JP5086765B2 - microscope - Google Patents

Description

  The present invention relates to a microscope, and more particularly to a novel high-performance and high-performance microscope that illuminates a stained sample with light of a predetermined wavelength and observes the sample with high spatial resolution.

  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 resulting from a specific optical transition. This principle will be described with reference to FIGS. FIG. 9 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 (S0 state) shown in FIG. 9 are excited by light of wavelength λ1. Thus, the first electronic excitation state (S1 state) shown in FIG. 10 is assumed. Next, excitation is performed in the same manner with light of another wavelength λ2, and a second electron excitation state (S2 state) shown in FIG. 11 is obtained. In this excited state, the molecule emits fluorescence or phosphorescence and returns to the ground state as shown in FIG.

  In the microscope method using the double resonance absorption process, an absorption image and a light emission image are observed using the absorption process of FIG. 11 and the fluorescence and phosphorescence emission of FIG. In this microscope method, first, the molecules constituting the sample are excited to the S1 state as shown in FIG. 10 with light having a resonance wavelength λ1 by laser light or the like. At this time, the number of molecules in the S1 state within the unit volume is It increases as the intensity of light to be applied 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. 11, 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.

  Further, when a deexcitation process by fluorescence or phosphorescence from the excited state of FIG. 11 is possible, the emission intensity is proportional to the number of molecules in the S1 state. Therefore, image contrast can be controlled even when used as a fluorescence microscope.

  Furthermore, the microscopy using the double resonance absorption process can not only control the above-described image contrast but also perform chemical analysis. That is, since the outermost valence electron orbit shown in FIG. 9 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 the sample is illuminated with a conventional single wavelength, it is possible to observe an absorption image or fluorescence image of a specific molecule to some extent. However, in general, some molecules have overlapping absorption band wavelength regions, so when illuminating a sample at a single wavelength, 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. 13 is a conceptual diagram of a double resonance absorption process in a molecule. A molecule in the ground state of S0 is excited to a first electronically excited state of S1 by light having a wavelength of λ1, and further a second of S2 by light having a wavelength of λ2. It shows a state of being excited in an electronically excited state. Note that FIG. 13 shows that the fluorescence from the S2 state of certain molecules is extremely weak.

  In the case of molecules having optical properties as shown in FIG. 13, a very interesting phenomenon occurs. FIG. 14 is a conceptual diagram of the double resonance absorption process as in FIG. 13, where the horizontal X-axis represents the spread of the spatial distance, and the light of the wavelength region λ2 and the spatial region A1 irradiated with the light of wavelength λ2 is not irradiated. A space area A0 is shown.

  In FIG. 14, a large number of molecules in the S1 state are generated by excitation of light of wavelength λ1 in the spatial region A0, and fluorescence emitted at wavelength λ3 is seen from the spatial region A0. However, in the spatial region A1, since the light of wavelength λ2 is irradiated, most of the molecules in the S1 state are immediately excited to the higher S2 state and no S1 state molecule exists. Such a phenomenon has been confirmed by several molecules. Thereby, in the spatial region A1, the fluorescence of the wavelength λ3 is completely lost, and the fluorescence from the S2 state is not originally present. Therefore, in the spatial region A1, the fluorescence itself is completely suppressed (fluorescence suppression effect), and only from the spatial region A0. Fluorescence will be emitted.

  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 into a micro beam by a condensing lens and scanned on an observation sample. The size of the micro beam at that time is determined by 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. 14, if the fluorescent region is suppressed by spatially superimposing two types of light of wavelength λ1 and wavelength λ2, for example, if attention is paid to the irradiation region of light of wavelength λ1, fluorescence The 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 also called pump light, and the light of wavelength λ2 is also called erase light. Therefore, by using this principle, it becomes possible to realize a super-resolution microscope, for example, a super-resolution fluorescence microscope, that uses a double resonance absorption process exceeding the diffraction limit.

JP-A-8-184552 JP 2001-100102 A

  However, the super-resolution microscope that has been proposed conventionally requires two-color laser beams having different wavelengths as the pump light and the erase light as described above. For example, when observing a sample stained with rhodamine 6G, which is a typical dye molecule, pump light having a wavelength of 532 nm and erase light having a wavelength of 600 nm are required. For this reason, in this type of super-resolution microscope, there is a concern that the configuration, adjustment, and the like are complicated.

  For example, in order to introduce pump light and erase light having different wavelengths into a microscope objective lens, a special wavelength dispersive element such as a dichroic mirror is used to reflect one of the pump light and erase light and transmit the other. , Spatially need to be adjusted coaxially.

  In order to detect fluorescence emitted from the sample, it is necessary to dispose an optical filter or a spectroscopic element that cuts pump light and erase light and transmits only fluorescence in front of the detector. Normally, a notch filter that can reliably block the pump light and erase light is placed in front of the detector, and a bandpass filter that transmits only the fluorescence wavelength is placed to cope with high-intensity erase light. Therefore, it is necessary to reliably cut the scattered light from the light source.

  Furthermore, it is necessary to design the microscope objective lens so that chromatic aberration does not occur with respect to pump light and erase light having different wavelengths.

  In addition, for example, when using an optical thin film coated on the reflecting surface in a single optical path of pump light or erase light, it is necessary to design the reflecting mirror so that the reflectance is optimized at the wavelength used. When used in a common optical path, it is necessary to design the reflectivity to be optimized for two different wavelengths. For this reason, there is a concern that the design and production of the reflection mirror is cumbersome and costly, and when assembling the illumination optical system, pay careful attention to the type of reflection mirror in the optical path so as not to make a mistake. There is a concern that assembly will be troublesome.

  Accordingly, an object of the present invention made in view of the above points is to provide a microscope that can be simplified in configuration, easily assembled, and can reliably exhibit a super-resolution effect.

The invention according to claim 1 for achieving the above object is a microscope for observing a desired molecule in a sample,
Light source means for emitting pump light for exciting the molecule from a stable state to a first excited state, and erase light for exciting the molecule from the first excited state to a second excited state;
An optical system for partially superimposing the pump light and the erase light from the light source means and condensing the sample on the sample;
Scanning means for scanning the sample by relatively moving the pump light and the erase light collected by the optical system and the sample;
Detecting means for detecting a light response signal generated from the sample by irradiation of the pump light and the erase light, and
The light source means includes
As the pump light, the photon flux when focused on the sample is 1 / (τσ _two ) ^1/2 or more when the two-photon absorption cross section of the molecule is σ _two and the fluorescence lifetime is τ. Emits pulsed light of wavelength,
The erase light is configured to emit a pulsed light having the same wavelength as the pump light and a photon flux when condensed on the sample is less than 1 / (τσ _two ) ^1/2. To do.

The invention according to claim 2 is the microscope according to claim 1,
The light source means includes
As the pump light, pulse light having a pulse width of less than 1 nanosecond is emitted,
The erase light is configured to emit pulsed light having a pulse width of 1 nanosecond or more.

The invention according to claim 3 is the microscope according to claim 1 or 2,
The light source means has one laser light source,
The pump light and the erase light pulse having different peak values are emitted from the laser light source.

The invention according to claim 4 is the microscope according to claim 1, 2, or 3,
The light source means is configured to emit the pump light prior to the emission of the erase light.

The invention according to claim 5 is the microscope according to claim 4,
A difference in emission timing between the pump light and the erase light is shorter than the fluorescence lifetime.

The invention according to claim 6 is the microscope according to any one of claims 1 to 5,
The light source means further includes a spatial modulation element that spatially modulates the erase light.

The invention according to claim 7 is the microscope according to claim 6,
The spatial modulation element performs spatial modulation so that the erase light is condensed in a hollow shape on the sample.

  According to the present invention, since the pulse light having the same wavelength and different peak values are used as the pump light and the erase light, the configuration can be simplified, the assembly can be easily performed, and the super-resolution effect can be surely exhibited. it can.

(Outline of the present invention)
First, prior to the description of the embodiments of the present invention, an outline of the present invention will be described. The microscope of the present invention utilizes a spectroscopic fluorescence suppression process and a two-photon excitation process. 1 and 2 are diagrams for explaining a fluorescence suppression process. In the fluorescence suppression process, as shown in FIG. 1, for example, when a nanosecond erase light pulse is irradiated, the fluorescence appears to be suppressed at once. However, as shown in FIG. 2, this fluorescence suppression effect can be understood as a stack of excitation / relaxation processes from the S1 state to the S2 state with an erase light pulse of the order of picoseconds.

  1 and 2, the excitation time from the S1 state to the S2 state is approximately several picoseconds, although it depends on the intensity of the erase light. The relaxation time is, for example, several picoseconds to several tens picoseconds. This dominant relaxation process on the time scale is a non-radiation process from the S2 state to the S1 state, and occurs with a high probability of about 90% according to Kasha's law. However, the remaining 10% is relaxed to the ground state without radiation by internal conversion or the like.

  In FIG. 1, this excitation process and relaxation process are repeated many cycles in nanoseconds. As a result, the probability of taking the S1 state is reduced, and molecular fluorescence is suppressed. In other words, even if a light pulse shorter than nanosecond, for example, a picosecond order, is irradiated, the above excitation / relaxation process occurs only for one cycle. There is almost no fluorescence suppression.

  On the other hand, when a light pulse shorter than nanosecond is irradiated, a nonlinear effect such as two-photon absorption appears, and another absorption process becomes dominant from the ground state. Usually, the excitation wavelength from the S1 state to the S2 state is considerably longer than the excitation wavelength from the S0 state to the S1 state, so that when the erase light intensity is low, the transition from the S0 state to the S1 state does not occur. Therefore, when the erase light intensity is low, no fluorescence emission is observed. However, irradiation with femtosecond pulsed light having a high peak value, which has been developed in recent years, can induce a two-photon absorption process without causing fluorescence suppression. This principle was used in two-photon microscopy.

  This two-photon absorption process generally occurs very efficiently when the excitation energy from the S0 state to the S1 state of the molecule is twice as large as the photon energy of the incident light. In addition, at this time, even if there is a resonance absorption band from the S1 state to the S2 state, the expression of fluorescence suppression can be ignored for the above reason.

  Focusing on this phenomenon, the excitation energy from the S0 state to the S1 state of the molecule is twice as large as the photon energy of the incident light, and it also resonates in the resonance absorption band from the S1 state to the S2 state. In this case, super-resolution microscopy with one color illumination light is possible.

  Looking at this phenomenon in the time domain, another consideration is possible. For example, as shown in FIG. 3, when a nanosecond pulse and a femtosecond pulse with a high peak value (crest value) are fused in a time domain, a two-photon process and a fluorescence suppression process are separated in the time domain. It is strongly expressed in each time domain. That is, at the moment when a femtosecond order pulse with a high peak value is irradiated, fluorescence emission due to a two-photon process is observed, and in the time domain when a nanosecond order pulse with a low peak value is irradiated, Suppression occurs.

For example, in the case of rhodamine 6G, a two-photon process can be caused by a pulsed light having a pulse width of 1 ps and a peak value of about 1 GW / cm ² , and fluorescence is generated by a pulsed light having a pulse width of 10 ns and a peak value of about 10 MW / cm ^2. An inhibitory effect can be induced. Therefore, for example, within the excitation lifetime of the S1 state, first, the sample is irradiated with a very short pulse light having a high peak value, for example, picosecond or less (hereinafter also referred to as short pulse pump light as appropriate), and then the short pulse is irradiated. If the sample is irradiated with long pulse light with a low peak value, for example, nanosecond order (hereinafter also referred to as long pulse erase light as appropriate) after pump light irradiation, excitation by irradiation with short pulse pump light is possible. The generated fluorescence can be suppressed. That is, by controlling the waveform of the laser pulse applied to the sample in the time domain, a super-resolution microscope that uses light of one color (single wavelength) can be configured.

  FIG. 4 is a schematic diagram for explaining the waveform control of the short pulse pump light and the long pulse erase light when a super-resolution microscope is configured using one color of light. As shown in FIG. 4, the short pulse pump light is shaped into a normal Gaussian beam and incident on a microscope objective lens (not shown) through the reflection mirror 1 and the beam combiner 2. Further, the long pulse erase light is spatially modulated by the spatial modulation element 3 so as to be condensed in a ring shape by the microscope objective lens, and is synthesized coaxially with the short pulse pump light by the beam combiner 2 to be a microscope objective lens. To enter.

  Thereby, on the sample surface, in the central region of the focal point where only the short pulse pump light is collected, strong fluorescence is emitted by the two-photon absorption process, that is, the region where the long pulse erase light is irradiated in duplicate. Fluorescence is suppressed at the edge of the collected pump light. In this way, by using one color of light and appropriately irradiating the sample with its laser pulse waveform in the time domain, the same super solution as in the case of using a conventionally proposed two-wavelength laser beam is used. An image effect can be expected.

  Here, when the sample is irradiated with light of a certain wavelength, the light contributing to two-photon excitation and the light contributing to fluorescence suppression depend on the photon flux (Ie) of the irradiated light. For example, when a ratio (Dip-ratio) indicating how much fluorescence is suppressed when a predetermined molecule is irradiated with laser light is R (Ie), this R (Ie) is expressed by the following equation (1). Can be obtained approximately (see, for example, the document “Opt.Eng.vol.44 (2005) 033602”).

  According to the above equation (1), R (Ie) is 1 in the limit where Ie is infinite, and in this state, it is understood that two-photon excitation is dominant and fluorescence suppression does not occur.

Differentiating (1) above, Ie = 1 / (τσ _two ) ^1/2 , that is, the fluorescence intensity R (Ie) from the molecule when fluorescence suppression is activated at the critical limit, is expressed by the following equation (2): The minimum value represented is taken and the fluorescence suppression effect is maximized.

From the above, the two-photon excitation begins to function effectively because the photon flux when focused on the sample is Ie = 1 / (τσ _two ) ^1/2 or more, and Ie = 1 / (τσ _two If the ratio is less than ^1/2 , the efficiency of two-photon excitation is poor and the fluorescence suppression effect works effectively. That is, in the state of less than Ie = 1 / (τσ _two ) ^1/2 , the fluorescence suppression function has priority.

For example, in Rhodamine 6G, when the wavelength of illumination light is 750 nm, σ _two is 2 × 10 ⁻⁴⁸ cm ⁴ s, σ _dip is 10 ⁻¹⁸ cm ² , and τ is 3.75 ns. The critical strength at this time is approximately 1.2 × 10 ²⁸ cm ⁻² s ⁻¹ . Therefore, under the condition of less than the critical intensity, for example, Ie = 8.2 × 10 ²⁷ cm ⁻² s ⁻¹ , R (Ie) is 0.04, and the illumination light in this case functions as erase light. In addition, when the photon flux is an order of magnitude larger than this and larger than the critical strength, for example, Ie = 5 × 10 ²⁹ cm ⁻² s ⁻¹ , R (Ie) is 0.5. Under this condition, the molecule emits light with two photons, and the illumination light in this case functions as pump light. As described above, the illumination light having a wavelength of 750 nm has a function of pump light and erase light with respect to rhodamine 6G, with a photon flux of Ie = 1 / (τσ _two ) ^1/2 as a boundary.

  Further, in order to generate a fluorescence-detectable molecule in the S1 state by two-photon excitation, excitation irradiation with a short pulse light of nanosecond or less is sufficient (for example, the document “Opt. Eng. Vol. 44 (2005 ) 033602)), in order to efficiently suppress the fluorescence by exciting the molecule in the S1 state to the S2 state, irradiation for a long time of nanosecond or more is required to equilibrate the excitation process as a stable state. These serve as a guide for the irradiation time of the pump light and the erase light.

  Hereinafter, embodiments of the present invention will be described by taking a super-resolution microscope for observing a sample stained with rhodamine 6G as an example.

  Here, in Rhodamine 6G, in water, for example, as shown in FIG. 5, the absorption wavelength region from the ground state of S0 to the S1 state extends from a wavelength of 480 nm to a wavelength of 540 nm with a wavelength of 530 nm as a central excitation wavelength. Further, the fluorescence band spreads over a wavelength region from a wavelength of 540 nm to a wavelength of 600 nm, centering on a wavelength of 560 nm. Furthermore, the absorption band from the S1 state to the S2 state develops in a relatively wide region from a wavelength longer than 500 nm to around 800 nm. In FIG. 5, the horizontal axis indicates the wavelength (nm), and the vertical axis indicates the absorptance / fluorescence efficiency.

  However, in recent years, a fluorescence spectrum that can be absorbed from the S0 state to the S1 state by a two-photon excitation process has been observed in the near-infrared region centered at a wavelength of 740 nm to 840 nm (for example, reference “A. Fischer.et.al.Appl.Opt.34.1989 (1995) ”). On the other hand, in the two-wavelength fluorescence spectroscopy using a nanosecond laser, the fluorescence suppression effect by absorption from the S1 state to the S2 state has been confirmed in the above wavelength region.

  Therefore, in the super-resolution microscope according to the embodiment of the present invention described below, laser light having a wavelength of 775 nm is used as pump light and erase light.

(First embodiment)
FIG. 6 is a schematic configuration diagram of a main part of the super-resolution microscope according to the first embodiment of the present invention. In the present embodiment, pump light and erase light are generated using two fiber laser light sources 11 and 21.

  The fiber laser light source 11 includes a semiconductor laser 12, a collimator lens 13, a coupling lens 14, a fiber amplifier 15, a coupling lens 16, and a harmonic wave crystal element 17. The semiconductor laser 12 uses an infrared semiconductor laser that emits infrared light having a wavelength of 900 nm. The laser light emitted from the semiconductor laser 12 is converted into parallel light by a collimator lens 13 and then passed through a coupling lens 14 and a fiber. Introduced into the amplifier 15.

  The fiber amplifier 15 uses, as a laser active medium, a fiber amplifier doped with, for example, erbium ions and having a core diameter of about 2 μm and a length of about 10 mm. An infrared laser having a wavelength of 900 nm from the semiconductor laser 12 is used as the fiber amplifier 15. By introducing light, a single mode laser beam having a wavelength of 1.55 μm is amplified and emitted. The fiber amplifier 15 can emit laser light having an output of 100 W to several tens of kW from the fiber end by optimizing its core structure and length.

  The laser light having a wavelength of 1.55 μm emitted from the fiber amplifier 15 is incident on the harmonic crystal element 17 through the coupling lens 16 and is subjected to double wave conversion, whereby the laser light having a wavelength of 775 nm is converted from the harmonic crystal element 17. Let it emit.

  On the other hand, the fiber laser light source 21 includes a semiconductor laser 22, a collimator lens 23, a coupling lens 24, a fiber amplifier 25, a coupling lens 26, and a harmonic wave crystal element 27, similarly to the fiber laser light source 11. Then, a laser beam having a wavelength of 775 nm is emitted from the double wave crystal element 27.

  In the present embodiment, the semiconductor laser 12 of the fiber laser light source 11 is driven by the control unit 31 via the pulse generator 32, and the short circuit having the time width of the femtosecond order as shown in FIG. Pulse light is emitted, and thereby pump light having a high peak value (peak value) having a wavelength of 775 nm is emitted from the harmonic crystal element 17.

  Further, the semiconductor laser 22 of the fiber laser light source 21 is driven by the control unit 31 via the pulse generator 33 in synchronization with the semiconductor laser 12 of the fiber laser light source 11, and the semiconductor laser 22 as shown in FIG. Long pulse light having a time width on the order of nanoseconds is emitted, and thereby, erase light with a low peak value (peak value) having a wavelength of 775 nm is emitted from the harmonic crystal element 27.

  The pump light emitted from the fiber laser light source 11 passes through the reflective optical element 34, the beam combiner 35, the half mirror 36 and the microscope objective lens 37, and is condensed on the sample 39 on the scanning stage 38. A desired molecule (here, rhodamine 6G) is excited.

  Further, the erase light emitted from the fiber laser light source 21 is phase-modulated so that a hollow beam is formed by a spiral phase plate 41 which is a spatial modulation element, and then passes through a reflection mirror 42 and pump light and space by a beam combiner 35. Then, they are synthesized coaxially, and condensed on a sample 39 on a scanning stage 38 through a half mirror 36 and a microscope objective lens 37.

  When the polarization direction of the pump light and the polarization direction of the erase light are orthogonal to each other, the beam combiner 35 is configured by a polarization beam splitter and synthesizes the pump light and the erase light spatially coaxially. In addition, the spiral phase plate 41, for example, as shown in an enlarged plan view in FIG. 7, the central portion of the quartz substrate is divided into eight octagonal shapes symmetrical with respect to the optical axis, and the divided area is divided into, for example, erase light. Etching is performed in a staircase pattern with a phase difference of λ / 8 so that the phase difference rotates around the optical axis by 2π with respect to the center wavelength. When erase light passes through the spiral phase plate 41, the phase is inverted at the target position of the optical axis.

  The scanning stage 38 is two-dimensionally moved in a plane orthogonal to the optical axis of the microscope objective lens 37 by the control unit 31 in synchronization with the irradiation of the pump light and the erase light onto the sample 39, thereby pumping the sample 39. Two-dimensional scanning with light and erase light.

  On the other hand, the fluorescence (light response signal) generated from the sample 39 by the irradiation of the pump light and the erase light is incident on the short pass filter 43 through the microscope objective lens 37 and the half mirror 36, where the pump light and the erase light are made to enter. The light is blocked, and only the fluorescence is guided to the projection lens 44 and received by the photomultiplier tube 45. The output of the photomultiplier tube 45 is taken into a computer (not shown) to obtain a two-dimensional fluorescence image. Since the fluorescence band of rhodamine 6G has a band of about 40 nm before and after peaking at a wavelength of 555 nm, the short pass filter 43 is configured to cut the long wavelength side of, for example, a wavelength of 600 nm or more, and has a wavelength of 775 nm. The scattered light of pump light or erase light is prevented from entering the photomultiplier tube 45.

  Therefore, the super-resolution microscope shown in FIG. 6 constitutes a light source means including two fiber light sources 11 and 21, and constitutes an optical system including a beam combiner 35, a half mirror 36 and a microscope objective lens 37, The scanning unit includes the scanning stage 38, and further includes the short-pass filter 43, the projection lens 44, and the photomultiplier tube 45 to configure the detecting unit.

When observing a sample stained with rhodamine 6G as in this embodiment, the two-photon absorption cross section σ _two (λ) when using light with a wavelength of 775 nm is about 200 × 10 ⁻⁵⁰ cm ⁴ s. It is. In this wavelength region, the fluorescence suppression cross section σ _dip (λ) that substantially contributes to fluorescence suppression from the S1 state is about 10 ⁻¹⁷ cm ² .

Here, when the pulse width t of the pump light is 5 ps, the photon density (photon flux: I) for generating molecules in the S1 state necessary for detection is estimated. If the probability of taking the S1 state for one molecule of rhodamine 6G is n ₁ (t), the rate equation is expressed by the following equation (3).

For example, if I = 10 ²⁹ cm ⁻² s ⁻¹ is substituted into the above equation (3), n ₁ (t) becomes 0.1, and sufficient fluorescence intensity can be obtained without saturation of the fluorescence phenomenon. I understand. This photon density is 85 W when converted to the intensity of the pump light when condensing with the numerical aperture of the microscope objective lens 37 being 1.4.

  Furthermore, the photon flux: Ie necessary for the expression of the super-resolution function is estimated. The ratio R (Dip-Ratio) of the fluorescence intensity with no erase light is given by the following equation (4).

Here, since the fluorescence lifetime τ in the S1 state of rhodamine 6G is 3.75 ns in methanol, for example, the pulse width of the erase light is set to 3.75 ns or more, for example, Ie = 10 ²⁶ cm ⁻² s ⁻ Assuming ¹ , R = 0.21. This value is a photon flux sufficient to develop a super-resolution effect. Similarly, when this photon flux is converted into the intensity of erase light when condensing with the numerical aperture of the microscope objective lens 37 being 1.4, it becomes 340 mW. With this erase light intensity, n ₁ (t) in the above equation (3) is 8 × 10 ⁻⁵ , and light emission due to two-photon excitation of the erase light itself can be completely ignored.

  The intensities of the pump light and erase light described above are the peak value (peak value) of the pulsed light emitted from the corresponding semiconductor lasers 12 and 22 and / or the core structure of the corresponding fiber amplifiers 15 and 25, respectively. Set the length appropriately controlled.

  As described above, according to the super-resolution microscope of the present embodiment, since pump light and erase light having the same wavelength are used, as a beam combiner that synthesizes these optical axes coaxially, for example, the polarization directions of both are orthogonal to each other. By doing so, it is possible to synthesize using an inexpensive polarization beam splitter without using an expensive element such as a dichroic mirror. In addition, it becomes easy to design each optical element such as the half mirror 36 and the microscope objective lens 37 in the common optical path of the pump light and the erase light, and the aberration is dramatically reduced without reducing the throughput of the entire optical system. It becomes possible.

  Further, it is known that the spatial resolution of the super-resolution microscope is given by the following equation (5) (for example, “Opt. Eng. Vol. 44 (2005) 033602”).

Therefore, in the present embodiment, τ: 3.75 ns, σ: 10 ⁻¹⁷ cm ² , Ie: 10 ²⁶ cm ⁻² s ⁻¹ , and λe: 775 nm. Therefore, the numerical aperture of the microscope objective lens 37 ( When NA) is 1.4 and the spatial resolution (Γ) is calculated, it is approximately 143 nm. On the other hand, the spatial resolution obtained by single irradiation of pump light, that is, two-photon excitation with a wavelength of 775 nm is approximately 338 nm from Rayleigh's equation (0.61 × λ / NA). According to this, the spatial resolution can be improved more than twice that of the conventional microscope.

(Second Embodiment)
FIG. 8 is a schematic configuration diagram of a main part of a super-resolution microscope according to the second embodiment of the present invention. In this embodiment, pump light and erase light are generated using one semiconductor laser 51 that emits infrared light having a wavelength of 755 nm. Therefore, under the control of the control unit 52, a drive pulse having a steep rise as shown in FIG. 3 is supplied from the pulse generator 53 to the semiconductor laser 51 to drive the semiconductor laser 51.

  In this way, when a driving pulse having a steep rise is supplied to the semiconductor laser 51, the emission chirp peak including emission at a wavelength of 755 nm is generated from the semiconductor laser 51 at the rise of emission with a very narrow time width on the order of picoseconds. appear. Thereafter, a long pulse with a peak value of nanosecond order stable at a wavelength of 755 nm appears. Such laser emission characteristics are general characteristics in semiconductor lasers.

  Laser light emitted from the semiconductor laser 51 is collimated by a collimator lens 55 and is incident on a nonlinear optical element 56 having wavelength dispersion characteristics. The nonlinear optical element 56 emits light with a high peak value (crest value) by rotating the plane of polarization by 90 degrees due to the optical Kerr effect and emits light with a low peak value. The polarization plane is emitted without rotating.

  That is, when a pulse light having a high peak value is incident, the nonlinear optical element 56 induces a higher-order nonlinear optical effect in the medium and instantaneously changes the refractive index distribution of the medium, thereby When the electric susceptibility, that is, the electrical stress tensor is changed, the phase of the incident light is changed significantly and the polarization plane is rotated by 90 degrees, and the pulse light having a low peak value is incident, the normal linearity The length and material of the optical medium are appropriately configured with respect to the wavelength and intensity of the light so that the incident light can be transmitted by a natural response. Such a nonlinear optical element 56 can be easily and inexpensively configured using an organic material such as ordinary quartz or plastic.

  The laser light emitted from the nonlinear optical element 56 is incident on the polarization beam splitter 57. For example, a short pulse whose polarization plane is rotated is reflected by the polarization beam splitter 57 and emitted as pump light, and the rotation of the polarization plane is received. The long pulse that does not exist is transmitted through the polarization beam splitter 57 and emitted as erase light. Therefore, the super-resolution microscope according to the present embodiment includes the semiconductor laser 51, the collimator lens 55, the nonlinear optical element 56, and the polarization beam splitter 57 to constitute a light source means.

  The pump light emitted from the polarization beam splitter 57 is collected on the sample 39 on the scanning stage 38 through the reflection optical element 34, the beam combiner 35, the half mirror 36, and the microscope objective lens 37 as in the first embodiment. The light is excited, thereby exciting the desired molecules in the sample 39.

  The erase light emitted from the polarization beam splitter 57 passes through the reflective optical elements 61 and 62 and is incident on the spiral phase plate 41 similar to that of the first embodiment, where the phase is set so that a hollow beam is formed. After modulation, the beam is combined with the pump light spatially coaxially by a beam combiner 35 including a polarizing beam splitter via a reflection mirror 42, and collected on a sample 39 on a scanning stage 38 via a half mirror 36 and a microscope objective lens 37. Shine.

  The scanning stage 38 is two-dimensionally moved in a plane orthogonal to the optical axis of the microscope objective lens 37 by the control unit 52 in synchronization with the pulse drive of the semiconductor laser 51, thereby moving the sample 39 with pump light and erase light. Two-dimensional scanning. Other configurations and operations are the same as those in the first embodiment, and thus description thereof is omitted here.

  According to the super-resolution microscope of the present embodiment, the same effect as that of the first embodiment can be obtained. In addition, the laser light emitted from one semiconductor laser 51 is converted into a nonlinear optical element according to the peak value. Since the polarization plane is selectively rotated 90 degrees by 56 and separated into the pump light and the erase light by the polarization beam splitter 57, the light source means is made more than in the case of the first embodiment. There is an advantage that it can be configured easily and inexpensively.

  In addition, this invention is not limited only to the said embodiment, Many deformation | transformation or a change is possible. For example, in the above embodiment, the sample 18 is two-dimensionally scanned by the scanning stage 17, but a two-dimensional mirror having two oscillating mirrors in the optical path between the pump light and the erase light that are coaxial. A galvano scanner can be arranged to perform two-dimensional scanning, or a one-dimensional scanning stage and a one-dimensional galvano scanner can be combined to perform two-dimensional scanning.

It is a figure for demonstrating the fluorescence suppression process of the molecule | numerator by the nanosecond erase light pulse. It is a figure for demonstrating the fluorescence suppression process of the molecule | numerator by the picosecond erase light pulse. It is a figure which shows the two-photon process and fluorescence suppression process explaining the principle of this invention. It is a schematic diagram explaining waveform control of short pulse pump light and long pulse light when a super-resolution microscope is configured using light of one color. It is a figure which shows the absorption wavelength band and fluorescence band in the case of observing rhodamine 6G. It is a principal part block diagram of the super-resolution microscope which concerns on 1st Embodiment of this invention. It is an enlarged plan view which shows the structure of the spiral phase plate shown in FIG. It is a schematic block diagram of the principal part of the super-resolution microscope which concerns on 2nd Embodiment of this invention. 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 shown in FIG. It is a conceptual diagram which shows the 2nd excitation state of the molecule | numerator shown in FIG. It is a figure which shows notionally the state which the molecule | numerator shown in FIG. 9 returns to a ground state from a 2nd excitation state. It is a conceptual diagram for demonstrating the double resonance absorption process in a molecule | numerator. It is a conceptual diagram for demonstrating the double resonance absorption process in a molecule | numerator.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Reflection mirror 2 Beam combiner 3 Spatial modulation element 11, 21 Fiber laser light source 12, 22 Semiconductor laser 13, 23 Collimator lens 14, 24 Coupling lens 15, 25 Fiber amplifier 16, 26 Coupling lens 17, 27 Double wave crystal element DESCRIPTION OF SYMBOLS 31 Control part 32, 33 Pulse generator 34 Reflective optical element 35 Beam combiner 36 Half mirror 37 Microscope objective lens 38 Scan stage 39 Sample 41 Spiral phase plate 42 Reflection mirror 43 Short pass filter 44 Projection lens 45 Photomultiplier tube 51 Semiconductor laser 52 Control unit 53 Pulse generator 55 Collimator lens 56 Non-linear optical element 57 Polarizing beam splitter 61, 62 Reflective optical element

Claims (7)

A microscope for observing a desired molecule in a sample,
Light source means for emitting pump light for exciting the molecule from a stable state to a first excited state, and erase light for exciting the molecule from the first excited state to a second excited state;
An optical system for partially superimposing the pump light and the erase light from the light source means and condensing the sample on the sample;
Scanning means for scanning the sample by relatively moving the pump light and the erase light collected by the optical system and the sample;
Detecting means for detecting a light response signal generated from the sample by irradiation of the pump light and the erase light, and
The light source means includes
As the pump light, the photon flux when focused on the sample is 1 / (τσ _two ) ^1/2 or more when the two-photon absorption cross section of the molecule is σ _two and the fluorescence lifetime is τ. Emits pulsed light of wavelength,
The erase light is configured to emit a pulsed light having the same wavelength as the pump light and a photon flux when condensed on the sample is less than 1 / (τσ _two ) ^1/2. Microscope. The light source means includes
As the pump light, pulse light having a pulse width of less than 1 nanosecond is emitted,
2. The microscope according to claim 1, wherein the erase light is configured to emit pulse light having a pulse width of 1 nanosecond or more. The light source means has one laser light source,
The microscope according to claim 1 or 2, wherein the pump light and the pulsed light of the erase light having different peak values are emitted from the laser light source.   The microscope according to claim 1, 2 or 3, wherein the light source means is configured to emit the pump light prior to the emission of the erase light.   The microscope according to claim 4, wherein a difference in emission timing between the pump light and the erase light is shorter than the fluorescence lifetime.   The microscope according to any one of claims 1 to 5, wherein the light source means further includes a spatial modulation element that spatially modulates the erase light.   The microscope according to claim 6, wherein the spatial modulation element spatially modulates the erase light so as to be condensed in a hollow shape on the sample.

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