomes unique to the molecule.
[0009]
Here, even when illuminating with a conventional single wavelength, it is possible to observe an absorption image or a fluorescence image of a specific molecule to some extent, but in general, the wavelength regions of the absorption bands of some molecules overlap, so However, accurate identification of the chemical composition of a sample is not possible.
[0010]
On the other hand, in the microscopy using the double resonance absorption process, molecules that absorb or emit at two wavelengths, wavelength λ1 and wavelength λ2, are limited, so that it is possible to identify the chemical composition of a sample more accurately than the conventional method. Become. Further, when exciting valence electrons, only light having a specific electric field vector with respect to the molecular axis is strongly absorbed. Even molecules can be used to identify the orientation direction.
[0011]
Recently, a fluorescence microscope having a high spatial resolution exceeding the diffraction limit using a double resonance absorption process has been proposed (for example, see Patent Document 2).
[0012]
FIG. 7 is a conceptual diagram of a double resonance absorption process in a molecule, in which a molecule in a ground state S0 is excited by light having a wavelength λ1 into S1, which is a first electronically excited state, and further, is excited by a light having a wavelength λ2. The state of being excited by the state S2 is shown. FIG. 7 shows that the fluorescence from S2 of a certain molecule is extremely weak.
[0013]
In the case of a molecule having optical properties as shown in FIG. 7, a very interesting phenomenon occurs. FIG. 8 is a conceptual diagram of the double resonance absorption process as in FIG. 7, in which the X axis on the horizontal axis represents the expansion of the spatial distance, and the spatial region A1 irradiated with light of wavelength λ2 and the light of wavelength λ2 are not irradiated. A spatial area A0 is shown.
[0014]
In FIG. 8, in the spatial region A0, a large number of molecules in the S1 state are generated by the excitation of the light having the wavelength λ1, and at this time, fluorescence emitted at the wavelength λ3 is seen from the spatial region A0. However, in the spatial region A1, since the light of the wavelength λ2 is irradiated, most of the molecules in the S1 state are immediately excited to the higher-order S2 state, and the molecules in the S1 state no longer exist. Such a phenomenon has been confirmed by several molecules. As a result, in the spatial region A1, the fluorescence of the wavelength λ3 completely disappears, and since the fluorescence from the S2 state is not originally present, the fluorescence itself is completely suppressed in the spatial region A1 (fluorescence suppressing effect), and only from the spatial region A0. Fluorescence will be emitted.
[0015]
This is extremely important when viewed from the microscope application field. That is, in a conventional scanning laser microscope or the like, a laser beam is condensed into a micro beam by a condenser lens to scan the observation sample, and the size of the micro beam at that time depends on the numerical aperture of the condenser lens and the wavelength. , And a higher spatial resolution cannot be expected in principle.
[0016]
However, in the case of FIG. 8, the two types of light of the wavelength λ1 and the wavelength λ2 are spatially well overlapped, and the fluorescent region is suppressed by irradiation with the light of the wavelength λ2. Focusing on the irradiation area, the fluorescent area can be 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 utilizing this principle, it becomes possible to realize a super-resolution microscope, for example, a fluorescence microscope, using a double resonance absorption process exceeding the diffraction limit.
[0017]
Furthermore, in order to enhance the super-resolution of the microscope, a fluorescent labeler molecule for fully utilizing the function of the super-resolution microscope and the timing of irradiating the sample with two lights of the wavelengths λ1 and λ2 to be used are disclosed. (For example, see Patent Document 3). This prior art has at least three quantum states including a ground state, and the transition when de-excited from a higher energy state excluding the first electronically excited state to the ground state is more a thermal relaxation process than a fluorescence relaxation process. A sample in which a fluorescent labeler molecule that stains various dominant molecules and a biomolecule that has been subjected to biochemical staining technology are chemically bonded is excited into the S1 state by light having a wavelength λ1 that excites the molecule to be stained. Subsequently, the light of wavelength λ2 is immediately excited to a higher quantum level to suppress the fluorescence from the S1 state. As described above, the spatial resolution can be improved by artificially suppressing the spatial fluorescent region using the optical properties of molecules.
[0018]
The optical properties of such molecules can be explained from a quantum chemical standpoint. That is, generally, each atom constituting the molecule is connected by σ or π bond. In other words, the molecular orbital of the molecule has a σ molecular orbital or a π molecular orbital, and electrons existing in these molecular orbitals play an important role of bonding each atom. Among them, the electron of the σ molecular orbit strongly bonds each atom, and determines the distance between atoms in the molecule which is the skeleton of the molecule. On the other hand, electrons in the π molecular orbital make little contribution to the bonding of each atom, but are rather bound to the whole molecule by an extremely weak force.
[0019]
In many cases, when an electron in a σ molecular orbital is excited by light, the interatomic distance of the molecule changes greatly, causing a large structural change including dissociation of the molecule. As a result, most of the energy given to the molecule by light changes to thermal energy due to the kinetic energy and structural change of the atom. Therefore, the excitation energy is not consumed in the form of light called fluorescence. In addition, since the structural change of a molecule occurs at a very high speed (less than picosecond), even if fluorescence occurs in the process, the life is extremely short.
[0020]
On the other hand, the electrons in the π molecular orbital structure hardly change even when excited, stay at a high quantum discrete level for a long time, emit fluorescence in the order of nanoseconds, and excite them. Has properties.
[0021]
According to quantum chemistry, a molecule has a π molecular orbit and a double bond is equivalent, and it is necessary to select a molecule with a large number of double bonds for the fluorescent labeler molecule used. It becomes. It has been confirmed that, even in a molecule having a double bond, a 6-membered ring molecule such as benzene or pyrazine has extremely weak fluorescence from the S2 excited state (for example, see Non-Patent Document 1).
[0022]
Therefore, if a molecule containing a six-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 the light is excited from the S1 state to the S2 state by photoexcitation. Can be easily suppressed, so that the super-resolution can be effectively used. In other words, if observation is performed by staining with these fluorescent labeler molecules, not only can the fluorescence image of the sample be observed with high spatial resolution, but also by adjusting the chemical groups of the side chains of the molecule, the biological sample can be analyzed. Since only a specific chemical tissue can be selectively stained, even the detailed chemical composition of the sample can be analyzed.
[0023]
In general, the double resonance absorption process occurs only when the two light wavelengths, polarization states, and the like satisfy specific conditions. Therefore, by using this, the structure of a molecule can be known in detail. 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 each of the two wavelengths of light and the orientation direction of molecules form a specific angle, the double resonance absorption process occurs. It happens strongly. Therefore, by simultaneously irradiating the sample with light of two wavelengths and rotating the polarization direction of each light, the extent of the disappearance of the fluorescence changes. Can also be obtained. This is also possible by adjusting the wavelengths of the two lights.
[0024]
As described above, according to the technique described in Patent Literature 3, it is understood that the technique has a high analysis capability in addition to the super-resolution. Further, by devising the irradiation timing of the two lights of the wavelength λ1 and the wavelength λ2, the S / N can be improved and the fluorescence can be suppressed effectively, and the super-resolution is more effectively exhibited. It is possible to do.
[0025]
As a specific example of such super-resolution microscopy, light of wavelength λ1 (particularly, laser light) that excites fluorescent labeler molecules from the S0 state to the S1 state is used as pump light, and wavelength λ2 that excites the S1 state to the S2 state is used. As shown in FIG. 9, pump light is emitted from a light source 81 and erase light is emitted from a light source 82, and the pump light is reflected by a dichroic mirror 83. The beam is condensed on the upper side, and the erase light is formed into a hollow beam by the phase plate 86, then transmitted through the dichroic mirror 83, spatially overlapped with the pump light, and condensed on the sample 85 by the annular optical system 84. The following has been proposed (for example, see Patent Document 4).
[0026]
According to this microscope, fluorescence other than near the optical axis where the intensity of the erase light becomes zero is suppressed, and as a result, a region narrower than the spread of the pump light (Δ <0.61 · λ1 / NA, NA Only the fluorescent labeler molecules present in the (numerical aperture of the optical system 84) are observed, and as a result, super-resolution is developed. As the phase plate 86 for converting the erase light into a hollow beam, for example, as shown in FIG. 10, a structure in which a phase difference π is provided at a point symmetric position with respect to the optical axis, or a liquid crystal surface is used. A liquid crystal spatial modulator can be used.
[0027]
[Patent Document 1]
JP-A-8-184552
[Patent Document 2]
JP 2001-100102 A
[Patent Document 3]
JP-A-11-95120
[Patent Document 4]
JP 2001-272345 A
[Non-patent document 1]
M. Fujii et. al. Chem. Phys. Lett. 171 (1990) 341
[0028]
[Problems to be solved by the invention]
As described above, the conventionally proposed super-resolution fluorescence microscope is excellent in various functions in observation performance. However, in such a microscope, two colors of laser light sources having different wavelengths are used, so that the light source requires further stability in order to sufficiently exhibit the performance of the microscope. That is, the degree of occurrence of the super-resolution phenomenon depends on how the erase light and the pump light are condensed and overlapped on the sample without displacement. In particular, when a pulsed laser is used as the light source, if the optical axis of the pump light and the optical axis of the erase light fluctuate independently for each laser shot, the area where fluorescence should not be suppressed originally This also suppresses the fluorescence from the light source, so that the resolution is not improved, but rather the fluorescence intensity is reduced and the image quality is degraded.
[0029]
Hereinafter, a case where the super-resolution fluorescence microscope is configured as shown in FIG. 11 will be described as an example. The super-resolution fluorescence microscope shown in FIG. 11 is disclosed in Patent Document 2 mentioned above, and observes a sample 100 stained with rhodamine 6G. In this microscope, a laser beam having a wavelength of 1064 nm emitted from a mode-locked Nd: YAG laser 101 is converted into a laser beam having a wavelength of 532 nm, which is a second harmonic, by a wavelength conversion element 102 made of beta barium borate (BBO) crystal. Then, the laser light is branched into two optical paths of transmitted light and reflected light by a half mirror 103, and the transmitted light is sequentially transmitted through dichroic mirrors 104 and 105 as pump light. Light is condensed on the sample 100 placed on the stage 107.
[0030]
The light reflected by the half mirror 103 is reflected by the reflection mirror 108 and then reflected by Ba (NO ₃ ) ₂ The laser light having a wavelength of 599 nm is converted into a laser beam having a wavelength of 599 nm by a Raman shifter 109 made of crystal, and the laser beam is reflected by a reflection mirror 110 as an erase light. It is arranged coaxially and condensed on the sample 100 by the objective lens 106 via the dichroic mirror 105.
[0031]
On the other hand, the fluorescence from the sample 100 is reflected by the dichroic mirror 105 via the objective lens 106, and then received by the photomultiplier 115 via the fluorescence condenser lens 112, the sharp cut filter 113, and the pinhole 114. . The pump light and the erase light projected on the sample 100 are received by the photomultiplier 116, and based on their outputs, the intensity of the laser light emitted from the Nd: YAG laser 101 is controlled so as to be constant. It has become.
[0032]
In the super-resolution fluorescence microscope having such a configuration, the following problem occurs particularly when the Nd: YAG laser 101 is pulse-oscillated.
1) The optical axis of the laser light fluctuates with each pulse oscillation.
2) Since the wavefront of the laser light is disturbed, it is difficult to apply the method to erase light on the premise of shape shaping by spatial modulation.
[0033]
In particular, regarding the above 1), if the laser light is guided along a long optical path, the optical axis of the laser light fluctuates even with fluctuations in the air, and the positional stability at the focal point further deteriorates. For this reason, in the worst case, the focusing area of the pump light and the focusing area of the erase light do not overlap, and the super-resolution effect may not appear at all.
[0034]
As a solution to the problem 1), it is conceivable to transmit a pulse laser beam using an optical fiber. However, in the case of a pulse laser, unlike a continuous wave laser, which is a so-called CW laser, although the time-average light intensity of the laser light is small, the pulse peak value may reach as much as megawatts. There are problems such as burning of the incident end face and generation of stimulated Raman light in the optical fiber. In particular, when stimulated Raman light is generated, the light becomes a background when observing a fluorescence signal from the sample, and the image quality of a microscope image is degraded.
[0035]
Accordingly, a main object of the present invention made in view of the above point is that a sample can be irradiated with the first light and the second light without causing a change in the optical axis while maintaining a good wavefront, and the sample is always stable. To provide a microscope capable of exhibiting a super-resolution phenomenon.
[0036]
[Means for Solving the Problems]
According to a first aspect of the present invention, there is provided a condensing optical system in which a first light from a first light source and a second light from a second light source are partially overlapped by an overlapping optical system. In a microscope that is condensed on a sample, and the optical response of the sample is detected by a photodetector,
The first light or the second light from at least one of the first light source and the second light source is guided to the superposition optical system as a uniform plane wave via an optical fiber optical system having an optical fiber and a collimator lens. It is characterized by having such a configuration.
[0037]
The invention according to claim 2 is the microscope according to claim 1, wherein at least one of the first light source and the second light source is a pulse light source.
[0038]
According to a third aspect of the present invention, in the microscope according to the first or second aspect, the wavelength of the first light is such that the molecules of the sample containing molecules having at least three electronic states including the ground state are converted to the ground state. Within the resonance absorption wavelength band for transitioning from to the first electronically excited state,
The wavelength of the second light is within a resonance absorption wavelength band that causes the molecule to transition from the first electronically excited state to another electronic state having a higher energy level.
[0039]
The invention according to claim 4 is the microscope according to any one of claims 1 to 3, wherein the optical fiber optical system is disposed between the optical fiber and the first light source or the second light source. Characterized in that it has a spatial filter described in the following.
[0040]
According to a fifth aspect of the present invention, in the microscope according to any one of the first to fourth aspects, the optical fiber optical system is arranged such that the first optical fiber is disposed between the optical fiber and the superposition optical system. It has a wavelength dispersion element that transmits light or second light.
[0041]
First, the principle of the present invention will be described. For example, as described in Suematsu and Iga: “Introduction to Optical Fiber Communication,” Ohmsha (1979), when light of a wavelength satisfying the conditions enters the single-mode optical fiber, the uniform The wavefront propagates, and a spherical wave whose wavefront is completely aligned is emitted from the emission end face. Therefore, if this light is collimated by, for example, a lens, parallel light having a uniform wavefront can be generated.If the parallel light having the uniform wavefront is formed into a hollow beam by a phase plate or a liquid crystal type spatial light modulator, It is possible to obtain an ideal hollow erase light. In addition, such an optical system using an optical fiber has a feature that fluctuation of the optical axis does not occur in principle if the emitting end of the optical fiber is mechanically fixed.
[0042]
However, for example, pulsed light emitted from a laser light source has a very poor spatial mode, does not have a so-called Gaussian profile with a uniform wavefront, and has various angles of divergence (divergence). Although it cannot be stopped down to the diffraction limit by light, it is common. For example, an optical fiber having a core diameter of about 5 μm corresponds to light having a wavelength of 600 nm. In this case, the focused diameter of the pulse light is much larger than the core diameter of the optical fiber.
[0043]
In super-resolution microscopy, in particular, it is preferable that the intensity of the erase light is large. However, in order to extract the erase light having a high intensity from the output end face of the energetic optical fiber, the laser light incident on the incident end face of the optical fiber is also increased. There is a need. At this time, light condensed on the incident end face at a shallow angle equal to or smaller than the critical angle can be extracted from the exit end face with an efficiency close to 100% although there is a small amount of attenuation. The light is scattered at the incident end face and cannot pass through the optical fiber. In addition, since this portion is always irradiated with strong light, damage due to burning occurs, and the entire incident end face of the optical fiber is burnt. This is because the higher-order modes that occupy most of the pulsed laser beam could not be confined within the core diameter of the optical fiber.
[0044]
Therefore, in a preferred embodiment of the present invention, an optical fiber optical system is provided with a spatial filter, and laser light from a laser light source is incident on the optical fiber via the spatial filter. Specifically, as shown in FIG. 1, the spatial filter 1 includes a condenser lens 2, a pinhole 3, a collimator lens 4, and a condenser lens 5, and laser light from a laser light source is condensed by the condenser lens 2. After being condensed on the pinhole 3, the light diffracted by the pinhole 3 is collimated by the collimator lens 4, and is matched with the solid angle taken in by the single mode optical fiber 6. Again. The size of the pinhole 3 is set based on the size of the diffraction limit determined by the focal length of the condenser lens 2 and the beam diameter of the incident laser beam.
[0045]
This makes it possible to take in all the light condensed in the core of the optical fiber 6 and take it out as a spherical wave. In addition, since excess light that causes burning of the end face of the optical fiber is cut by the pinhole 3 of the spatial filter 1, the optical fiber 6 is not burned during the measurement, so that it cannot be used. Therefore, even if the intensity of the laser light itself is increased in order to extract the high-intensity light emitted from the optical fiber, the optical fiber 6 does not burn out.
[0046]
The stimulated Raman light generated in the optical fiber can be dealt with by applying a very simple filter optical system. For example, in the super-resolution fluorescence microscope described in Patent Document 4, a notch filter that cuts wavelength components of pump light and erase light is generally arranged in front of a detector that detects fluorescence from a sample. When stimulated Raman light is generated in the optical fiber, the notch filter is configured such that the emission spectrum band of the stimulated Raman light is located within the pass blocking wavelength band of the notch filter.
[0047]
Alternatively, an optical fiber optical system is configured by disposing a wavelength dispersion element that transmits pump light or erase light of a desired wavelength on the emission end side of the optical fiber. As the wavelength dispersion element, a narrow-band bandpass filter, an interference filter using a multilayer film, a diffraction grating, or the like can be used.
[0048]
In a preferred embodiment of the present invention, in order to more reliably cut the stimulated Raman light, the wavelength of the pump light or the erase light is centered as a wavelength dispersion element on the emission end face side of the optical fiber that transmits the pump light or the erase light. An optical fiber optical system is configured by arranging a band-pass filter having a narrow band having a wavelength, and the pump light or the erase light emitted from the optical fiber and transmitted through the band-pass filter is projected on the sample. Further, a notch filter having a pass blocking wavelength band wider than the pass wavelength band of the above band pass filter is arranged in front of the detector for receiving the light emitted from the sample, and the light transmitted through the notch filter is detected. To receive the light.
[0049]
In this way, not only stimulated Raman light generated in the optical fiber but also pump light and erase light can be reliably prevented from entering the detector, and only fluorescence from the sample can be received. The background can be reduced. As the notch filter disposed in front of the detector, for example, a dielectric multilayer mirror of a direct incidence design or the like can be used.
[0050]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, an embodiment of the microscope according to the present invention will be described in detail with reference to FIG.
[0051]
FIG. 2 is a diagram showing a main configuration of the optical system of the microscope according to the embodiment of the present invention. The microscope according to the present embodiment is a laser scanning type super-resolution fluorescence microscope for observing a biological sample stained with rhodamine 6G.
[0052]
Here, Rhodamine 6G has the maximum absorption by excitation from the ground state (S0) to the first electronically excited state (S1) in the excitation wavelength band of 530 nm. Further, an absorption band capable of being excited from the first electronically excited state (S1) to a higher energy electronically excited state exists in a wavelength range of 600 to 650 nm.
[0053]
Therefore, in the present embodiment, a pulsed Nd: YAG laser 11 is used as a first light source, and its second harmonic (532 nm) is used as pump light (first light). The second light source is composed of a pulsed Nd: YAG laser 12 and Ba (NO ₃ ) ₂ A Raman shifter 13 made of a crystal, wherein the second harmonic of the pulsed Nd: YAG laser 12 is converted into a laser beam having a wavelength of 599 nm by the Raman shifter 13, and the laser beam having a wavelength of 599 nm is erased ( Second light).
[0054]
The pulse Nd: YAG lasers 11 and 12 can oscillate picosecond laser pulses at a repetition frequency on the order of MHz by adjusting the design parameters of the laser resonator. For example, the GE-100 series manufactured by Time-Bandwidth, Switzerland, can oscillate pulse light having a pulse width of 6 psec with an average output of 50 mW at a repetition frequency of 100 MHz as a standard specification. This corresponds to the energy of one pulse corresponding to 500 pJ. Also, the repetition frequency can be adjusted between 25 MHz and 1 GHz by changing the design of the resonator.
[0055]
The pump light from the pulsed Nd: YAG laser 11 passes through a pump light optical fiber optical system 15 and is incident on a beam combiner 16 composed of a cuvette type half mirror constituting a superposition optical system. The pumping optical fiber optical system 15 includes an optical coupling unit 21 having a spatial filter shown in FIG. 1, a polarization-maintaining single mode fiber 22, a collimator lens 23, and a narrow band having a central wavelength of 532 nm, which is a wavelength dispersion element. , And the pump light from the pulsed Nd: YAG laser 11 is made incident on the core of the polarization-maintaining single-mode fiber 22 via the optical coupling section 21 to be propagated. After the pump light emitted from the wavefront preserving single mode fiber 22 is shaped into parallel light by the collimator lens 23, the light is incident on the beam combiner 16 via the band pass filter 24. As the bandpass filter 24, a commercially available narrow band filter (for example, manufactured by Barr Associates Inc., USA) having a center wavelength of 532 nm and a pass wavelength bandwidth of 1 nm formed of a dielectric multilayer film can be used.
[0056]
As described above, when the pump light from the pulsed Nd: YAG laser 11 is made incident on the polarization-maintaining single-mode fiber 22 via the optical coupling section 21 having the spatial filter shown in FIG. Since only the component can be selected and incident on the polarization-maintaining single-mode fiber 22, the light emitted from the polarization-maintaining single-mode fiber 22 is converted into parallel light by the collimator lens 23, so that the pump light of the uniform plane wave is obtained. Can be obtained. Further, if the pump light from the collimator lens 23 is made incident on a narrow band band-pass filter 24 having a center wavelength of 532 nm, even if stimulated Raman light is generated in the Passage can be blocked by the pass filter 24, and pump light of a desired wavelength (532 nm) can be obtained.
[0057]
On the other hand, the erase light from the Raman shifter 13 is made incident on the phase plate 26 as shown in FIG. 10 through the optical fiber optical system 25 for erase light, where it is made into a hollow beam and made incident on the beam combiner 16 to be combined with the pump light. Synthesize coaxially. The optical fiber optical system 25 for the erase light, like the optical fiber optical system 15 for the pump light, includes an optical coupling section 27 having a spatial filter shown in FIG. 1, a polarization-maintaining single-mode fiber 28, and a collimator lens 29. A narrow band pass filter 30 having a center wavelength of 599 nm, which is a wavelength dispersing element, and the erase light from the Raman shifter 13 is applied to the core of the polarization-maintaining single mode fiber 28 via the optical coupling unit 27. The erase light emitted from the polarization-maintaining single-mode fiber 28 is shaped into a collimated beam by a collimator lens 29, and then enters the phase plate 26 via the band-pass filter 30. As the bandpass filter 30, a narrow band filter having a center wavelength of 599 nm and a pass wavelength bandwidth of 1 nm formed of, for example, a dielectric multilayer film is used.
[0058]
As described above, the erase light from the Raman shifter 13 is incident on the polarization-maintaining single-mode fiber 28 via the optical coupling unit 27 having the spatial filter shown in FIG. A uniform spherical wave erase light can be obtained by converting the light emitted from the polarization-maintaining single mode fiber 28 into a parallel light by the collimator lens 29. Further, by irradiating the erase light from the collimator lens 29 to the narrow-band bandpass filter 30 having a center wavelength of 599 nm, even if stimulated Raman light is generated in the polarization-maintaining single-mode fiber 28, it is transmitted to the bandpass filter. The passage of the light can be blocked by the filter 30, and the erase light of a desired wavelength (599 nm) can be obtained.
[0059]
The pump light and the erase light which are coaxially combined by the beam combiner 16 are transmitted through a beam separator 31 composed of a cuvette-type half mirror, and thereafter, are galvanomirrors 32 and 33 which can swing about axes orthogonal to each other. And sequentially condensed on the surface of the biological sample 35 stained with rhodamine 6G by the objective lens 34, and the condensing point is moved by swinging the galvanometer mirrors 32 and 33 to two-dimensionally scan the sample surface. . In FIG. 2, for simplification of the drawing, the galvanometer mirrors 32 and 33 are shown as swinging about axes parallel to each other.
[0060]
Also, the fluorescence emitted from the sample 35 by the irradiation of the pump light and the erase light is collimated by the objective lens 34, then follows a path opposite to the incident optical path, and enters the beam separator 31 through the galvanomirrors 33 and 32, The fluorescence reflected by the beam separator 31 is focused on a pinhole 37 by a focusing lens 36.
[0061]
The pinhole 37 is disposed at a confocal position with respect to the fluorescent light emitting point of the sample 35, and the fluorescence transmitted through the pinhole 37 is transmitted through the pump light cut notch filter 38 and the erase light cut notch filter 39, respectively. After removing the pump light and the erase light, the light is received by the photomultiplier tube 40 as a photodetector to obtain a fluorescence output signal. The fluorescence output signal is stored in a video frame memory of a computer (not shown) for controlling the microscope. Then, a two-dimensional fluorescence image of the sample 35 is displayed on a monitor (not shown) in real time. The galvanomirrors 32 and 33 swing in synchronization with the video rate of the fluorescent two-dimensional image displayed on the monitor.
[0062]
The pass blocking wavelength band of the pump light cut notch filter 38 is made wider than the pass wavelength band of the band pass filter 24 in the pump light optical fiber optical system 15. Similarly, the pass blocking wavelength band of the erase light cut notch filter 39 is set wider than the pass wavelength band of the band pass filter 30 in the optical fiber optical system 25 for erase light. The pump light cut notch filter 38 and the erase light cut notch filter 39 are a single commercially available filter (for example, manufactured by Barr Associates Inc., USA) that blocks passage of light in a wavelength band including the pump light and the erase light. Can also be configured.
[0063]
As described above, in the present embodiment, the pump light from the pulse Nd: YAG laser 11 is made incident on the beam combiner 16 via the optical fiber optical system 15 for pump light, and the erase light from the Raman shifter 13 is erased. The light is made to enter the phase plate 26 through the optical fiber optical system 25 for light, is made into a hollow beam here, and is made to enter the beam combiner 16 so as to be coaxially combined with the pump light. Can be overlapped on the surface of the sample without fluctuations of the sample, and the super-resolution effect can be reliably obtained. In addition, since the pulse Nd: YAG laser 11, the pulse Nd: YAG laser 12, and the Raman shifter 13 can be arranged separately from the optical bench having the objective lens 34, there is an advantage that the degree of freedom in designing a microscope can be increased.
[0064]
Further, in the optical fiber optical system 15 for pump light, a spatial filter is provided on the incident end face side of the polarization-maintaining single mode fiber 22, and the collimator lens 23 is provided on the output end face side. Not only can it be extracted and converted into a uniform plane wave, but also extra light that causes burns on the end face of the optical fiber can be cut. Similarly, in the optical fiber optical system 25 for erasing light, a spatial filter is provided on the incident end face side of the polarization-maintaining single mode fiber 28, and a collimator lens 29 is provided on the emitting end face side. Can be taken out and converted into a uniform plane wave, and excess light that causes burning of the end face of the optical fiber can be cut. Therefore, in particular, in the case of the erase light, the hollow beam can be accurately formed by the phase plate 26, the super-resolution can be reliably exhibited, and the intensity of the laser light itself is increased in order to extract the high-intensity erase light. Even so, the polarization-maintaining single-mode fiber 28 is not burned.
[0065]
Further, the optical fiber optical system 15 for pump light is provided with a narrow-band bandpass filter 24 on the emission end face side of the polarization-maintaining single mode fiber 22 so that only the pump light of a desired wavelength is transmitted. In the optical fiber optical system 25 for erasing light, a narrow-band bandpass filter 30 is provided on the emission end face side of the polarization-maintaining single mode fiber 28 so as to transmit only erasing light of a desired wavelength. Even if stimulated Raman light is generated in the wavefront preserving single mode fibers 22 and 28, it can be reliably prevented from passing through. Therefore, it is possible to prevent the stimulated Raman light from becoming a background when observing the fluorescence signal from the sample 35. Moreover, in the present embodiment, the pump light cut notch filter 38 having a pass blocking wavelength band wider than the pass wavelength band of the band pass filter 24 and the pass wavelength band of the band pass filter 30 are located ahead of the photomultiplier tube 40. Since the erase light cut notch filter 39 having a wide passband wavelength band is arranged, not only the stimulated Raman light generated in the polarization-maintaining single mode fibers 22 and 28 but also the pump light and the erase light are transmitted to the photomultiplier tube 40. Since it is possible to reliably prevent the incident light and to receive only the fluorescence from the sample 35, it is possible to obtain a microscope image with good image quality.
[0066]
It should be noted that the present invention is not limited only to the above-described embodiment, and various modifications or changes can be made. For example, when stimulated Raman light is generated in the polarization-maintaining single mode fibers 22 and 28, the emission spectrum band of the stimulated Raman light is within the pass blocking wavelength band of the pump light cut notch filter 38 and the erase light cut notch filter 39. , The bandpass filters 24 and 30 can be omitted. Further, the phase modulation element for shaping the erase light into a hollow beam is not limited to the above-described phase plate, and a liquid crystal spatial light modulator or other known elements can be used. Further, in the above embodiment, the pump light and the erase light are pulsed light, but the present invention can be effectively applied to a case where one or both of them are continuous waves.
[0067]
Additional items
1. The first light from the first light source and the second light from the second light source are partially overlapped by the superimposing optical system and condensed on the sample by the condensing optical system. In a microscope that detects with a photodetector,
The first light or the second light from at least one of the first light source and the second light source is guided to the superposition optical system as a uniform plane wave via an optical fiber optical system having an optical fiber and a collimator lens. A microscope characterized by having such a configuration.
2. 2. The microscope according to claim 1, wherein at least one of the first light source and the second light source is a pulse light source.
3. The wavelength of the first light is within a resonance absorption wavelength band that causes the molecules of the sample including molecules having at least three electronic states including the ground state to transition from the ground state to the first electronically excited state,
The wavelength of the second light is within a resonance absorption wavelength band that causes the molecule to transition from the first electronically excited state to another electronic state having a higher energy level. Microscope as described.
4. The microscope according to any one of additional items 1 to 3, wherein the optical fiber optical system includes a spatial filter disposed between the optical fiber and the first light source or the second light source. .
5. The optical fiber optical system further includes a wavelength dispersion element disposed between the optical fiber and the superposition optical system, the wavelength dispersive element transmitting the first light or the second light. The microscope according to any one of claims 1 to 4.
6. The notch filter according to claim 1, wherein a notch filter is disposed in front of the photodetector so as to allow light emitted by the optical response of the sample to pass therethrough and to prevent passage of the first light or the second light. The microscope according to any one of the preceding claims.
7. The optical fiber optical system has a wavelength dispersion element that transmits the first light or the second light disposed between the optical fiber and the superposition optical system, and is provided in front of the photodetector. A notch filter for passing light emitted by the optical response of the sample and blocking the passage of the first light or the second light, and setting a pass blocking wavelength band of the notch filter to a pass wavelength of the wavelength dispersive element. 5. The microscope according to any one of additional items 1 to 4, wherein the microscope is wider than the band.
8. 8. The microscope according to additional item 5 or 7, wherein the wavelength dispersion element includes a bandpass filter.
9. 8. The microscope according to claim 6, wherein the notch filter comprises a multilayer film wavelength dispersion element.
10. 8. The microscope according to claim 6, wherein the notch filter comprises a direct-incidence type multilayer film reflecting mirror.
[0068]
【The invention's effect】
As described above, according to the present invention, at least one of the first light from the first light source and the second light from the second light source passes through the optical fiber optical system having the optical fiber and the collimator lens. The first light and the second light are partially superimposed by a superimposing optical system, are converged on a sample by a condensing optical system, and the optical response of the sample is detected by a photodetector. Therefore, it is possible to irradiate the sample with the first light and the second light without changing the optical axis while maintaining a good wavefront, and it is possible to always exhibit the super-resolution phenomenon stably.
[Brief description of the drawings]
FIG. 1 is a diagram for explaining the principle of the present invention.
FIG. 2 is a diagram illustrating a main configuration of an optical system of a microscope according to an embodiment of the present invention.
FIG. 3 is a conceptual diagram showing an electronic structure of a valence orbit of molecules constituting a sample.
FIG. 4 is a conceptual diagram showing a first electronically excited state of the molecule of FIG. 3;
FIG. 5 is a conceptual diagram showing a second electronically excited state.
FIG. 6 is a conceptual diagram showing a state of returning from a second electronically excited state to a ground state.
FIG. 7 is a conceptual diagram for explaining a double resonance absorption process in a molecule.
FIG. 8 is a conceptual diagram for explaining a double resonance absorption process.
FIG. 9 is a diagram showing a configuration of an example of a conventional super-resolution microscope.
FIG. 10 is a plan view showing a configuration of a phase plate shown in FIG. 9;
FIG. 11 is a diagram showing a configuration of another example of a conventional super-resolution microscope.
[Explanation of symbols]
1 Spatial filter
2 Condensing lens
3 Pinhole
4 Collimator lens
5 Condensing lens
6 Single mode optical fiber
11,12 pulse Nd: YAG laser
13 Raman shifter
15 Optical fiber optical system for pump light
16 Beam combiner
21,27 Optical coupling part
22,28 polarization-maintaining single-mode fiber
23, 29 Collimator lens
24, 30 bandpass filter
25 Optical fiber optical system for erase light
26 Phase plate
31 beam separator
32,33 Galvanometer mirror
34 Objective lens
35 samples
36 Condenser lens
37 Pinhole
38 Pump light cut notch filter
39 Erase light cut notch filter
40 Photomultiplier tube

Claims (5)

The first light from the first light source and the second light from the second light source are partially overlapped by the superimposing optical system and condensed on the sample by the condensing optical system. In a microscope that detects with a photodetector,
The first light or the second light from at least one of the first light source and the second light source is guided to the superposition optical system as a uniform plane wave via an optical fiber optical system having an optical fiber and a collimator lens. A microscope characterized by having such a configuration. The microscope according to claim 1, wherein at least one of the first light source and the second light source is a pulse light source. The wavelength of the first light is within a resonance absorption wavelength band that causes the molecules of the sample including molecules having at least three electronic states including the ground state to transition from the ground state to the first electronically excited state,
The wavelength of the second light is within a resonance absorption wavelength band that causes the molecule to transition from the first electronically excited state to another electronic state having a higher energy level. Microscope as described. The microscope according to any one of claims 1 to 3, wherein the optical fiber optical system includes a spatial filter disposed between the optical fiber and the first light source or the second light source. . 5. The optical fiber optical system according to claim 1, further comprising a wavelength dispersive element that transmits the first light or the second light disposed between the optical fiber and the superposition optical system. The microscope according to any one of claims 1 to 4.

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