Stimulated emission depletion microscopy system

Abstract

To provide a stimulated emission depletion microscopy system that shortens scanning time while maintaining sufficient spatial resolution.SOLUTION: A stimulated emission depletion microscopy system controls a wavefront of stimulated emission depletion light into a circular shape using a wavefront control optical system 5 and controls a wavefront of excitation light into a flattened shape using a wavefront control optical system 6. By scanning, in a y-direction, the stimulated emission depletion light whose wavefront is controlled by a deflector 7, a stimulated emission depletion light group STLG arranged in parallel along the y-direction is generated. The stimulated emission depletion light group STLG and the excitation light EXLF, combined by a dichroic filter 8, are scanned in a xy-direction by a two-dimensional deflector 9, enabling simultaneous excitation and de-excitation of a plurality of regions within a single frame time of a detector and allowing fluorescence signals to be detected. Before the stimulated emission depletion light and excitation light are combined by the dichroic filter 8, the stimulated emission depletion light is scanned in the y-direction by the deflector 7 at a speed higher than the non-resonant frequency of the two-dimensional deflector 9.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a stimulated emission depletion microscope system. [Background technology]

[0002] There are several super-resolution optical microscope techniques that can capture images on the scale of several tens of nanometers, one of which is the stimulated emission depletion (STED) microscope technique described in Non-Patent Document 1. As described in Non-Patent Document 2, STED technology has undergone further evolution to improve resolution and is used in the fields of biology and bioanalysis.

[0003] In bioanalysis, it is incorporated into next-generation DNA sequencing technology, and is an essential technology for performing high-speed sequencing, as described in Patent Document 1. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2019-520596 [Non-patent literature]

[0005] [Non-Patent Document 1] Stefan W. Hell et.al., “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy”, Opt. Lett. 19, pp.780-782 (1994) [Non-patent document 2] IC Hernandez et.al., “Gated STED microscopy with time-gated single-photon avalanche diode”, Biomedical Optics Express, 6, pp.2258-2267 (2015) Summary of the Invention [Problem to be solved by the invention]

[0006] In STED microscopes, excitation, de-excitation, and detection are repeated in a focal region determined by the wavefront of the light beam and the objective lens NA. The region illuminated with a circular excitation light is then again illuminated with a coaxial ring-shaped de-excitation light. The region illuminated by both the excitation and de-excitation light de-excites to the ground state immediately after excitation, while the region illuminated only by the excitation light but not the de-excitation light emits a fluorescent signal, which is detected by a detector. Two-dimensional scanning is required to image the entire sample, and galvanometer mirrors, piezo-driven mirrors, stepping motors, and other devices are used.

[0007] There are several scanning methods. For example, consider the case where the objective lens's field of view is controlled by a galvanometer mirror and the sample is moved by a stepping motor. If we call the galvano resonance direction the x direction and the non-resonance direction the y direction, scanning occurs in the x direction according to the resonance frequency. In the y direction, the frequency can often be set within a certain frequency range, such as DC to 500 Hz, and the frequency is slower than the resonance frequency. When sweeping in the x direction, a line of fluorescence with a width equivalent to the inner diameter of the de-excitation light ring is generated, regardless of the focused size of the excitation light, and is detected according to the detector configuration.

[0008] The scanning time for the field of view depends on the galvano's resonant frequency and the set frequency in the non-resonant direction, and there is a problem that the longer it takes, the larger the target area to be measured, particularly depending on the resonant frequency.

[0009] One approach to solving this problem is to create a matrix of excitation light and de-excitation light, perform excitation, de-excitation, and detection collectively using multiple lights arranged in a matrix, and move the matrix as a whole, as described in Patent Document 1. While this method increases the scanning speed, it requires optical elements for each light beam of different wavelengths, and also requires a strong light source when creating a de-excitation light matrix from a single light source, or else presents another problem of weakening each de-excitation light.

[0010] The intensity of the de-excitation light is very important in an STED microscope system. The fluorescence wavelength is λ, the numerical aperture of the objective lens is NA, and the intensity of the de-excitation light is I. STED max , the excitation efficiency of the emitting ensemble is I sat Then, the resolution Δr is as shown in equation (1). STED max / I sat Since reflects the degree of saturation of the stimulated emission process, the stronger the de-excitation light intensity, the better the lateral resolution. Therefore, weakening the de-excitation light intensity to improve chromatic aberration for multiple light wavelengths or the scanning speed of the system can be counterproductive.

[0011]

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[0012] The present invention has been made to solve the above problems, and an object of the present invention is to provide a stimulated emission depletion microscope system that shortens the scanning time while maintaining sufficient spatial resolution. [Means for solving the problem]

[0013] In order to solve the above-mentioned problems, the stimulated emission suppression microscope system of the present invention includes a first light source that generates stimulated emission suppression light, a second light source that generates excitation light, a first wave-front control element that controls the wavefront of the stimulated emission suppression light to be circular, a second wave-front control element that controls the wavefront of the excitation light to be flat, a first deflection element that scans the stimulated emission suppression light from the first wave-front control element in the y direction which is a non-resonance direction, a first dichroic filter that combines the stimulated emission suppression light from the first deflection element and the excitation light from the second wave-front control element, and a first dichroic filter that scans the combined light in the y direction and in the x direction which is a resonance direction perpendicular to the y direction. the excitation light, the stimulated emission suppression light, and the excitation light combined by the first dichroic filter are scanned in the x and y directions by the second deflector, thereby simultaneously exciting and de-exciting a plurality of regions within one frame time of the detector and detecting a fluorescence signal. Other aspects of the present invention will be described in the embodiments described later. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide a stimulated emission depletion microscope system that shortens the scanning time while maintaining sufficient spatial resolution. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a block diagram showing the configuration of an STED microscope system according to an embodiment of the present invention. [Figure 2] 2 is an explanatory diagram showing deformation, deflection, and multiplexing of excitation light and stimulated denaturation (STED) light according to the first embodiment. FIG. [Figure 3] FIG. 1 is a schematic diagram showing the operation of an acousto-optic deflector (AOD) and the deflection of light. [Figure 4A]This is a diagram (part 1) of the arrangement of the flattened excitation light (EXLF) and the STED light group (STLG). [Figure 4B] This is the layout diagram (part 2) of the flattened excitation light (EXLF) and the STED light group (STLG). [Figure 4C] This is the layout diagram (part 3) of the flattened excitation light (EXLF) and the STED light group (STLG). [Figure 5] This is an image depicting the two-dimensional scanning of the flat excitation light (EXLF) and the STED light group (STLG) within the field of view of the objective lens. [Figure 6] This is a simulation diagram showing the irradiation and sweep of the flat excitation light (EXLF) and STED light group (STLG) at the detector. [Figure 7] 10A and 10B are explanatory diagrams showing deformation, deflection, and multiplexing of excitation light and STED light according to the second embodiment. [Figure 8] FIG. 1 is an image diagram showing the operation of an electro-optic deflector (EOD). DETAILED DESCRIPTION OF THE INVENTION

[0016] This specification provides several examples of STED microscope systems. The descriptions in this specification are merely typical examples and do not limit the scope of the claims or application examples in any sense. In addition, in the description of the drawings, the same elements are designated by the same reference numerals, and duplicate descriptions are omitted.

[0017] First Embodiment FIG. 1 is a block diagram showing the configuration of an STED microscope system 100 according to this embodiment. The STED microscope system 100 is an apparatus for acquiring fluorescent images of, for example, a cell specimen or a flow cell used in DNA sequencing. As shown in FIG. 1, the STED microscope system 100 according to this embodiment includes an STED light source 1 (first light source), an excitation light source 2 (second light source), a high-speed shutter 3, an oscillator 4, a wavefront control optical system 5 (first wavefront control element), a wavefront control optical system 6 (second wavefront control element), a deflector 7 (first deflection element), a dichroic filter 8 (first dichroic filter), a dichroic filter 10 (second dichroic filter), a two-dimensional deflector 9 (second deflection element), an objective lens 11, a target specimen 12, a moving platform 13, an imaging optical system 14, a detector 15, a controller 16, and an image display 17. All embodiments have roughly the same configuration.

[0018] First, the features of the main equipment in the STED microscope system 100 will be described. The deflector 7 (first deflection element) deflects the stimulated emission depletion light (STED light STL) in the y direction at intervals of several microseconds to several milliseconds, which is much shorter than one second, to generate a stimulated emission depletion light group (STED light group STLG). The STED light STL is converted into a ring shape by an optical element or a wavefront controller, and then the STED light STL is scanned in the y direction at intervals of several microseconds to several milliseconds by the deflector 7, and propagates as the STED light group STLG to the specimen to be observed. The zero intensity regions STZ (see Figures 4A to 4C) in the STED light group STLG do not overlap, but are adjacent or dispersed and do not overlap. As mentioned above, for example, the galvano resonance direction is the x direction, and the non-resonance direction is the y direction.

[0019] The wavefront control optical system 6 (second wavefront control element) controls the wavefront of the excitation light to be long and flat in the y direction, and irradiates the area where it overlaps with the stimulated emission depletion light group (STED light group STLG). The excitation light EXL is transformed into a long and flat shape in the y direction by the optical element or wavefront control optical system 6, and then propagates to the specimen, overlapping with the flat excitation light EXLF and the STED light group STLG. The size of the flat excitation light EXLF is such that it can irradiate all of the zero intensity regions STZ (see Figures 4A to 4C) (STZG) in the STED light group.

[0020] The stimulated emission depletion light and the excitation light are combined by a dichroic filter 8 (first dichroic filter). Before the stimulated emission depletion light and the excitation light are combined, the deflector 7 (first deflection element) scans the stimulated emission depletion light in the y direction, thereby scanning at a speed faster than the non-resonant frequency of the two-dimensional deflector 9 (second deflection element). After the STED light group STLG and the flat excitation light EXLF are combined by an optical element such as a dichroic filter, they are manipulated by a two-dimensional deflection element, as in a conventional microscope system, so that the specimen is illuminated as if with a band-shaped STED light group STLG and flat excitation light EXLF. This method prevents degradation of the intensity of a single STED light beam even when the STED light group STLG is combined.

[0021] In other words, the two-dimensional deflector 9, which is the second deflection element, scans the combined light in both the y direction and the x direction, which is the resonance direction perpendicular to the y direction (xy direction). This configuration makes it possible to shorten the scanning time compared to a system that does not have this configuration.

[0022] That is, stimulated emission depletion microscope systems can achieve shorter scan times while maintaining sufficient spatial resolution.

[0023] 2 shows an optical system according to the first embodiment. Description will be made with reference to FIG. The light emitted from the STED light source 1 and excitation light source 2 passes through a predetermined optical system, is combined by a dichroic filter 8 (first dichroic filter), and is then irradiated onto a target specimen 12 using a predetermined movement mechanism and an objective lens 11. Of the light returned from the target specimen 12, only the fluorescent FLL is selected by a dichroic filter 10 (second dichroic filter), passes through an imaging optical system 14, is detected by a detector 15, is processed by a controller 16, and is output as image data to an image display 17. A MEMS mirror, a galvanometer mirror, a piezoelectric mirror, or the like is used for the two-dimensional deflector 9, but in any system, the resonance direction is called the x-direction and the non-resonance direction is called the y-direction.

[0024] Returning to Figure 1, the STED light source 1 is a light source system that generates light that becomes the STED light STL, and generates light with high coherence such as laser light. The STED light source 1 has a mechanism for generating continuous wave (CW) light or pulsed light, and the pulse width and repetition frequency are controlled by signals input from an oscillator 4. The wavelength, intensity, pulse width, and repetition frequency of the light that becomes the STED light STL are determined by the target cell specimen, etc. and its label. The light output from the STED light source system is converted into ring-shaped STED light STL by a wavefront control optical system 5.

[0025] The excitation light source 2 is a light source that generates light that becomes the excitation light EXL, and generates coherent light, such as laser light, or incoherent light such as LED light or halogen light. The excitation light EXL is CW light or pulsed light, and depending on the specifications of the excitation light source 2, it can be pulsed using a high-speed shutter 3. The high-speed shutter 3 is controlled by an input signal from an oscillator 4. The characteristics of the excitation light EXL, such as the wavelength width and intensity, are determined by the target cell specimen and its label, and the high-speed shutter 3 can be omitted depending on the measurement content and conditions.

[0026] In the first embodiment, the wavefront controller is equipped with spatial light modulators (Liquid Crystal On Silicon-Spatial Light Modulators; LCOS-SLMs) 5a and 6a shown in Fig. 2. Note that similar effects can be obtained using optical elements that control the wavefront or phase, such as fixed holograms, instead of spatial light modulators. For example, a Powell lens may be used to control the excitation light, and an axicon lens pair may be used to control the STED light.

[0027] The deflector 7 is equipped with an acousto-optical deflector (AOD) 7a. The acousto-optical deflector 7a is controlled by an input signal from the oscillator 4 (see Figure 1), and when the input frequency changes, the deflection (diffraction) direction of the first-order light changes due to the Raman-Nurse effect or the Bragg effect. However, the rise of the output light in response to a change in the input signal is sufficiently fast.

[0028] Figure 3 is a schematic diagram showing the operation of an acousto-optic deflector (AOD) and the deflection of light. Let f be the input signal, λ be the wavelength of the STED light, and v be the sound velocity in the crystal inside the acousto-optic deflector 7a. a Then, the light is diffracted as shown in Figure 3, and the diffraction angle θ B is as shown in equation (2).

[0029]

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[0030] Let us consider the case where the crystal of the acousto-optic deflector 7a is tellurium dioxide (TeO2). There are several other crystals for AOMs, including lead molybdate (PbMoO4) in the visible light region. The sound velocity in tellurium dioxide crystal, v, is a is 617 m / s, and if the wavelength λ of the STED light is 500 nm and the modulation frequency f is 80 MHz, then θ B From equation (2), θ = 1.8576 degrees. Bis changed to, for example, 2.0 degrees, the modulation frequency f is calculated to be 86.132 MHz using formula (2). On the target specimen 12, the parallel size of the STED light group STLG is in a conjugate relationship with the entrance pupil of the objective lens 11, and therefore the size is converted by the magnification of the objective lens 11. The parallel size of the STED light group STLG is determined by changing the modulation frequency f according to the distance from the acousto-optic deflector 7a to the objective lens 11 and the conditions limited by the wavefront control optical system 6.

[0031] FIG. 4A is a diagram (part 1) of the arrangement of flattened excitation light (flattened excitation light, EXLF) and STED light groups (STLG). FIG. 4B is a diagram (part 2) of the arrangement of flattened excitation light (flattened excitation light, EXLF) and STED light groups (STLG). FIG. 4C is a diagram (part 3) of the arrangement of flattened excitation light (flattened excitation light, EXLF) and STED light groups (STLG). The dotted lines in FIGS. 4A to 4C show the arrangement of the STED light groups STLG deflected in different directions depending on the input signal. Two or more signal frequencies are input to the acousto-optic deflector 7a, and the light is deflected to different positions depending on the frequency. That is, the deflector 7 (first deflection element) controls the deflection angle for different input signals.

[0032] The STED microscope system 100 (stimulated emission depletion microscope system) of this embodiment is characterized in that the zero light intensity regions in the stimulated emission depletion light group are arranged in parallel in the y direction, with each region being arranged without overlapping.

[0033] Zero-intensity regions (non-de-excitation regions) in the STED light beams are deflected without overlapping and are detected as independent areas. The sum of the oscillation frequency change time of the oscillator 4 and the rise time of the acousto-optic deflector 7a is several microseconds to several milliseconds, which is faster than the response time of the detector 15 and much shorter than one second. Therefore, the STED light beams are aligned in the y direction at short time intervals. Incidentally, the detector 15 in this embodiment is configured to detect multiple fluorescent images per second. In other words, the response time of the detector 15 is longer than the sum of the oscillation frequency change time of the oscillator 4 and the rise time of the acousto-optic deflector 7a.

[0034] The solid lines in Figures 4A to 4C show the arrangement of the flat excitation light EXLF relative to the STED light group STLG. The excitation light EXL is converted into flat excitation light EXLF that is long in the y direction by the spatial light modulator 6a, and is oriented in approximately the same direction as the STED light group STLG. The size of the flat excitation light EXLF is large enough to irradiate all of the non-de-excitation regions STZG in the STED light group STLG. However, for reasons of signal-to-noise (SN), it is desirable that the size of the flat excitation light EXLF be smaller than that of the STED light group STLG, but this is not always the case. Furthermore, for the sake of measurement, the polarization direction of the STED light group STLG and the flat direction of the flat excitation light EXLF do not need to be perfectly aligned in the y direction; they only need to be roughly oriented in the y direction (see Figure 4C).

[0035] That is, the wavefront control optical system 6 (second wavefront control element) controls the wavefront of the excitation light to be long and flat in the y direction, and irradiates an area overlapping with the stimulated emission inhibition light group.

[0036] The dichroic filter 8 is superimposed with a group of stimulated emission suppression light beams irradiated at intervals of several microseconds to several milliseconds, which is much shorter than one second, and the modified excitation light beam, and then scanned by a two-dimensional deflector 9 (second deflection element).

[0037] Figure 5 is an image diagram depicting two-dimensional scanning of the flat excitation light EXLF and the STED light group STLG within the field of view of the objective lens 11. Scanning in the x direction is performed by resonant scanning of the two-dimensional deflector 9, and scanning in the y direction is interpolated by non-resonant scanning of the two-dimensional deflector 9 as well as scanning of the flat excitation light EXLF and the STED light group STLG.

[0038] Figure 6 is a schematic diagram showing the irradiation and sweep of the flat excitation light EXLF and the STED light group STLG on the detector 15. The detector 15 is assumed to be a two-dimensional CMOS sensor (Complementary Metal Oxide Semiconductor; CMOS), but other two-dimensional sensors such as a CCD sensor (Charge Coupled Device; CCD) can also be used. In a two-dimensional sensor, pixels are arranged in an array, and the shape of the detection surface is generally rectangular or square.

[0039] When the detection surface is rectangular, aligning the long side with the resonance direction (x direction) of the two-dimensional deflector 9 allows the flat excitation light EXLF and the STED light group STLG to scan per frame as shown in Figure 6, enabling efficient imaging. The fluorescence FLL emitted from the de-excitation region STZG in the STED light group STLG becomes the actual detection signal, and the number of pixels receiving the FLL emitted from one de-excitation region STZ may be greater than one. The field of view of the objective lens 11 is scanned two-dimensionally as described above, and the measurement area is expanded in the z direction perpendicular to the xy plane, or by using the moving platform 13 if the target specimen 12 is larger than the field of view.

[0040] The detector 15 is a two-dimensional detector and can detect signals emitted from multiple regions within a single frame of the detector 15. The two-dimensional deflector 9 (second deflection element) can scan in both the x-direction and the y-direction in a time shorter than a single frame time of the detector 15.

[0041] As described above, according to the first embodiment, it is possible to provide a stimulated emission depletion microscope system that shortens the scanning time while maintaining sufficient spatial resolution.

[0042] Second Embodiment FIG. 7 is an explanatory diagram showing the deformation, deflection, and combination of excitation light and STED light according to the second embodiment. FIG. 7 shows the optical system according to the second embodiment, in which the wavefront of the STED light is changed by an axicon lens pair 5b, and the wavefront of the excitation light source 2 is changed by a Powell lens 6b. Note that this wavefront change can also be achieved by other optical elements, such as an LCOS (Liquid Crystal on Silicon) spatial light modulator or a fixed hologram. After passing through a predetermined optical system, the STED light STL and excitation light EXL are combined by a dichroic filter 8 (first dichroic filter). Then, a target specimen 12 is illuminated using a predetermined movement mechanism and an objective lens 11. Of the light returning from the specimen, only the fluorescence FLL is selected by a dichroic filter 10 (second dichroic filter). The light passes through an imaging optical system 14, is detected by a detector 15, is processed by a controller 16, and is output as image data to an image display 17.

[0043] In the second embodiment, the deflector 7 is equipped with an electro-optical deflector (EOD) 7b. The electro-optical deflector 7b is controlled by a signal voltage input from the oscillator 4, and the deflection (diffraction) direction changes due to the Pockels effect, which changes the refractive index within the crystal depending on the input voltage. If a uniaxial crystal (the crystal axis is called the c-axis) is used as the crystal, applying a voltage in the c-axis direction will only change the refractive index in the direction parallel to the c-axis. If the c-axis is defined as the z0 direction and the directions perpendicular to it are defined as the x0 and y0 directions, when STED light enters the crystal parallel to the x0 or y0 direction, the propagation can be considered similar to that in an isotropic medium.

[0044] Figure 8 is an image diagram showing the operation of an electro-optic deflector (EOD). Considering a trapezoidal crystal with two right angles on the side where the STED light is incident, the propagation of the STED light within the crystal is diffracted as shown in Figure 8 using Snell's law shown in formula (3). The input voltage applied in the c-axis (z0) direction is V, and the refractive index within the crystal is n o (x0 or y0 direction), n e (z0 direction), one value of the dielectric tensor of a uniaxial crystal is r 13 Then, the refractive index n by the electro-optic deflector 7b is x0 (=n y0 =n0) is as shown in equation (4).

[0045]

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[0046] Let us consider the case where a lithium niobate (LiNbO3) single crystal is used as the electro-optic deflector 7b. Consider a trapezoidal crystal with two right angles on the incident side as shown in Figure 8, with the acute angle being α. When the STED light STL enters the crystal from a space with a refractive index of n0 at an angle of θ0, and the angle between the line perpendicular to the edge of the crystal on the output side and the direction of the emitted STED light is θ, the diffraction angle can be calculated using equation (5).

[0047]

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[0048] For example, in the case of the electro-optic deflector 7b sealed in dry nitrogen (refractive index 1.0003), the refractive index n of the lithium niobate crystal for the STED wavelength λ of 500 nm is o =2.3410, n e =2.2457, electro-optic constant r 13 =9.6×10 -12m / V, and the sum of the incident angle θ0 and the acute angle α of the trapezoidal crystal is θ0 + a = 60 degrees. By using formulas (4) and (5), the applied voltage V = 5.533 × 10 when the diffraction angle θ = 89 degrees. 9 V, when θ=90 degrees, the applied voltage V=5.528×10 9 It is required to be V.

[0049] As described above, the stimulated emission depletion microscope system of this embodiment has the following features. The stimulated emission depletion microscope system includes a first light source (STED light source 1) that generates stimulated emission depletion light, a second light source (excitation light source 2) that generates excitation light, a first wave-front control element (wave-front control optical system 5) that controls the wavefront of the stimulated emission depletion light into a circular shape, a second wave-front control element (wave-front control optical system 6) that controls the wavefront of the excitation light into a flattened shape, a first deflection element (deflector 7) that scans the stimulated emission depletion light from the first wave-front control element in the y direction, which is a non-resonant direction, a first dichroic filter (dichroic filter 8) that combines the stimulated emission depletion light from the first deflection element and the excitation light from the second wave-front control element, and a first dichroic filter that scans the combined light in the y direction and in a coaxial direction perpendicular to the y direction. The apparatus is characterized in that it comprises a second deflecting element (two-dimensional deflector 9) that scans in both the x-direction, which is the sound direction, and the x-direction, a second dichroic filter (dichroic filter 10) that separates the excitation light, stimulated emission suppression light, and fluorescence response from the specimen, and a detector 15 that detects multiple fluorescence images per second, and the first deflecting element scans the stimulated emission suppression light in the y-direction to generate a group of stimulated emission suppression light parallel to the y-direction, and the stimulated emission suppression light and excitation light combined by the first dichroic filter are scanned in the x- and y-directions by the second deflecting element, thereby simultaneously exciting and de-exciting multiple regions within one frame time of the detector 15 and detecting fluorescence signals.

[0050] As a result, the stimulated emission depletion microscope system can reduce the imaging time of the target specimen by almost simultaneously irradiating the target specimen with excitation light controlled to be flat and stimulated emission depletion light that scans at intervals of several microns to milliseconds, which is much shorter than one second, and detecting multiple regions simultaneously. In other words, according to the disclosure, it is possible to provide a stimulated emission depletion microscope system that further reduces the scanning time while maintaining sufficient spatial resolution. [Explanation of symbols]

[0051] 1 STED light source (first light source) 2. Excitation light source (second light source) 3. High-speed shutter 4. Oscillators 5. Wavefront control optical system (first wavefront control element) 5a Spatial Light Modulator 5b Axicon Lens Pair 6 Wavefront control optical system (second wavefront control element) 6a Spatial Light Modulator 6b Powell Lens 7 Deflector (first deflection element) 7a Acousto-optic deflector 7b Electro-optic deflector 8 Dichroic filter (first dichroic filter) 9 Two-dimensional deflector (second deflection element) 10 Dichroic filter (second dichroic filter) 11 Objective Lens 12 Target specimens 13 Moving Platform 14 Imaging optical system 15 detectors 16 Controller 17 Image display 100 STED Microscope System (Stimulated Emission Depletion Microscope System)

Claims

1. a first light source that generates stimulated emission suppressed light; a second light source that generates excitation light; a first wave-front control element that controls the wavefront of the stimulated emission suppression light to have a circular shape; a second wave-front control element that controls the wavefront of the excitation light to be flat; a first deflection element that scans stimulated emission suppressed light from the first wave-front control element in a y direction that is a non-resonance direction; a first dichroic filter that combines the stimulated emission suppression light from the first deflection element and the excitation light from the second wave-front control element; a second deflection element that scans the combined light in both the y direction and an x ​​direction that is a resonance direction perpendicular to the y direction; a second dichroic filter that separates the excitation light, the stimulated emission suppression light, and the fluorescence emitted from the specimen; a detector that detects a plurality of fluorescent images per second; the first deflection element scans the stimulated emission suppression light in a y direction to generate a group of stimulated emission suppression light beams arranged in parallel in the y direction; The stimulated emission suppression light and the excitation light combined by the first dichroic filter are scanned in the x and y directions by the second deflection element, A stimulated emission depletion microscope system characterized in that multiple regions are excited and de-excited simultaneously within one frame time of the detector, and fluorescent signals are detected.

2. Before the stimulated emission suppression light and the excitation light are combined by the first dichroic filter, the stimulated emission suppression light is scanned in the y direction by the first deflection element, thereby scanning at a speed higher than a non-resonant frequency of the second deflection element.

2. The stimulated emission depletion microscope system according to claim 1.

3. The first deflection element deflects the stimulated emission suppression light in the y direction at intervals of several microseconds to several milliseconds, thereby generating a stimulated emission suppression light group.

2. The stimulated emission depletion microscope system according to claim 1.

4. The zero light intensity regions in the stimulated emission suppression light group are arranged in parallel in the y direction, with each region being arranged without overlapping.

2. The stimulated emission depletion microscope system according to claim 1.

5. the second wave-front control element controls the wavefront of the excitation light to be elongated and flat in a y direction, Irradiating an area overlapping with the stimulated emission suppression light group.

2. The stimulated emission depletion microscope system according to claim 1.

6. The detector is a two-dimensional detector that detects signals emitted from multiple regions within a single frame of the detector.

2. The stimulated emission depletion microscope system according to claim 1.

7. The first deflection element controls the deflection angle in response to different input signals.

2. The stimulated emission depletion microscope system according to claim 1.

8. The second deflection element scans in both the x and y directions in a time shorter than a single frame time of the detector.

2. The stimulated emission depletion microscope system according to claim 1.

9. The stimulated emission suppression light group is irradiated at intervals of several microseconds to several milliseconds; The deflection element is scanned by the second deflection element while superimposing the deflected excitation light.

2. The stimulated emission depletion microscope system according to claim 1.

Images