JP2005266705A - Super-resolution microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a super-resolution microscope capable of always stably demonstrating a super-resolution effect with a simple and inexpensive constitution. <P>SOLUTION: A continuous oscillation layer 12 and an acousto-optical modulator 14 are provided to constitute a second light source means which emits at least a second pulse light beam (erase light pulse), and coherent light from the continuous oscillation laser 12 has the intensity modulated into a false pulse by the acousto-optical modulator 14, and a first pulse light beam (pump light pulse) is emitted from a first light source means (11, 13, and 15) at least a time t after the start of voltage application to the acousto-optical modulator 14, wherein t is 0.65×D/v-(L2-L1)/c, and thus the super-resolution microscope which has high S/N and high spatial resolution and is superior in practicality is realized with an inexpensive constitution. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a high-performance and high-function 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, for example. The present invention relates to an improvement of the light source device.

  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. 4 shows the electronic structure of the valence electron orbit of the molecules constituting the sample. First, the electrons in the valence electron orbit of the molecule in the ground state (S0 state) shown in FIG. 4 are excited by light of wavelength λ1. Thus, the first electronic excitation state (S1 state) shown in FIG. Next, excitation is similarly performed with light of another wavelength λ2, and a second electron excitation state (S2 state) shown in FIG. 6 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. 5 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. 5 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. 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 the deexcitation process by fluorescence or phosphorescence in the excited state of FIG. 6 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 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 valence electron orbit shown in FIG. 4 has an energy level unique to each molecule, the wavelength λ1 varies 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. 8 is a conceptual diagram of a double resonance absorption process in a molecule. A molecule in the ground state S0 is excited by light having a wavelength λ1 to S1, which is a first electronically excited state, and further excited by a second electron by light having a wavelength λ2. The state of being excited by the state S2 is shown. FIG. 8 shows that the fluorescence from S2 of certain molecules is extremely weak.

  In the case of molecules having optical properties as shown in FIG. 8, a very interesting phenomenon occurs. FIG. 9 is a conceptual diagram of the double resonance absorption process, as in FIG. 8. The horizontal X axis represents the spread of the spatial distance, and the light of the wavelength region λ2 and the light of the wavelength region λ2 is not irradiated. A space area A0 is shown.

  In FIG. 9, in the spatial region A0, a large number of molecules in the S1 state are generated by excitation of light of wavelength λ1, and at this time, 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. 9, two types of light of wavelength λ1 and wavelength λ2 are spatially superposed 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. 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.

  Furthermore, by devising the irradiation timing of the two lights of wavelength λ1 and wavelength λ2, a condition that can improve S / N and effectively suppress fluorescence is proposed. It can also be expressed more effectively (see, for example, Patent Document 3).

  As a specific example of such super-resolution microscopy, light of wavelength λ1 (especially laser light) that excites fluorescent labeler molecules from the S0 state to the S1 state is used as pump light, and wavelength λ2 that excites the S1 state to the S2 state. As shown in FIG. 10, the light is emitted from the light source 81 and the erase light is emitted from the light source 82, and the pump light is reflected by the dichroic mirror 83 and then collected by the condensing optical system 84. The erasing light is condensed on the phase plate 86 and then hollowed by the phase plate 86, and then transmitted through the dichroic mirror 83 and spatially superimposed on the pump light so as to be condensed on the sample 85 by the condensing optical system 84. Have been proposed (see, for example, Patent Document 4).

  According to this microscope, fluorescence other than the vicinity of the optical axis where the intensity of the erase light becomes zero is suppressed, and as a result, a region narrower than the spread of the pump light (Δ <0.61 · λ1 / NA, where NA is a condensed light) Only the fluorescent labeler molecules present in the numerical aperture) of the optical system 84 are observed, and as a result, super-resolution is developed. As the phase plate 86 for converting the erase light into a hollow beam, for example, as shown in FIG. 11, a configuration in which a phase difference π is given at a point-symmetrical position with respect to the optical axis, or a liquid crystal surface is used. An optical spatial modulator or a deformable mirror that controls the shape of the mirror itself by a wavelength order can be used.

  On the other hand, as a light source device applied to the above-described super-resolution microscope, the present inventors do not use an expensive pulsed laser, for example, an inexpensive continuous oscillation excellent in beam position stability and wavefront uniformity. A (CW) laser is used, and a laser beam emitted from the CW laser is modulated by an acousto-optic modulator using an acousto-optic crystal to generate a pseudo pulse (for example, a patent application) 2003-109018).

  Here, the acousto-optic crystal exhibits an acousto-optic effect (for example, deflection, diffraction, modulation, phase shift, frequency shift, birefringence, etc.) due to a refractive index change induced by the propagation of sound waves. The following three types of effects appear depending on the wavelength of sound and light when varying the refractive index with the speed of sound.

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 mth-order diffracted light diffracted by the acoustic wave of frequency f by the acousto-optic crystal is shifted in frequency by mf due to the Doppler effect, and the diffraction angle θm is sinθm = where λ is the wavelength of the light and Λ is the wavelength of the sound wave. mλ / Λ.

The acousto-optic modulator used for laser beam deflection and modulation applies this effect, and FIG. 12 shows its principle configuration. As shown in FIG. 12, the acoustooptic modulator 91 is configured by adhering a transducer 93 to an acoustooptic crystal 92, and applies a voltage to the transducer 93 to generate an ultrasonic wave and propagate the acoustooptic crystal 92. Diffracts incident light, and FIG. 12 shows diffraction by Bragg reflection. The acoustooptic modulator 19 can modulate up to 25 MHz (band) if, for example, a PbMoO ₄ crystal or a TeO ₂ crystal is used as the acoustooptic crystal 92.

  If the acousto-optic modulator 91 is inserted into the optical path of the CW laser and the transducer 93 is electrically controlled to propagate the sound wave intermittently to the acousto-optic crystal 92, a pulse light source suitable for the super-resolution microscope system can be obtained. Can be realized. That is, pulsing can be performed while maintaining the excellent beam quality of CW lasers such as He / Ne lasers, Ar lasers, Kr lasers, and Ti sapphire lasers. In addition, any pulse oscillation frequency can be selected by electrical control. For example, the pulse oscillation timing and pulse width of the pump light and the erase light can be easily and electrically controlled. It is possible to efficiently induce the fluorescence suppression effect that forms the following.

As a result, it is possible to provide a high-performance microscope with improved super-resolution and 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.
JP-A-8-184552 JP 2001-100102 A JP-A-11-95120 JP 2001-272345 A

  However, according to subsequent experimental studies by the present inventors, it has been found that the super-resolution microscope using the previously developed light source device has the following points to be improved.

  That is, the acousto-optic modulator diffracts incident light by propagating the sound wave to the acousto-optic crystal and modulates the light intensity of the diffracted light. Determined by speed. For example, the time t when the intensity of the modulated light from the start of voltage application to the transducer is almost halved is t = 0, where D is the beam diameter of the incident light to the acoustooptic crystal and v is the speed of sound in the acoustooptic crystal. (Refer to “Illustrated Optical Device Dictionary”, Optronics, Inc., July 10, 1996, p. 32).

  Therefore, during t = 0.65 × D / v from the start of voltage application (hereinafter also referred to as a transient change time zone), the maximum diffraction efficiency is not reached, and a partial region of the beam is diffracted. Thus, the wave front of the diffracted light is disturbed.

  For this reason, in the super-resolution microscope, if the pump light and the erase light are directly overlapped in the time and space domain and projected onto the sample surface, a transient change time zone will be included in the time domain. In the time zone, the intensity of the erase light may not exceed a certain threshold value that induces the fluorescence suppression effect, and the expression of the super-resolution effect may be significantly weakened.

  In addition, when the erase light is spatially modulated and shaped into a hollow beam, the uniformity of the original wavefront becomes an important factor, and if there is some disturbance of the wavefront, the hollow is symmetrical with respect to the optical axis. I can't get a beam. For this reason, since the wavefront of the diffracted light changes greatly in the transient change time zone, the spatial modulation of the erase light becomes uncertain, and as a result, the spatial fluorescence erasure becomes insufficient on the sample focusing surface. Thus, not only the resolution is decreased, but the total amount of the fluorescence signal may be weakened.

  Accordingly, an object of the present invention made in view of such circumstances is to provide a super-resolution microscope that can always express a super-resolution effect stably with a simple and inexpensive configuration.

The invention according to claim 1, which achieves the above object, provides a first coherent excitation method for exciting a molecule from a ground state to a first electronically excited state with respect to a sample including a molecule having at least three electronic states including a ground state. First light source means for emitting the pulse light of
Second light source means for emitting coherent second pulsed light for exciting the molecule from the first electronically excited state to a second electronically excited state having a higher energy level;
An optical system for condensing and irradiating the sample with the first pulsed light and the second pulsed light partially overlapped;
Scanning means for scanning the sample by relatively moving the light condensed and irradiated 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,
At least the second light source means has a continuous wave laser, and an acousto-optic modulator that modulates the intensity of the coherent light from the continuous wave laser by applying a voltage and emits it as the second pulsed light,
The optical path length from the first light source means to the sample is L1, the optical path length from the second light source means to the sample is L2, and the beam diameter of coherent light entering the acoustooptic modulator from the continuous wave laser Is D, the sound velocity in the acoustooptic modulator is v, and the speed of light is c, at least t = 0.65 × D / v− (L2−L1) / c from the start of voltage application to the acoustooptic modulator. Thereafter, the first pulsed light is emitted from the first light source means.

  According to a second aspect of the present invention, in the super-resolution microscope according to the first aspect, when the pulse time of the first pulsed light emitted from the first light source means is tp, the acousto-optic modulator is provided. The voltage application to the acousto-optic modulator is terminated at least after t = 0.65 × D / v− (L2−L1) / c + tp from the start of voltage application. is there.

The invention according to claim 3 is the super-resolution microscope according to claim 1 or 2, wherein the first light source means modulates the intensity of the continuous wave laser and the coherent light from the continuous wave laser by applying a voltage. And an acousto-optic modulator that emits the first pulsed light,
The acousto-optics of the first light source means after at least t = 0.65 × D / v- (L2-L1) / c from the start of voltage application to the acousto-optic modulator of the second light source means. The present invention is characterized in that voltage application to the modulator is started.

The invention according to claim 4 is the super-resolution microscope according to claim 1, 2, or 3, wherein the acoustooptic modulator has a PbMoO ₄ crystal or a TeO ₂ crystal as an acoustooptic crystal. It is.

  First, the principle of the present invention will be described with reference to the time chart shown in FIG.

  FIG. 1 shows a state in which erase light and pump light are obtained using an independent CW laser and an acousto-optic modulator (AOM), respectively. FIG. 1 (a) shows the pulse shown in FIG. 1 (b). FIG. 1C shows the diffraction intensity of the erase light pulse obtained when the voltage waveform is applied to the erase light AOM. FIG. 1C shows the pulse voltage waveform shown in FIG. 1D applied to the pump light AOM. The diffraction intensity of the pump light pulse obtained at this time is shown.

In FIG. 1A, T _R (E) is a transient change time zone of the erase light, and is a time zone where the diffraction intensity of the erase light is weak and the wave front is disturbed. T _USE (E) is a time zone in which the sound wave is completely propagated to the acousto-optic crystal, the diffraction efficiency is maximized, and the wavefront is stable. T _D (E) is a time zone in which the diffracted light leaks due to the remaining sound wave even after the voltage application to the AOM is completed, and the diffraction intensity of the erase light is the same as T _R (E). Is a time zone where the wave front is disturbed.

Further, in FIG. 1C, T _R (P) is a transient change time zone of the pump light, which is a time zone in which the diffraction intensity of the pump light is weak and the wave front is disturbed. Further, T _D (P) is a time zone in which diffracted light leaks due to the remaining sound wave even after the voltage application to the AOM is finished, and similarly to T _R (P), the diffraction intensity of the pump light. Is a time zone where the wave front is disturbed. In FIG. 1C, there may be a time zone T _USE (P) corresponding to T _USE (E) in FIG.

Regarding the erase light, the time zone of T _R (E) and T _D (E) cannot be used for the erase light of the super-resolution microscope because the intensity is insufficient and the wave front is disturbed. Therefore, the only available time zone is T _USE (E).

On the other hand, with respect to the pump light, since the uniformity of the beam intensity and the uniformity of the wave front are not pursued, the time zone of T _R (P) and T _D (P) can be used for the super-resolution microscope, The time when T _R (P) and T _D (P) are combined can be set as the irradiation time T _USE (P) of the pump light pulse. Of course, a time zone corresponding to T _USE (E) may be provided, and pump light diffracted in that time zone may be used.

As is clear from FIG. 1, in the super-resolution microscope, since it is the time zone of T _USE (E) that can be effectively used as erase light, the irradiation time T _USE (P) of the pump light pulse is Ideally, it will be completed within the time zone of T _USE (E). During this time, the erase light has sufficient intensity and can be spatially modulated into a complete hollow beam shape.

  Therefore, for example, if the irradiation timing as shown in the time chart of FIG. 1 is realized on the sample surface where the pump light and the erase light arrive, the super-resolution microscope using the AOM can be optimized and the resolution can be improved. The effect can be maximized.

Here, as shown in FIG. 1, the irradiation time T _USE (P) of the pump light pulse is set within the time zone of T _USE (E) while avoiding the time zones of T _R (E) and T _D (E). Specific means for overlapping include electrical means and optical means.

  As an electrical means, for example, a clock generator generates an AOM control pulse signal, and the control pulse signal is input to a digital counter circuit typified by a time gate generator or a delay generator, so that the control pulse signal rises or falls Then, a pulse having an arbitrary length is re-oscillated after a certain period of time, thereby driving the AOM and extracting the diffracted light at an arbitrary timing. Instead of the digital counter circuit, an analog circuit module such as an RC circuit or a cable delay can be used to obtain a pulse having an arbitrary length for driving the AOM from the control pulse signal.

  Moreover, as an optical means, those irradiation timings can be adjusted by changing the optical path length of pump light and / or erase light. For example, in air, a delay of about 1 ns is possible at about 30 cm, so changing the spatial arrangement of optical components, adding mirrors, etc., changing the optical path length, adjusting the irradiation timing, or passing an optical fiber Thus, the irradiation timing can be adjusted by changing the optical path length.

In this way, using the electric means or the optical means, the irradiation time T _USE (P) of the pump light pulse is set to be T _USE (E) while avoiding the time zones T _R (E) and T _D (E). If it is made to overlap within this time zone, in the super-resolution microscope, it is possible to realize sample illumination conditions that can maximize the resolution improvement effect.

  According to the first aspect of the present invention, the second light source means for emitting at least the second pulsed light (erase light pulse) includes the continuous wave laser and the acoustooptic modulator, and the continuous wave laser is used. The coherent light is intensity-modulated with an acousto-optic modulator to form a pseudo pulse, and at least after t = 0.65 × D / v− (L2−L1) / c from the start of voltage application to the acoustooptic modulator. Since the first pulsed light (pump light pulse) is emitted from one light source means, it is possible to realize a super-resolution microscope excellent in practicality with high S / N and spatial resolution with an inexpensive configuration. it can. In particular, if a gas laser with a good wavefront and position stability is used as a continuous wave laser, beam shaping of erase light pulses and spatial resolution degradation due to beam position deviation can be prevented, so super-resolution and resolution An excellent super-resolution microscope can be realized. Therefore, it is possible to provide a super-resolution microscope with good workability for general users who are not specialized in lasers, particularly biological researchers.

According to the invention of claim 2, when the pulse time of the pump light pulse is tp, at least t = 0.65 × D / v− (L2−L1) / c + tp from the start of voltage application to the acoustooptic modulator. Since the voltage application to the acousto-optic modulator is terminated later, the irradiation time tp of the pump light pulse is completed within T _USE (E) of FIG. 1 where the erase light pulse has sufficient intensity. Can do. Therefore, the super-resolution microscope can be optimized, and the effect of improving the resolution can be maximized.

  According to the invention of claim 3, the second light source means for emitting the pump light pulse also includes the continuous wave laser and the acoustooptic modulator, and the coherent light from the continuous wave laser is converted into the acoustooptic modulator. The intensity of the pump light is converted into a pseudo pulse, and at least after t = 0.65 × D / v− (L2−L1) / c from the start of voltage application to the acoustooptic modulator for erase light, the pump light Since the application of voltage to the acousto-optic modulator is started, a super-resolution microscope excellent in practicality with a high S / N and spatial resolution can be realized with a more inexpensive configuration.

According to the invention of claim 4, since the PbMoO ₄ crystal or the TeO ₂ crystal is used as the acousto-optic crystal of the acousto-optic modulator, the rise response time for the input electric pulse can be shortened to about 5 nsec, which is equivalent to the nanosecond pulse laser. A repetition frequency of the order of MHz can be obtained with the pulse width.

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

  FIG. 2 is a diagram showing a main configuration of the optical system of the super-resolution microscope in one embodiment of the present invention. The microscope according to the present embodiment is a super-resolution fluorescence microscope in which the intensity of both CW pump light and erase light is modulated by an acousto-optic modulator to irradiate the sample. Hereinafter, a case where a biological sample stained with rhodamine 6G is observed will be described as an example.

  Rhodamine 6G has an 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 the second electronic excited state (S2) 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, the He / Ne laser 11 that continuously oscillates laser light having a wavelength of 543.5 nm is used as a light source that generates pump light, and laser light having a wavelength of 647.1 nm is continuously used as a light source that generates erase light. An oscillating Kr laser 12 is used.

The pump light from the He / Ne laser 11 and the erase light from the Kr laser 12 are respectively incident on acousto-optic modulators (AOM) 13 and 14 having the same configuration as in FIG. 12, and these AOMs 13 and 14 are supplied to a clock generator. Based on the reference clock from 35, the corresponding gate generators 15 and 16 control each to generate a pseudo pulse. That is, the first light source means is configured to include a continuous oscillation He / Ne laser 11, AOM 13, and gate generator 15, and the second light source means includes a continuous oscillation Kr laser 12, AOM 14, and gate generator 16. And adjusting the gate width and delay time in the gate generators 15 and 16 to optimize the overlapping state of T _USE (P) and T _USE (E) shown in FIG. A pulse voltage is applied to the AOMs 13 and 14 under conditions that can effectively induce a resolution effect, that is, a pulse width, a period, and a delay time, and incident light is transmitted when the voltage is applied.

  The pump light that passes through the AOM 13 is reflected by the beam combiner 17 and the half mirror 18 and condensed on the sample 20 by the objective lens 19. The erase light transmitted through the AOM 14 is incident on the phase plate 21 and spatially modulated into a hollow beam, then reflected by the reflection mirror 22 and incident on the beam combiner 17 to be synthesized coaxially with the pump light. The sample 20 is condensed through the half mirror 18 and the objective lens 19.

  For example, as shown in FIG. 3, the phase plate 21 is configured by dividing a quartz substrate into eight parts around the optical axis and giving each region a phase difference of λ / 8 stepwise with respect to the wavelength of the erase light. . More specifically, the optical path length is controlled by eroding the quartz substrate by chemical etching. By passing such a phase plate 21, the erase light has a phase distribution having an opposite phase with respect to the optical axis on the beam surface, and as a result, erase light having a light intensity of zero at the center of the optical axis is obtained. can get.

  The sample 20 is placed on a sample moving stage 23 which is a scanning means, and the sample moving stage 23 is driven two-dimensionally, whereby the sample 20 is two-dimensionally scanned with pump light and erase light.

  On the other hand, the fluorescence emitted from the sample 20 by the irradiation of the pump light and the erase light passes through the objective lens 19 and then the notch filter 24 for removing the wavelength component of the pump light (wavelength 543.5 nm) and the wavelength component of the erase light (wavelength 647). .1 nm) through a notch filter 25 to be incident on the half mirror 26, and the fluorescence from the sample 20 reflected by the half mirror 26 is condensed at the center of the pin hole 28 by the lens 27, and the pin hole 28 is The transmitted fluorescence is incident on the photomultiplier 30 serving as detection means by the lens 29 and detected. Here, the pinhole 28 is disposed at the confocal position and functions as a spatial filter. This is because only fluorescence emitted from a specific depth portion in the sample 20 is emitted from other than the sample 20, for example, at the same time as it acts to increase the S / N of the measurement by cutting off fluorescence and scattered light from the optical system. The optical sectioning function is selected.

  The fluorescence that has passed through the half mirror 26 is imaged by a CCD camera 32 having an imaging lens 31 so that a fluorescent spot image can be directly observed and used for focusing the objective lens 19 and the like.

In the above configuration, if the AOM 13 or 14 uses, for example, a PbMoO ₄ crystal as the acousto-optic crystal, the rise response time with respect to the input electric pulse from the corresponding gate generator 15 or 16 can be set to about 5 nsec. As the erase pulse pseudo-pulse light, a repetition frequency of the order of MHz can be obtained with a pulse width equivalent to that of the nanosecond pulse laser.

In the present embodiment, as shown in FIG. 1, the pulse width of the erase light applied to the sample 20 is longer than that of the pump light, and when the pump light is applied to the sample 20, the erase light is also applied simultaneously. As described above, the AOMs 13 and 14 are driven and controlled by the corresponding gate generators 15 and 16 to adjust the phase and frequency of the pseudo pulses of the generated pump light and erase light. In particular, regarding the phase, a phase difference during modulation of about several nanoseconds occurs due to individual differences of PbMoO ₄ crystals, and the expression of the fluorescence suppression effect is optimized by the gate generators 15 and 16. In this way, since the pump light and the erase light can be reliably overlapped in the time domain, the fluorescence suppression effect can be reliably induced and super-resolution can be realized.

  Here, the irradiation conditions of the erase light and the pump light are, for example, by removing the notch filters 24 and 25 and monitoring the scattered light from the sample surface, for example, receiving the scattered light with the photomultiplier 30 and outputting the output signal. The gate generators 15 and 16 may be used for adjustment while monitoring with an oscilloscope. If it does in this way, it can adjust reliably, after grasping correctly irradiation timing of pump light and erase light in a sample side.

  Alternatively, instead of the adjustment by the gate generators 15 and 16, using an electrical delay means for adjusting the length of the cable connecting the clock generator 35 and the gate generators 15 and 16 so as to satisfy the conditions described below, By using an optical delay means that adjusts the delay amount of the reference clock input from the clock generator 35 to the gate generators 15 and 16 or inserts an optical cable of an appropriate length in the optical path of the erase light or pump light, Alternatively, the irradiation conditions of the erase light and the pump light can be adjusted by adjusting the optical path length of the pump light.

  That is, in adjusting the irradiation conditions of the erase light and the pump light, the optical path length L1 from the He / Ne laser 11 as the pump light source to the sample 20 and the optical path from the Kr laser 12 as the erase light source to the sample 20 Since there is an optical path difference with the length L2, there is a light arrival time difference corresponding to (L2−L1) / c in the air when the light velocity is c. The AOMs 13 and 14 are in a transient change time zone from the start of voltage application to t = 0.65 × D / v, and the erase light becomes stable diffracted light through this transient change time zone.

Therefore, in consideration of the above, in order to prevent the pump light from overlapping in the transient change time zone, as shown in FIG. 1, the rise of the pulse voltage with respect to the AOM 13 for the pump light is changed for the erase light. From the rise of the pulse voltage with respect to the AOM 14, t = 0.65 × D / v− (L 2 −L 1) / c is adjusted. Further, when the irradiation time of the pump light pulse is T _USE (P), the fall of the pulse voltage for the AOM 14 is after t = 0.65 × D / v− (L2−L1) / c + T _USE (P). .

In this way, the irradiation time T _USE (P) of the pump light pulse can be overlapped within the time zone of T _USE (E) where stable diffracted light of the erase light pulse can be obtained. In the fluorescence microscope, the resolution improving effect can be maximized.

  It should be noted that the present invention is not limited to the above-described embodiment, and many variations or modifications can be made. For example, in the above embodiment, both the pump light and the erase light of CW are intensity-modulated by the acousto-optic modulator and pseudo-pulsed, but only the erase light is pseudo-pulsed using the acousto-optic modulator, The pump light can be configured to obtain pulsed light using a pulsed laser without using an acousto-optic modulator. Even in this case, the cost can be reduced as compared with the case where a pulsed laser is used for both pump light and erase light. Moreover, it can replace with an acousto-optic modulator and can also be pseudo-pulsed using an electro-optic element. Also in this case, since there is a transient change time zone and problems such as wavefront disturbance occur, the optical path length and the response time of the electro-optic element are measured, and the timing of the pump light and erase light on the sample surface is described with reference to FIG. By adjusting as described above, the effect of developing super-resolution can be enhanced.

It is a time chart for demonstrating the principle of this invention. It is a schematic block diagram of the super-resolution fluorescence microscope which shows one embodiment of this invention. It is a figure which shows the structure of the phase plate shown in FIG. 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 figure which shows the structure of an example of the conventional super-resolution microscope. It is a top view which shows the structure of the phase plate shown in FIG. It is a figure which shows the fundamental structure of the acousto-optic modulator which can be used for the super-resolution microscope which concerns on this invention.

Explanation of symbols

11 He / Ne laser 12 Kr laser 13,14 Acousto-optic modulator (AOM)
15, 16 Gate generator 17 Beam combiner 18, 26 Half mirror 19 Objective lens 20 Sample 21 Phase plate 22 Reflecting mirror 23 Sample moving stage 24, 25 Notch filter 27, 29 Lens 28 Pinhole 30 Photomal 31 Imaging lens 32 CCD camera 35 Clock generator

Claims (4)

First light source means for emitting a coherent first pulsed 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;
Second light source means for emitting coherent second pulsed light for exciting the molecule from the first electronically excited state to a second electronically excited state having a higher energy level;
An optical system for condensing and irradiating the sample with the first pulsed light and the second pulsed light partially overlapped;
Scanning means for scanning the sample by relatively moving the light condensed and irradiated 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,
At least the second light source means has a continuous wave laser, and an acousto-optic modulator that modulates the intensity of the coherent light from the continuous wave laser by applying a voltage and emits it as the second pulsed light,
The optical path length from the first light source means to the sample is L1, the optical path length from the second light source means to the sample is L2, and the beam diameter of coherent light entering the acoustooptic modulator from the continuous wave laser Is D, the sound velocity in the acoustooptic modulator is v, and the speed of light is c, at least t = 0.65 × D / v− (L2−L1) / c from the start of voltage application to the acoustooptic modulator. The super-resolution microscope is characterized in that the first pulsed light is emitted from the first light source means.   When the pulse time of the first pulsed light emitted from the first light source means is tp, at least t = 0.65 × D / v− (L2− from the start of voltage application to the acoustooptic modulator. The super-resolution microscope according to claim 1, wherein the application of the voltage to the acousto-optic modulator is terminated after L1) / c + tp. The first light source means includes a continuous wave laser, and an acoustooptic modulator that modulates the intensity of the coherent light from the continuous wave laser by applying a voltage and emits the first pulsed light,
The acousto-optics of the first light source means after at least t = 0.65 × D / v- (L2-L1) / c from the start of voltage application to the acousto-optic modulator of the second light source means. The super-resolution microscope according to claim 1 or 2, wherein voltage application to the modulator is started. _4. The super-resolution microscope according to claim 1, wherein the acoustooptic modulator has a PbMoO ₄ crystal or a TeO ₂ crystal as an acoustooptic crystal.

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