JP2006058477A - Ultra-high resolution microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an ultra-high resolution microscope capable of easily and accurately performing an optical adjustment of pumping light and erasing light, and capable of surely accomplishing ultra-high resolution effect. <P>SOLUTION: In the ultra-high resolution microscope, the pumping light emitted from a pumping light source 31 and the erasing light emitted from an erasing light source 32 are partly overlaid on top of each other by an optical system 72, condensed on a sample 74, and scanned by scanning means 52 and 53, and an optical reply signal from the sample 74 is detected by a detecting means 58, wherein the pumping light emitted from the light source 31 and the erasing light emitted from the light source 32 are coupled on the axis by an optical fiber 35. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microscope, particularly a high-performance, high-performance super-resolution microscope that obtains high spatial resolution by illuminating a stained sample with light of a plurality of wavelengths from a highly functional laser light source. The sample is irradiated with light of the first color of wavelength, the molecules in the sample are transitioned from the ground state to another quantum state, and the sample is irradiated with light of the second color different from the wavelength of the first color. The present invention relates to an optical microscope for observing various optical responses.

  Optical microscope technology is old and various types of microscopes have been developed. In recent years, more advanced microscope systems have been developed due to advances in peripheral technologies such as laser technology and electronic image technology.

  Against this background, we propose a highly functional microscope that not only controls the contrast of the resulting image but also enables chemical analysis using the double resonance absorption process that occurs when the sample is illuminated with light of multiple wavelengths. (For example, refer to Patent Document 1).

This microscope selects a specific molecule using double resonance absorption and observes absorption and fluorescence caused by a specific optical transition. This principle will be described with reference to FIGS. FIG. 16 shows the electronic structure of the valence orbital of the molecules constituting the sample. First, the electrons in the valence orbital of the molecule in the ground state (S ₀ state) shown in FIG. 16 are excited by light of wavelength λ1. to, the first electronic excited state shown in FIG. 17 _{(S 1} state). Next, similarly excited by the light of another wavelength .lambda.2, and second electronic excited state shown in FIG. 18 (S ₂ state). In this excited state, the molecule emits fluorescence or phosphorescence and returns to the ground state as shown in FIG.

In the microscopy using the double resonance absorption process, an absorption image and a light emission image are observed using the absorption process of FIG. 17 and the fluorescence and phosphorescence emission of FIG. In this microscope method, first, the molecules constituting the sample are excited to the S ₁ state by laser light or the like with the light having the resonance wavelength λ1, as shown in FIG. 17. At this time, the number of molecules in the S ₁ state within the unit volume is excited. Increases as the intensity of the irradiated 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. 18, the linear absorption for the resonance wavelength λ2 that is subsequently irradiated. The coefficient depends on the intensity of the light having the wavelength λ1 irradiated first. That is, the linear absorption coefficient with respect to the wavelength λ2 can be controlled by the intensity of the light with the wavelength λ1. This indicates that the contrast of the transmitted image can be completely controlled by the light of the wavelength λ1 when the sample is irradiated with the light of the two wavelengths of the wavelength λ1 and the wavelength λ2 and a transmission image by the wavelength λ2 is taken.

When the deexcitation process by fluorescence or phosphorescence in the excited state of FIG. 18 is possible, the emission intensity is proportional to the number of molecules in the S ₁ state. Therefore, image contrast can be controlled even when used as a fluorescence microscope.

  Furthermore, the microscope method using a double resonance absorption process enables not only the above-described image contrast control but also chemical analysis. That is, since the outermost shell valence electron orbit shown in FIG. 16 has an energy level unique to each molecule, the wavelength λ1 differs depending on the molecule, and the wavelength λ2 is also unique to the molecule.

  Here, even when illuminating with a conventional single wavelength, it is possible to observe an absorption image or fluorescence image of a specific molecule to some extent, but generally the wavelength regions of absorption bands of several molecules overlap. It is impossible to accurately identify the chemical composition of the sample.

  On the other hand, in the microscope method using the double resonance absorption process, the molecules that absorb or emit light are limited by the two wavelengths of λ1 and λ2, so that the chemical composition of the sample can be identified more accurately than the conventional method. Become. In addition, when valence electrons are excited, only light having a specific electric field vector with respect to the molecular axis is strongly absorbed. Therefore, if the polarization direction of the wavelengths λ1 and λ2 is determined and an absorption or fluorescence image is taken, the same Even molecules can be used to identify the orientation direction.

  Recently, a fluorescence microscope having a high spatial resolution exceeding the diffraction limit using a double resonance absorption process has also been proposed (see, for example, Patent Document 2).

FIG. 20 is a conceptual diagram of a double resonance absorption process in a molecule. A molecule in the ground state S ₀ is excited by light having a wavelength λ1 to S ₁ which is a first electronically excited state, and is further excited by light having a wavelength λ2. shows a state that is excited to S ₂ is an electron excited state. FIG. 20 shows that the fluorescence from S _{2 of} certain molecules is extremely weak.

In the case of a molecule having optical properties as shown in FIG. 20, a very interesting phenomenon occurs. Figure 21 is a conceptual diagram of a likewise double resonance absorption process and FIG. 20, X-axis of abscissa represents the spread of spatial distance, the light of the spatial region A ₁ and the wavelength λ2 was irradiated with light of wavelength λ2 irradiated shows a spatial region a ₀ which is not.

In Figure 21, the molecules of S ₁ state is generated number by the excitation light in the spatial domain A ₀ the wavelength .lambda.1, fluorescence is observed to emit light at a wavelength λ3 from the spatial region A ₀ at that time. However, the spatial area A _1, since irradiated with light of wavelength .lambda.2, most molecules of S ₁ state immediately be excited to higher S ₂ state, molecules of S ₁ state is no longer present. Such a phenomenon has been confirmed by several molecules. Thus, the spatial region A _1, the fluorescence of the wavelength λ3 is completely eliminated, and since the fluorescent is not originally from S ₂ state, the spatial area A ₁ fully fluorescence itself is suppressed (fluorescence suppression effect), the spatial region It will be emitted only fluorescence from the a _0.

  This is extremely important when considered from the field of application of a microscope. That is, in a conventional scanning laser microscope or the like, a laser beam is condensed on a micro beam by a condensing lens and scanned on an observation sample. The size of the micro beam at that time depends on the numerical aperture and wavelength of the condensing lens. The diffraction limit is determined by, and in principle, no further spatial resolution can be expected.

  However, in the case of FIG. 21, two types of light of wavelength λ1 and wavelength λ2 are superposed spatially and the fluorescent region is suppressed by irradiation with light of wavelength λ2, for example, the light of wavelength λ1. Focusing on the irradiation region, the fluorescent region 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. Hereinafter, the light of wavelength λ1 is called pump light, and the light of wavelength λ2 is called erase light. Therefore, by utilizing this principle, it becomes possible to realize a super-resolution microscope using a double resonance absorption process exceeding the diffraction limit, for example, a super-resolution fluorescence microscope.

  FIG. 22 is a configuration diagram of a main part of an optical system of a conventionally proposed super-resolution microscope. This super-resolution microscope is premised on a normal laser scanning fluorescence microscope, and mainly includes three independent units, that is, a light source unit 110, a scan unit 130, and a microscope unit 150.

  The light source unit 110 includes, for example, an LD-excited mode-locked Nd: YAG laser 111 that emits pump light that is coherent light having a wavelength of 532 nm, a Kr laser 112 that emits erase light that is coherent light having a wavelength of 647 nm, and an erase light space. A modulation phase plate 113 and a beam combiner 114 for fusing erase light and pump light are provided. As shown in FIG. 23, the phase plate 113 is vapor-deposited with an optical thin film that is adjusted so that the phase of the erase light that has passed at a position symmetrical to the optical axis is reversed. In FIG. 23, there are four independent regions around the optical axis, and the phases differ by a quarter with respect to the erase light wavelength. If the light that has passed through the phase plate 113 is collected, the electric field is canceled on the optical axis and hollow erase light is generated.

  The scan unit 130 passes the pump light and the erase light sharing the same optical axis emitted from the light source unit 110 through the half mirror 131 and then swings in a two-dimensional direction by the two galvanometer mirrors 132 and 133. The light is scanned and emitted to a microscope unit 150 (to be described later), and the fluorescence detected by the microscope unit 150 is branched by the half mirror 131 along the path opposite to the forward path. Light is received by the photomultiplier tube 138 through the projection lens 134, the pinhole 135, and the notch filters 136 and 137. In FIG. 22, in order to simplify the drawing, the galvanometer mirrors 132 and 133 are shown swingable in the same plane. The notch filters 136 and 137 are for removing pump light and erase light mixed in the fluorescence. The pinhole 135 is an important optical element forming a confocal optical system, and allows only the fluorescence emitted from a specific tomographic plane in the observation sample to pass.

  The microscope unit 150 is a so-called normal fluorescent microscope, and includes molecules having at least three electronic states including at least a ground state by the objective lens 152 by reflecting the pump light and the erase light incident from the scan unit 130 by the half mirror 151. The fluorescent light emitted from the observation sample 153 is condensed on the observation sample 153 and collimated again by the objective lens 152 and reflected by the half mirror 151, so that the half mirror 151 is returned to the scan unit 130 at the same time. Part of the passing fluorescence is guided to the eyepiece lens 154 so that it can be visually observed as a fluorescence image.

According to this super-resolution fluorescence microscope, fluorescence other than the vicinity of the optical axis where the intensity of the erase light becomes zero is suppressed on the focusing point of the observation sample 153, and as a result, a region narrower than the spread of the pump light (Δ < Only fluorescent labeler molecules existing at 0.61 · λ1 / NA, where NA is the numerical aperture of the objective lens 152) are observed, and as a result, super-resolution is exhibited. Therefore, if the fluorescence signal is measured while scanning the pump light and the erase light with the scan unit 130, a super-resolution two-dimensional fluorescence image can be obtained.
JP-A-8-184552 JP 2001-100102 A

  However, the conventionally proposed super-resolution fluorescence microscope has the following problems, particularly regarding the optical adjustment of the pump light and the erase light.

  In other words, in a normal laser scanning microscope, it is sufficient to align a single wavelength laser beam with the optical axis of the system as a guide, but in a super-resolution fluorescence microscope, the central portion of the pump light on the imaging plane of the objective lens The key technology is to erase the fluorescence at the edge of the collected pump light by combining the center of the erase light with the center of the erase light. The fluorescence at the central portion of the pump light is also erased, and as a result, the overall fluorescence intensity is lowered. As a result, the spatial resolution is not improved at all, and only the S / N is deteriorated.

  Therefore, in the conventional super-resolution fluorescence microscope, the pump light and the erase light are individually adjusted by an independent optical system so that the condensed positions of the pump light and the erase light are accurately adjusted to the focus of the objective lens. Yes.

  However, the size of the pump light and the erase light that are narrowed down to the diffraction limit is several hundred nm, and the alignment accuracy exceeds the order of at least 100 nm. It is extremely difficult to adjust and accurately adjust the focus of the objective lens.

  Accordingly, an object of the present invention made in view of such circumstances is to provide a super-resolution microscope that can easily and accurately perform optical adjustment of pump light and erase light and that can reliably exhibit a super-resolution effect. There is.

The invention according to claim 1, which achieves the above object, emits pump light for exciting the molecule from the ground state to the first electronically excited state with respect to a sample including molecules having at least three electronic states including the ground state. A pump light source to
An erase light source that emits erase light for exciting the molecule from the first electronic excited state to a second electronic excited state having a higher energy level;
An optical system that partially superimposes the pump light and the erase light and collects the light on the sample;
Scanning means for scanning the sample by relatively moving the light collected by the optical system and the sample;
In a super-resolution microscope having detection means for detecting a light response signal generated from the sample by light irradiation from the optical system,
It has an optical fiber which coaxially couples the pump light emitted from the pump light source and the erase light emitted from the erase light source.

  The invention according to claim 2 is the super-resolution microscope according to claim 1, characterized in that the optical fiber has a core diameter capable of exciting a single mode with respect to the erase light.

  The invention according to claim 3 is the super-resolution microscope according to claim 1 or 2, wherein the pump light emitted from the pump light source and the erase light emitted from the erase light source are each independently modulated in intensity. It is characterized by having an intensity modulation means.

  The invention according to claim 4 is the super-resolution microscope according to claim 3, wherein the intensity modulation means includes an electro-optic modulation element or an acousto-optic modulation element.

  The invention according to claim 5 is the super-resolution microscope according to claim 3 or 4, wherein the pump light intensity modulating means and the erase light intensity modulating means are independently controlled. To do.

  The invention according to claim 6 is the super-resolution microscope according to any one of claims 1 to 5, wherein the pump light and the erase light emitted from the optical fiber are reduced in size to an effective pupil diameter of the optical system. And a collimator lens for parallel light.

  The invention according to claim 7 is the super-resolution microscope according to any one of claims 1 to 6, wherein the pump light emitted from the pump light source and the erase light emitted from the erase light source are It has a deflecting means for adjusting the optical axis independently.

  The invention according to claim 8 is the super-resolution microscope according to any one of claims 1 to 7, wherein a spectral region that transmits the pump light emitted from the optical fiber and reflects the erase light, and a pump It has a spectral element in which a spectral region that reflects light and transmits erase light is spatially independent.

  A ninth aspect of the present invention is the super-resolution microscope according to any one of the first to eighth aspects, further comprising spatial modulation means for spatially modulating the erase light emitted from the optical fiber. Is.

The invention according to claim 10 is the super-resolution microscope according to any one of claims 1 to 7, wherein a spectral region that transmits the pump light emitted from the optical fiber and reflects the erase light, and a pump A spectral region that reflects light and transmits erased light has a spectral element that is spatially independent, and has spatial modulation means that spatially modulates erased light emitted from the optical fiber,
The spectral element has spectral characteristics that are symmetric with respect to the optical axis within the effective pupil plane of the optical system, and the optical axis centers of the spatial modulation means and the spectral element coincide with each other. is there.

  The invention according to claim 11 is the super-resolution microscope according to claim 8 or 10, characterized in that the spectral region of the spectral element is concentrically divided with respect to the optical axis. .

  The invention according to claim 12 is the super-resolution microscope according to claim 8, 10 or 11, wherein the spectroscopic element is formed of an optical multilayer film.

  A thirteenth aspect of the present invention is the super-resolution microscope according to the ninth or tenth aspect, wherein the spatial modulation means is formed by coating an optical substrate that is transparent to erase light or coating an optical thin film. It is characterized by this.

  The invention according to claim 14 is the super-resolution microscope according to claim 10, wherein the spectral element and the spatial modulation means are formed on the same substrate.

  The invention according to claim 15 is the super-resolution microscope according to claim 14, wherein the spectroscopic element is formed on one surface of the substrate, and the spatial modulation means is formed on the other surface. It is what.

  The invention according to claim 16 is the super-resolution microscope according to any one of claims 1 to 15, wherein the pump light emitted from the pump light source and the erase light emitted from the erase light source are: Each has a fixed wavelength.

  The invention according to claim 17 is the super-resolution microscope according to any one of claims 1 to 16, wherein the pump light source and the erase light source are each a gas laser, an ion laser, or a solid-state laser. It is characterized by being.

  The invention according to claim 18 is the super-resolution microscope according to any one of claims 1 to 17, wherein each of the pump light source and the erase light source is a continuous wave laser. It is.

The invention according to Claim 19 emits first coherent light that excites the molecule from the ground state to the first electronically excited state with respect to a sample containing molecules having at least three electronic states including the ground state. 1 light source,
A second light source that emits second coherent light that excites the molecule from the first electronically excited state to a second electronically excited state having a higher energy level;
An optical system that partially superimposes the first coherent light and the second coherent light and focuses the light on the sample;
Scanning means for scanning the sample by relatively moving the light collected by the optical system and the sample;
In a super-resolution microscope having detection means for detecting a light response signal generated from the sample by light irradiation from the optical system,
An optical fiber that coaxially couples the first coherent light emitted from the first light source and the second coherent light emitted from the second light source is provided.

  The characteristic configuration of the super-resolution microscope of the present invention will be described below with reference to FIG. FIG. 1 shows a configuration of a light source unit of a super-resolution microscope. Pump light that is first coherent light from a pump light source 1 that is a first light source and erase light source that is a second light source. The erase light that is the second coherent light from 2 is intensity-modulated by the intensity modulation means 3 and 4 and converted into a pseudo pulse, respectively, and then incident from the two incident ends of the branch fiber 5 that is an optical fiber. The light is coupled and emitted from one exit end. The pump light and erase light that are optically coupled by the branch fiber 5 are converted into parallel beams by the collimator lens 6, and then only the erase light is spatially modulated through the spectroscopic element 8 and the spatial modulation means 9. As shown at 22, the sample is guided to the microscope unit through the scanning unit and irradiated onto the observation sample.

  Here, the first characteristic configuration in the super-resolution microscope of the present invention is that an optical fiber including the branch fiber 5 is provided. Specifically, a so-called single mode fiber that can excite only a single mode with respect to the erase light wavelength is used as the branch fiber 5, and the erase light and the pump light are optically transmitted to the two incident ends of the branch fiber 5. Into and optically couple. That is, the core diameter of the branch fiber 5 is set so that only the single mode can be excited with respect to the erase light. Therefore, the erase light emitted from the branch fiber 5 is completely a spherical wave. On the other hand, pump light may be excited in a higher-order mode. In general, in the case of a super-resolution microscope, the wavelength of pump light for fluorescent light excitation is slightly smaller than the wavelength of erase light. Since it is short (10% to 20%), there are very few high-order mode components, and the pump light emitted from the branch fiber 5 can be regarded as a substantially spherical wave.

  Therefore, if the erase light and the pump light which are optically coupled from the branch fiber 5 are converted into parallel beams by the same collimator lens 6 having no chromatic aberration, the light beams are completely adjusted with the optical axis adjusted to be completely coaxial. It has a uniform wavefront. In addition, if this parallel beam is condensed by the microscope objective lens, the condensing point of the pump light and the erase light can be made to completely coincide with one point in the three-dimensional space in the micrometer region. As described above, by using the branch fiber 5, the optical adjustment of the pump light and the erase light can be easily and accurately performed as compared with the conventional case where the pump light and the erase light are individually adjusted by the independent optical system. Therefore, the super-resolution effect can be surely exhibited.

  Next, the second characteristic configuration of the super-resolution microscope of the present invention is that it comprises a spectroscopic element 8 and spatial modulation means 9 for erase light. That is, in the present invention, since the use of the branch fiber 5 as described above is assumed, it is necessary to spatially modulate only the erase light among the coaxial erase light and pump light. Specifically, in order to effectively erase the fluorescent region with erase light and improve the spatial resolution, it is necessary to shape the beam into a hollow beam. On the other hand, with respect to the pump light, it is indispensable to focus on one point on the sample surface without undergoing beam shaping.

  The spectroscopic element 8 is configured, for example, as shown in FIG. 2, and the spatial modulation means 9 is configured, for example, as shown in FIG. The spectral element 8 shown in FIG. 2 has at least two regions 8A and 8B having different spectral characteristics on a flat substrate. The region 8A does not transmit pump light but transmits only erase light, and the region 8B has On the contrary, it has a spectral characteristic that allows the pump light to pass through and does not allow the erase light to pass through. These regions 8A and 8B, specifically, an optical multilayer film are formed.

  When the spectroscopic element 8 is configured in this manner, for example, in the region 8A, the pump light is reflected by interference under a direct incidence condition, and the erase light is transmitted without satisfying the interference condition. In the region 8B, the erase light is reflected by interference under the condition of normal incidence, and the pump light is transmitted without satisfying the interference condition. For example, as shown in FIG. 2, when the regions 8A and 8B are concentrically patterned and the coaxial pump light and the erase light are transmitted, the erase light passes through the central circular region 8A, and the pump light is It passes through the outer annular zone 8B. Therefore, as shown in FIG. 3, in the subsequent stage of the spectroscopic element 8, there are four independent regions having the same beam diameter as the erase light and giving a phase difference by π / 2 with respect to the erase light wavelength around the optical axis. If the spatial modulation means 9 composed of a phase plate having the above is arranged and the erase light is spatially modulated, only the erase light can be beam shaped without spatially modulating the outer pump light.

On the other hand, the pump light is a ring-shaped beam. When this light is condensed by a microscope objective lens, the intensity profile of the pump of wavelength λ on the xy imaging plane is a Bessel linear function J ₁ (z). Is expressed as follows.

  Here, as shown in FIG. 4, ρ indicates the light shielding rate of the annular zone in the pupil surface radial direction of the pump light, the inner diameter (radius) of the annular zone pupil is r, and the outer diameter (radius) is R. Is represented by ρ = r / R.

  FIG. 5 shows an intensity profile on the imaging surface of the pump light by the microscope objective lens. A thick line indicates an intensity profile of a ring-shaped beam, and a thin line indicates an intensity profile of a normal circular beam. As apparent from FIG. 5, the annular pump light has a weak peak in the side lobe, but has a strong peak in the center of the optical axis, unlike a normal circular beam. Therefore, when such pump light is condensed on the sample surface, the fluorescence is suppressed at the periphery of the pump light that emits fluorescence, so the fluorescent spot is the size below the diffraction limit of the pump light, that is, the super-resolution size. become. By using the spectroscopic element 8 and the spatial modulation means 9 devised in this way, the pump light and the erase light can be optically adjusted with complete coaxiality, so that the system adjustment work is markedly compared with the conventional super-resolution microscope. It becomes easy.

  Further, the third characteristic configuration of the super-resolution microscope of the present invention is an intensity modulation means 3 for modulating the intensity of the pump light from the pump light source 1 and the erase light from the erase light source 2 to generate pseudo pulses, respectively. And 4 in the point. That is, conventionally, a pulse laser or the like is used as the pump light source 1 and the erase light source 2, but in the present invention, a continuous oscillation (CW) laser such as an ion laser, a gas laser, or a solid laser is used. Then, the laser light is intensity-modulated by an intensity modulation means having an acousto-optic element and an electro-optic element to form a pseudo pulse.

  Here, the acousto-optic device uses an effect (for example, deflection, diffraction, modulation, phase shift, frequency shift, birefringence, etc.) exerted by sound waves on light. In this acousto-optic device, the optical refractive index is fluctuated with a sound wave in a period of sound speed, and the following three types of effects appear depending on the wavelengths of sound and light.

1) When the wavelength of the sound is long, if a sufficiently thin light beam is passed in a direction perpendicular to the traveling direction of the sound, a refractive index gradient is generated, the light beam is bent, and the light beam is deflected and scanned at the sound frequency.
2) When the wavelength of the sound is shortened, the sound wave acts as a diffraction grating for the light beam.
3) When the wavelength of the sound is further shortened to the light wavelength order, diffraction does not occur at normal incidence, and strong Bragg diffraction occurs at a specific incident angle.

  The m-th order diffracted light by the sound wave of frequency f shifts the frequency by mf due to the Doppler effect. The diffraction angle θm is given by sin θm = mλ / Λ, where λ is the wavelength of light and Λ is the wavelength of the sound wave.

The acousto-optic modulator used for laser light deflection and modulation applies this effect, and FIG. 6 shows its principle configuration. As shown in FIG. 6, the acoustooptic modulator 11 is configured by adhering a piezoelectric element 13 to an acoustooptic element 12, and generates an ultrasonic wave by the piezoelectric element 13 to propagate the acoustooptic element 12. Incident light is diffracted by making light incident on the acousto-optic element 12, and FIG. 6 shows diffraction by Bragg reflection. The acoustooptic modulator 11 can modulate up to a 10 MHz band if, for example, a PbMoO ₄ or TeO ₂ crystal is used as the acoustooptic element 12.

  The electro-optic element utilizes the phenomenon that the refractive index of the material changes when an electric field is applied to the material from the outside. The Pockels effect is used when the change in refractive index is proportional to the applied electric field. It is said. In the piezoelectric crystal, when the frequency of the applied electric field is smaller than the piezoelectric resonance frequency, the elastic deformation of the crystal due to the piezoelectric effect follows the change in the applied electric field, and the refractive index changes due to the elastic deformation simultaneously. On the other hand, when the frequency is high, the elastic deformation of the piezoelectric crystal cannot follow, and this case is called a bound electro-optic effect. This electro-optical effect can be applied to intensity modulation of light.

  FIG. 7 shows a principle configuration of an electro-optic modulator using an electro-optic element. The electro-optic modulator 15 is a Pockels cell that uses the Pockels effect, and includes a polarizer 16, an electro-optic element 17, a quarter-wave plate 18, and a detector that are sequentially arranged from the light incident side to the light emitting side. The photon 19 is included, and the polarizer 16 and the analyzer 19 are orthogonal to each other in polarization axis. The electro-optic element 17 uses a piezoelectric crystal having a large Pockels effect such as KDP (potassium phosphate dihydrate), and an electric field is selectively connected to the piezoelectric crystal via an electrode (not shown). Apply. As a connection method of the power source 20, that is, an electric field application method, there are a horizontal light intensity modulation method in which the light is applied in a direction perpendicular to the traveling direction of light and a vertical light modulation method in which an electric field is applied in parallel. FIG. 7 shows the case of the vertical light modulation method. In this case, a ring-shaped electrode or a transparent electrode is provided on the incident end face and the exit end face of the electro-optic element 17 so as not to block the optical path. 20 are selectively connected.

  In the electro-optic modulator 15 shown in FIG. 7, when the piezoelectric crystal of the electro-optic element 17 is a uniaxial crystal, birefringence occurs when an electric field is applied in the optical axis direction by the power source 20, and the electro-optics passes through the polarizer 16. The linearly polarized light incident on the element 17 is converted into elliptically polarized light, enters the analyzer 19 through the quarter-wave plate 18, and the component in the polarization axis direction of the analyzer 19 is transmitted. In contrast, when no electric field is applied to the electro-optic element 17, the electro-optic element 17 is uniaxial and does not transmit any light. In this way, by selectively applying an electric field to the electro-optic element 17, incident light can be modulated, and, for example, optical switching on the order of nanoseconds becomes possible.

  The acousto-optic modulator 11 or the electro-optic modulator 15 is inserted into the optical path of the CW laser and electrically controlled, thereby realizing a pulse light source suitable for the super-resolution microscope system. As a result, it is possible to perform pulsing while maintaining the excellent beam quality of CW lasers such as He / Ne lasers, Ar lasers, Kr lasers, and Ti sapphire lasers. In addition, since any oscillation frequency can be selected by electrical control, for example, the pulse oscillation timing and pulse width of pump light and erase light can be controlled easily and electrically, thereby providing the basis for a super-resolution microscope. It is possible to efficiently induce the effect of suppressing fluorescence.

  In particular, in the present invention, since the intensity modulation means is inserted between the laser light source and the optical fiber, the distortion of the wavefront generated by the intensity modulation means can be removed with an optical fiber that can excite only a single mode. Can be efficiently expressed. As a result, it is possible to improve the super-resolution and provide a high-performance microscope differentiated in resolution. In addition, it is possible to use a gas laser such as a He / Ne laser that has already been widely introduced in laser scanning microscope systems and has a proven track record, and it is possible to provide a user-friendly microscope system that is more practical.

  By combining these three characteristic configurations well, we can realize a super-resolution microscope that can be adjusted with an unprecedented system, simplifying and simplifying the product assembly process, and introducing the product. It is possible to provide infinite convenience in maintenance at the site.

  According to the present invention, the pump light emitted from the pump light source and the erase light emitted from the erase light source are coupled on the same axis using an optical fiber excellent in positional stability and wavefront uniformity. As described above, the optical adjustment of these lights can be performed easily and accurately while maintaining the uniformity of the wave fronts of the pump light and the erase light, and the super-resolution effect can be surely exhibited.

  Hereinafter, embodiments of a super-resolution microscope according to the present invention will be described in detail with reference to the drawings.

(First embodiment)
FIG. 8 is a configuration diagram of the main part of the optical system of the super-resolution microscope according to the first embodiment of the present invention. The super-resolution microscope according to the present embodiment mainly includes three independent units, that is, a light source unit 30, a scan unit 50, and a microscope unit 70.

  The light source unit 30 is a pump light source 31 that is a first light source that generates CW pump light that is first coherent light, and a second light source that generates CW erase light that is second coherent light. An erase light source 32, an electro-optic modulator (EOM) 33 made of a KDP crystal that is an intensity modulation means for pseudo-pulsing CW pump light from the pump light source 31, and CW erase from the erase light source 32 An EOM 34 made of a KDP crystal, which is an intensity modulation means for converting light into a pseudo-pulse, and a branch fiber 35, which is an optical fiber that emits optically coupled pump light and erase light that are pseudo-pulsed by the EOMs 33 and 34 on the same axis. A collimator lens 36 that converts the pump light and erase light that are optically coupled by the branch fiber 35 into parallel beams, and There is a spectroscopic element 38 that splits the pump light and the erase light that are converted into parallel beams by the collimator lens 36, and a spatial modulation means 39 that spatially modulates only the erase light that has passed through the spectroscopic element 38. The pump light and erase light having passed through 39 are made incident on the scan unit 50.

  Hereinafter, a case where a biological sample stained with rhodamine 6G is observed will be described as an example. Rhodamine 6G has a strong absorption band excited from the ground state (S0) to the first electronically excited state (S1) in the vicinity of the wavelength of 530 nm, and from the first electronically excited state (S1) in the wavelength range of 600 nm to 650 nm. , It has been confirmed that there is a double resonance absorption band excited in an electronically excited state (Sn) having a higher energy level (for example, E. Sahar and D. Treves: IEEE, J. Quantum Electron., QE-13,692 (1977)).

  Therefore, in the present embodiment, an Nd: YAG solid-state laser is used as the pump light source 31, and the light of the second harmonic wave having a wavelength of 532 nm is used as the pump light. Further, the erase light source 32 uses a Kr laser to convert the light having a wavelength of 647.1 nm into erase light.

  The pump light and erase light generated from the pump light source 31 and the erase light source 32 are respectively introduced into the EOMs 33 and 34 via single mode fibers and are intensity-modulated. That is, the EOMs 33 and 34 are applied with a voltage with a desired pulse width and cycle, for example, from an independent pulse generator (not shown), whereby the incident light is modulated by the electro-optic effect when the voltage is applied, and the EOMs 33 and 34 are By passing, pump light and erase light are emitted as pseudo pulse light, respectively. Note that the pulse width and timing of the voltage applied to the EOMs 33 and 34 are adjusted so that the pseudo-pulse light generation conditions can induce the fluorescence suppression effect under the optimum conditions.

  The pump light and erase light that have passed through the EOMs 33 and 34 are introduced into the branch fiber 35. The diameter of the branch fiber 35 is set so that the single mode is excited with respect to the erase light. Although the diameter of the pump light is slightly larger, the probability that the higher-order mode is excited is extremely low. Therefore, the pump light that can be transmitted through the branch fiber 35 can be regarded as a single mode. By this branch fiber 35, the pump and erase light are optically coupled coaxially and emitted as a spherical wave from the same point light source position, that is, from the exit end of the branch fiber 35.

  The pump light and erase light emitted from the branch fiber 35 are collimated completely coaxially by the collimator lens 36. The collimator lens 36 is corrected for chromatic aberration with respect to the wavelengths of the pump light and the erase light, such as a doublet lens, and the beam diameter thereof is the pupil diameter of an objective lens 72 in the microscope unit 70 described later. It matches. In the present embodiment, the effective pupil diameter is 8 mm.

  The pump light and erase light collimated by the collimator lens 36 sequentially pass through the spatial modulation means 39 including the spectroscopic element 38 and the phase plate. The spectroscopic element 38 has a planar structure as shown in FIG. In the present embodiment, since the effective pupil diameter, that is, the beam diameter of the objective lens 72 is 8 mm, the pump light (wavelength 532 nm) that is directly incident on the region 38A having a diameter of 6 mm is reflected to erase light (wavelength 647). .1 nm) is formed, and the remaining outer ring zone region 38B is formed with an optical filter that transmits the pump light and reflects the erase light. The optical filters in the regions 38A and 38B may be an optical multilayer film in which thin films having different refractive indexes are alternately stacked in order to enhance coherence.

  Spatial modulation means 39 is composed of a phase plate that phase-modulates only erase light. As shown in FIG. 10, the spatial modulation means 39 continuously forms a circular region having a diameter of 6 mm once around the optical axis. An etching pattern for modulating the erase light by 2π phase is formed. Therefore, when erase light is transmitted through the spatial modulation means 39, the phase difference of the erase light becomes π at the optical axis symmetric position in the beam plane. A hollow beam suitable for a resolution microscope can be obtained.

  Note that the spatial modulation means 39 shown in FIG. 10 is made of a quartz substrate, and is divided equally into eight regions 39A to 39H around the optical axis, stepwise by λ / 8 with respect to the wavelength of the erase light. An etching pattern that gives a phase difference is formed. More specifically, the optical path length is controlled by etching the quartz substrate by a chemical etching method. The specific etching depth is shown in Table 1.

  By passing this spatial modulation means 39, only the erase light is phase-modulated so that the light intensity becomes zero at the center of the optical axis. On the other hand, the ring-shaped pump light passes through the outside of the etched portion of the spatial modulation means 39 and is transmitted without undergoing any phase modulation.

  The pump light and erase light transmitted through the spectroscopic element 38 and the spatial modulation means 39 enter the scan unit 50.

  The scan unit 50 includes half mirrors 51, two galvanometer mirrors 52 and 53, a projection lens 54, a pinhole 55, notch filters 56 and 57 for removing pump light and erase light, and a photomultiplier tube 58. . The microscope unit 70 is a so-called normal fluorescence microscope, and includes a half mirror 71, an objective lens 72, and an eyepiece lens 73.

  The pump light and erase light incident on the scan unit 50 from the light source unit 30 pass through the half mirror 51, are then deflected in two dimensions by the two galvanometer mirrors 52 and 53, and are incident on the microscope unit 70. The light is reflected by the mirror 71 and focused by the objective lens 72 onto the observation sample 74 including molecules having at least three electronic states including the ground state, and is two-dimensionally scanned on the sample surface. In FIG. 8, in order to simplify the drawing, the galvanometer mirrors 52 and 53 are shown swingable in the same plane.

  On the other hand, the fluorescence emitted from the observation sample 74 due to the irradiation of the observation sample 74 with the pump light and the erase light follows the optical path opposite to the incident laser light and enters the half mirror 51 of the scan unit 50, Reflected by the half mirror 51. The fluorescence reflected by the half mirror 51 is collected by the projection lens 54 and transmitted through the pinhole 55, and the pump light and erase light are removed by the notch filters 56 and 57 and received by the photomultiplier tube 58. . The pinhole 55 is placed at a confocal position with the condensing point on the observation sample 74, thereby transmitting only the fluorescence emitted from a specific depth portion in the observation sample 10 simultaneously with the spatial filter function 3. It is designed to perform the dimensional space decomposition function. The fluorescence signal data obtained from the photomultiplier tube 58 is stored in a memory in the computer in synchronization with the scanning of the galvanometer mirrors 52 and 53, whereby a two-dimensional fluorescence microscopic image of the observation sample 74 is obtained. The light that has passed through the half mirror 71 of the microscope unit 70 is imaged by the eyepiece lens 73, whereby the fluorescent spot image can be directly observed, and can be used for adjustment of the observation position, focusing, and the like. .

  As described above, in this embodiment, CW lasers are used as the pump light source 31 and the erase light source 32, respectively, and the pump light and erase light from these CW lasers are pseudo-pulsed by the EOMs 33 and 34 made of KDP crystals. ing. Here, since the KDP crystal has a rise time response to an input electric pulse of about 5 nsec, a repetition frequency of the order of MHz can be obtained with a pulse width equivalent to that of a nanosecond pulse laser.

  Here, the pseudo pulses of the pump light and the erase light by the EOMs 33 and 34 are set so that the pulse width of the erase light is longer than that of the pump light, as shown in FIG. Thereby, the overlap of the pump light and the erase light in the time domain is ensured, and the fluorescence suppression effect can be reliably induced. For this purpose, preferably, the EOMs 33 and 34 are controlled by independent pulse generators as described above to adjust the phases and frequencies of the pseudo pulses of the pump light and the erase light. In particular, regarding the phase, a phase difference at the time of modulation of about several nanoseconds is generated due to individual differences of KDP crystals, and the expression of the fluorescence suppression effect is optimized by the corresponding pulse generator.

In the present embodiment, EOMs 33 and 34 are used as intensity modulation means, and the pump light and erase light from the pump light source 31 and erase light source 32 each made of a CW laser are intensity-modulated into pseudo pulses. In place of the EOM 33 and / or 34 as intensity modulation means, pseudo-pulses can be obtained by using an acousto-optic element. For example, if a TeO ₂ crystal is used as an acoustooptic device, the laser resistance threshold is also high, so that erase light having a relatively high intensity can be reliably pulsed.

(Second Embodiment)
FIG. 12 is a main part configuration diagram of the optical system of the super-resolution microscope according to the second embodiment of the present invention. In the present embodiment, the configuration of the light source unit 30 is different from that of the first embodiment, and other configurations are the same as those of the first embodiment. Therefore, the same members as those in the first embodiment are the same. Reference numerals are assigned and explanations thereof are omitted.

  In the present embodiment, pulse lasers are used as the pump light source 31 and the erase light source 32, respectively. Here, similarly to the first embodiment, when observing the biological sample 74 stained with rhodamine 6G, an Nd: YAG pulse laser is used as the pump light source 31, and the wavelength of the double wave of 532 nm is used. Coherent light is used as pump light. The erase light source 32 uses an Nd: YAG pulse laser, and uses coherent light having a fundamental wavelength of 1064 nm as erase light. Accordingly, in this case, the erase light is subjected to fluorescence suppression using the double resonance absorption band of rhodamine 6G in the near infrared region.

  The pump light emitted from the pump light source 31 is incident on a beam combiner 43 such as a half mirror cube through the deflection mirrors 41a and 41b and the condenser lens. Similarly, the erase light emitted from the erase light source 32 is incident on the beam combiner 43 via the deflecting mirrors 44a and 44b and the condenser lens 45, and is coupled coaxially with the pump light by the beam combiner 43. The pump light and erase light combined by the beam combiner 43 are transmitted through an optical fiber 46 that excites a single mode with respect to the erase light, and then converted into a parallel beam by a collimator lens 36. Further, a spectroscopic element 47 and a phase plate The light is incident on the scan unit 50 through the spatial modulation means 48 consisting of: Other configurations and operations are the same as those in the first embodiment.

  That is, in the present embodiment, the pair of deflection mirrors 41a, 41b, 44a provided independently of the pump light source 31 and the erase light source 32 without using the branch fiber of the first embodiment. 44b, the pump light and the erase light are directly introduced into one common optical fiber 46. Specifically, pump light and erase light are spatially optically adjusted by deflection mirrors 41a, 41b, 44a and 44b, and are coaxially coupled by a beam combiner 43, and their beam diameters are determined by the pump light source. The condensing lenses 42 and 45 provided independently for the light source 31 and the erase light source 32 are adjusted so that the incident efficiency to the optical fiber 46 is maximized.

  Here, the spatial modulation means 48 can be configured in the same manner as the spatial modulation means 39 shown in FIG. 10, but it can also be a ring type as shown in FIG. That is, the inner circular region 48A is etched on the quartz substrate so as to give phase modulation to the erase light by π, and the other outer annular region 48B does not change the phase of the erase light. To do. Therefore, if the erase light is condensed through the spatial modulation means 48, the wavefront components whose phases are inverted are mixed, so that a donut-shaped spot having an electric field intensity of zero is obtained at the central portion on the focal point.

  If such a spatial modulation means 48 is used, the structure of the spectroscopic element 47 is simplified as shown in FIG. That is, a dichroic mirror that reflects the pump light and transmits the erase light only by the circular region 48A inside the spatial modulation means 48 can be obtained. Specifically, an optical thin film having the aforementioned spectral characteristics may be coated corresponding to the circular region 48A inside the spatial modulation means 48. Further, as shown in FIG. 15A, a spectroscopic element 49A as shown in FIG. 15B is formed on the surface of the quartz substrate 49, and the phase as shown in FIG. The region 49B can be formed to integrate the spectroscopic element and the spatial modulation means. In this way, the number of parts can be reduced, the cost can be reduced, and optical adjustment can be easily performed.

It is a figure for demonstrating the characteristic structure in the super-resolution microscope of this invention. It is a figure which shows the spectroscopic element of FIG. Similarly, it is a figure which shows a spatial modulation means. Similarly, it is a figure for demonstrating the light-shielding rate of the annular zone part in the pupil surface radial direction of pump light. It is a figure which shows the intensity profile in the imaging surface of the pump light by a microscope objective lens. It is a figure which shows the fundamental structure of an acousto-optic modulator. It is a figure which shows the fundamental structure of an electro-optic modulator. It is a principal part block diagram of the optical system of the super-resolution microscope which concerns on 1st Embodiment of this invention. It is a figure which shows the spectroscopic element of FIG. Similarly, it is a figure which shows a spatial modulation means. Similarly, it is a figure which shows the pseudo pulse of pump light and erase light. It is a principal part block diagram of the optical system of the super-resolution microscope which concerns on 2nd Embodiment of this invention. It is a figure which shows the spatial modulation means of FIG. Similarly, it is a figure which shows a spectroscopic element. It is a figure for demonstrating the modification of the spectroscopic element of FIG. 12, and a spatial modulation means. It is a conceptual diagram which shows the electronic structure of the valence orbit of the molecule | numerator which comprises a sample. It is a conceptual diagram which shows the 1st excitation state of the molecule | numerator of FIG. Similarly, it is a conceptual diagram which shows a 2nd excitation state. Similarly, it is a conceptual diagram which shows the state which returns from a 2nd excitation state to a ground state. It is a conceptual diagram for demonstrating the double resonance absorption process in a molecule | numerator. Similarly, it is a conceptual diagram for explaining a double resonance absorption process. It is a principal part block diagram of the optical system of the super-resolution microscope proposed conventionally. It is a figure which shows the phase plate of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Pump light source 2 Erase light source 3, 4 Intensity modulation means 5 Branch fiber 6 Collimator lens 8 Spectrometer 9 Spatial modulation means 30 Light source unit 31 Pump light source 32 Erase light source 33, 34 Electro-optic modulator (EOM)
35 branch fiber 36 collimator lens 38 spectroscopic element 39 spatial modulation means 41a, 41b, 44a, 44b deflection mirror 42, 45 condensing lens 43 beam combiner 46 optical fiber 47 spectroscopic element 48 spatial modulation means 50 scan unit 51 half mirror 52, 53 Galvano mirror 54 Projection lens 55 Pinhole 56, 57 Notch filter 58 Photomultiplier tube 70 Microscope unit 71 Half mirror 72 Objective lens 73 Eyepiece 74 Observation sample

Claims (19)

A pump light source that emits pump light for exciting the molecule from the ground state to the first electronic excited state with respect to a sample including molecules having at least three electronic states including the ground state;
An erase light source that emits erase light for exciting the molecule from the first electronic excited state to a second electronic excited state having a higher energy level;
An optical system that partially superimposes the pump light and the erase light and collects the light on the sample;
Scanning means for scanning the sample by relatively moving the light collected by the optical system and the sample;
In a super-resolution microscope having detection means for detecting a light response signal generated from the sample by light irradiation from the optical system,
A super-resolution microscope comprising: an optical fiber that coaxially couples pump light emitted from the pump light source and erase light emitted from the erase light source.   The super-resolution microscope according to claim 1, wherein the optical fiber has a core diameter capable of exciting a single mode with respect to the erase light.   3. The super-resolution according to claim 1, further comprising independent intensity modulation means for modulating the intensity of the pump light emitted from the pump light source and the erase light emitted from the erase light source. microscope.   4. The super-resolution microscope according to claim 3, wherein the intensity modulation means includes an electro-optic modulation element or an acousto-optic modulation element.   5. The super-resolution microscope according to claim 3, wherein the pump light intensity modulating means and the erase light intensity modulating means are independently controlled.   6. The collimator lens according to claim 1, further comprising a collimator lens that makes the pump light and the erase light emitted from the optical fiber parallel to a size of an effective pupil diameter of the optical system. Super-resolution microscope.   7. A deflecting unit for independently adjusting an optical axis of pump light emitted from the pump light source and erase light emitted from the erase light source. The super-resolution microscope described.   A spectral element that spatially and independently provides a spectral region that transmits pump light emitted from the optical fiber and reflects erase light; and a spectral region that reflects pump light and transmits erase light A super-resolution microscope according to any one of claims 1 to 7.   The super-resolution microscope according to claim 1, further comprising a spatial modulation unit that spatially modulates erase light emitted from the optical fiber. A spectral element that spatially and independently provides a spectral region that transmits pump light emitted from the optical fiber and reflects erase light; and a spectral region that reflects pump light and transmits erase light And spatial modulation means for spatially modulating the erase light emitted from the optical fiber,
The spectral element has spectral characteristics which are symmetric with respect to the optical axis in an effective pupil plane of the optical system, and the optical axis centers of the spatial modulation means and the spectral element coincide with each other. The super-resolution microscope as described in any one of 1-7.   The super-resolution microscope according to claim 8 or 10, wherein the spectral region of the spectral element is divided concentrically with respect to the optical axis.   The super-resolution microscope according to claim 8, 10 or 11, wherein the spectroscopic element comprises an optical multilayer film.   The super-resolution microscope according to claim 9 or 10, wherein the spatial modulation means is configured by coating an optical substrate transparent to erase light with an etching or optical thin film coating.   The super-resolution microscope according to claim 10, wherein the spectroscopic element and the spatial modulation means are formed on the same substrate.   The super-resolution microscope according to claim 14, wherein the spectroscopic element is formed on one surface of the substrate, and the spatial modulation means is formed on the other surface.   The super-resolution according to claim 1, wherein the pump light emitted from the pump light source and the erase light emitted from the erase light source have fixed wavelengths. microscope.   The super-resolution microscope according to any one of claims 1 to 16, wherein the pump light source and the erase light source are each a gas laser, an ion laser, or a solid-state laser.   The super-resolution microscope according to claim 1, wherein each of the pump light source and the erase light source is a continuous wave laser. A first light source that emits a first coherent light that excites the molecule from a ground state to a first electronically excited state with respect to a sample including molecules having three electronic states including at least a ground state;
A second light source that emits second coherent light that excites the molecule from the first electronically excited state to a second electronically excited state having a higher energy level;
An optical system that partially superimposes the first coherent light and the second coherent light and focuses the light on the sample;
Scanning means for scanning the sample by relatively moving the light collected by the optical system and the sample;
In a super-resolution microscope having detection means for detecting a light response signal generated from the sample by light irradiation from the optical system,
A super-resolution microscope comprising an optical fiber that coaxially couples the first coherent light emitted from the first light source and the second coherent light emitted from the second light source. .

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