Microscope apparatus and image acquisition method

Abstract

This microscope is provided with an excitation light output part, an optical system, and a harmonic detection part. The excitation light output part outputs excitation light. The time waveform of the light intensity of the excitation light includes the nth root (where n is an integer of 2 or more) of a linear function of a sine wave. The maximum value of the light intensity of the excitation light exceeds the saturation excitation intensity in an observed object. The optical system irradiates the observed object with excitation light outputted from the excitation light output part. Fluorescence is produced in the observed object by n-photon excitation due to irradiation of the excitation light. The harmonic detection part detects a second harmonic included in the time waveform of the light intensity of the fluorescence.

Description

[Technical Field]

[0001] This disclosure relates to a microscope apparatus and an image acquisition method. [Background technology]

[0002] Patent Document 1 discloses a saturated excitation (SAX) microscope. In this document, the laser light, which is the excitation light, is modulated so that the time change in intensity of the laser light becomes a cosine wave.

[0003] Non-patent documents 1 and 2 disclose a combination of a SAX microscope and a two-photon excitation microscope. In these documents, the intensity of the excitation light is modulated so that the time evolution of the excitation light intensity is sinusoidal. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2006 / 061947 [Non-patent literature]

[0005] [Non-Patent Document 1] Sandeep Chakraborty et al., "Saturated two-photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth", Journal of Biophotonics, August 2018 [Non-Patent Document 2] Anh Dung Nguyen et al., "3D super-resolved in vitro multiphoton microscopy by saturation of excitation", Optics Express Volume 23, Issue 17 pp.22667-22675 (2015) [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Generally, in a SAX microscope, the object to be observed is excited by irradiating it with excitation light having a sinusoidal time waveform, causing it to emit fluorescence. At the same time, the peak intensity of the excitation light is made greater than the saturation excitation intensity of the object to saturate the fluorescence peak intensity. Then, harmonic components contained in the time waveform of the fluorescence, such as the second harmonic, are detected, and an observation image is created based on these harmonic components. This makes it possible to improve the spatial resolution of the observation image.

[0007] In multiphoton excitation microscopy, long-wavelength ultrashort pulsed light, such as near-infrared light, is used as excitation light to irradiate the object to be observed. This induces multiphoton excitation, such as two-photon excitation, in the object, and the resulting fluorescence is detected to create an observation image. Because this multiphoton excitation microscope uses long-wavelength light with excellent object penetration properties, it becomes possible to observe, for example, the deep parts of biological tissue non-invasively.

[0008] By combining a SAX microscope, which possesses the advantages described above, with a multiphoton excitation microscope, a microscope that combines these advantages can be realized. However, in n-photon excitation (where n is an integer greater than or equal to 2), the fluorescence intensity is proportional to the nth power of the excitation light intensity. Therefore, when an object to be observed is irradiated with excitation light having a sinusoidal time waveform, and n-photon excitation occurs in the object, the time waveform of the fluorescence emitted from the object will be proportional to the nth power of the sine wave. If an observation image is to be obtained based on fluorescence intensity having such a time waveform, it becomes necessary to detect high-order harmonics such as the third harmonic and the fifth harmonic, as described in Patent Document 2, for example. Since there is a limit to the frequency range of the fluorescence detection device, if high-order harmonics are to be detected, the frequency of the excitation light must be reduced, which increases the time required to create the observation image. Alternatively, it becomes necessary to introduce equipment with a high upper limit on the frequency range, which increases costs.

[0009] This disclosure has been made in view of the above problems and aims to provide a microscope apparatus and image acquisition method that have the advantages of both SAX microscopes and multiphoton excitation microscopes and that can create observation images using relatively low-order harmonics. [Means for solving the problem]

[0010] To solve the problems described above, the microscope apparatus according to this disclosure comprises an excitation light output unit, an optical system, and a harmonic detection unit. The excitation light output unit outputs excitation light. The time waveform of the excitation light intensity includes the nth root of a linear function of a sine wave (where n is an integer greater than or equal to 2). The maximum value of the excitation light intensity exceeds the saturation excitation intensity in the object being observed. The optical system irradiates the object being observed with the excitation light output from the excitation light output unit. The harmonic detection unit detects the second harmonic included in the time waveform of the fluorescence intensity generated in the object being observed by n-photon excitation due to irradiation with excitation light.

[0011] The image acquisition method according to this disclosure includes an excitation light output step, an excitation light irradiation step, a harmonic detection step, and an image generation step. In the excitation light output step, excitation light is output. The time waveform of the excitation light intensity includes the nth root of a linear function of a sine wave (n is an integer greater than or equal to 2). The maximum value of the excitation light intensity exceeds the saturation excitation intensity at the object being observed. In the excitation light irradiation step, the excitation light output in the excitation light output step is irradiated onto the object being observed. In the harmonic detection step, the second harmonic included in the time waveform of the fluorescence intensity generated at the object being observed by n-photon excitation due to irradiation with excitation light is detected. In the image generation step, an observation image of the object being observed is generated based on the second harmonic.

[0012] In these microscopes and image acquisition methods, the time waveform of the excitation light intensity contains the nth root of a linear sinusoidal function. As mentioned above, in n-photon excitation, the fluorescence intensity is proportional to the nth power of the excitation light intensity. Therefore, when an object is irradiated with excitation light having a time waveform containing the nth root of a linear sinusoidal function, and n-photon excitation occurs in the object, the time waveform of the fluorescence emitted from the object is proportional to the linear sinusoidal function, not the nth power of the sinusoidal function. Therefore, similar to a general SAX microscope, observation images can be obtained based on lower-order harmonics, such as the second or third harmonic. Thus, with these microscopes and image acquisition methods, it is not necessary to reduce the frequency of the excitation light due to the frequency range constraints of the fluorescence detection device, thus avoiding the need for longer image creation times. Alternatively, it is possible to use equipment with a lower frequency range, leading to cost reduction.

[0013] In the microscope apparatus described above, the excitation light output unit may include a light source that outputs pulsed light and an intensity-modulated optical modulator that modulates the pulsed light output from the light source to generate excitation light. Similarly, in the image acquisition method described above, the excitation light output step may include an intensity modulation step that modulates the pulsed light to generate excitation light. With such a configuration and method, excitation light containing the nth root of a linear function of a sine wave in the time waveform of the light intensity can be easily generated. In this case, the optical modulator may be an AO modulator.

[0014] In the microscope apparatus described above, the light source may be a laser light source. Similarly, in the image acquisition method described above, the pulsed light may be laser light. This makes it possible to generate excitation light with a high light intensity capable of causing n-photon excitation using a simple configuration.

[0015] In the above-described microscope apparatus and image acquisition method, the minimum value within each period of the time waveform of the excitation light intensity may be greater than 0, or it may be greater than 0.1% of the maximum signal that can be received in the harmonic detection unit or harmonic detection step and less than 20%. At and around the minimum value within each period of the time waveform of the excitation light intensity, the intensity of the fluorescence generated is small, and the detection result is greatly affected by noise. By making the minimum value within each period of the time waveform of the excitation light intensity greater than 0, the effect of noise can be reduced, the detection accuracy of the second harmonic can be improved, and the observed image can be made clearer. Alternatively, by making the minimum value within each period of the time waveform of the excitation light intensity greater than 0.1% of the maximum signal that can be received in the harmonic detection unit or harmonic detection step and less than 20%, the effect of noise can be reduced, the detection accuracy of the second harmonic can be improved, and the observed image can be made clearer. When the time waveform of the excitation light intensity includes the square root of a linear function of a sine wave, the rate of change of light intensity around the minimum value within each period is larger compared to when the time waveform of the excitation light intensity is a sine wave. In other words, the time waveform of the light intensity becomes steep near the minimum value within each period. This rate of change is greatest when the minimum value within each period is 0. By making the minimum value within each period greater than 0, the rate of change of the light intensity near the minimum value within each period can be reduced, and the steepness of the time waveform can be mitigated. Alternatively, by making the minimum value within each period greater than 0.1% and less than 20% of the maximum signal that can be received in the harmonic detection unit or harmonic detection step, the rate of change of the light intensity near the minimum value within each period can be reduced, and the steepness of the time waveform can be mitigated. Therefore, shaping the time waveform of the excitation light intensity in the excitation light output unit, in particular, shaping the time waveform near the minimum value within each period becomes easier.

[0016] In the above microscope apparatus, the harmonic detection unit may include a photodetection device that generates a signal corresponding to the light intensity of the fluorescence generated in the observation object, and a lock-in amplifier that inputs a signal from the photodetection device and outputs a second harmonic included in the time waveform of the signal. Similarly, in the above image acquisition method, the harmonic detection step may include a step of generating a signal corresponding to the light intensity of the fluorescence generated in the observation object, and a step of outputting a second harmonic included in the time waveform of the signal. With these configurations and methods, the second harmonic can be detected simply and accurately.

[0017] In the above microscope apparatus, the harmonic detection unit may further detect a third harmonic included in the time waveform of the light intensity of the fluorescence generated in the observation object. Similarly, in the above image acquisition method, in the harmonic detection step, a third harmonic included in the time waveform of the light intensity of the fluorescence generated in the observation object may be further detected. Then, in the image generation step, an observation image of the observation object may be generated based on one or both of the second harmonic and the third harmonic. In this case, an observation image using the harmonic suitable for the observation object among the second harmonic and the third harmonic can be generated simply.

[0018] In the above microscope apparatus and image acquisition method, the time waveform of the light intensity of the excitation light output from the excitation light output unit includes the square root of a linear function of a sine wave, and the harmonic detection unit or the harmonic detection step may detect a second harmonic included in the time waveform of the light intensity of the fluorescence generated in the observation object by two-photon excitation due to the irradiation of the excitation light.

[0019] The excitation light output device according to the present disclosure is used in a fluorescence microscope that generates an observation image based on the temporal waveform of the light intensity of fluorescence generated in an observation object by two-photon excitation by irradiation with excitation light. This excitation light output device outputs excitation light in which the temporal waveform of the light intensity includes the square root of a linear function of a sine wave and the maximum value of the light intensity exceeds the saturation excitation intensity in the observation object. The excitation method according to the present disclosure is an excitation method in a fluorescence microscope that generates an observation image based on the temporal waveform of the light intensity of fluorescence generated in an observation object by two-photon excitation by irradiation with excitation light. In this excitation method, excitation light in which the temporal waveform of the light intensity includes the square root of a linear function of a sine wave and the maximum value of the light intensity exceeds the saturation excitation intensity in the observation object is irradiated onto the observation object to excite the observation object.

[0020] In these excitation light output devices and excitation methods, the temporal waveform of the excitation light intensity includes the square root of a linear function of a sine wave. As described above, in two-photon excitation, the fluorescence intensity is proportional to the square of the excitation light intensity. Therefore, when excitation light having a temporal waveform including the square root of a linear function of a sine wave is irradiated onto an observation object to cause two-photon excitation in the observation object, the temporal waveform of the fluorescence output from the observation object is proportional to a linear function of a sine wave, rather than the square of a sine wave. Therefore, an observation image can be obtained based on a harmonic wave having a lower order, such as the second harmonic wave or the third harmonic wave. Therefore, according to these excitation light output devices and excitation methods, since it is not necessary to reduce the frequency of the excitation light due to the constraint of the frequency range of the device for detecting fluorescence, it is possible to avoid an increase in the time required to create an observation image. Alternatively, since a device with a low frequency range can be used, it leads to cost reduction.

Advantages of the Invention

[0021] According to the present disclosure, it is possible to provide a microscope device and an image acquisition method that have the advantages of both a SAX microscope and a multi-photon excitation microscope and can create an observation image using a harmonic wave having a relatively low order.

Brief Description of the Drawings

[0022] [Figure 1]Figure 1 shows the configuration of a microscope apparatus. [Figure 2] Figure 2 shows the time waveform of the excitation light. [Figure 3] Figure 3 shows the configuration of the optical scanner. [Figure 4] Figure 4 is a flowchart illustrating the operation of the microscope device. [Figure 5] Part 5(a) of Figure 5 conceptually shows the fluorescence intensity distribution in a SAX microscope. Part 5(b) of Figure 5 conceptually shows the difference between the theoretical and measured fluorescence intensity values. [Figure 6] Part 6(a) of Figure 6 conceptually shows the time waveform of the excitation light intensity. Part 6(b) of Figure 6 conceptually shows the time waveform of the fluorescence intensity. [Figure 7] Part 7(a) of Figure 7 conceptually shows the time waveform of the excitation light intensity. Part 7(b) of Figure 7 conceptually shows the time waveform of the fluorescence intensity. [Figure 8] Part 8(a) of Figure 8 is a graph showing the result of Fourier transforming the time waveform of the fluorescence light intensity measured in a microscope. Part 8(b) of Figure 8 is a graph showing the result of Fourier transforming the time waveform of the fluorescence intensity measured when the time waveform of the excitation light intensity is a sine wave. [Figure 9] Part 9(a) of Figure 9 is a graph showing the relationship between the light intensities of the fundamental and second harmonics of fluorescence measured in a microscope and the relative intensity of the excitation light. Part 9(b) of Figure 9 is a graph showing the relationship between the light intensities of the fundamental, second, and third harmonics of fluorescence measured when the time waveform of the excitation light intensity is a sine wave and the relative intensity of the excitation light. [Figure 10] Figure 10 is a conceptual diagram showing the time waveform of the excitation light. [Figure 11] Figure 11 is a conceptual graph showing the relationship between the applied voltage and output light intensity of a typical AO modulator. [Figure 12] Figure 12 is a schematic diagram showing the configuration of a light irradiation device according to the second embodiment. [Figure 13] Figure 13 shows the polarization direction of azimuth polarization in a plane perpendicular to the optical axis. [Figure 14] Sections (a) to (h) of Figure 14 show the focused image of azimuth-polarized light. [Figure 15] Figure 15 shows a spiral phase pattern. [Figure 16] Sections (a) to (h) of Figure 16 show the focused image when phase modulation by a helical phase pattern is applied to azimuth-polarized light. [Figure 17] Figure 17 shows an example of the ring mask configuration as viewed from the optical axis direction. [Figure 18] Sections (a) to (e) of Figure 18 show examples of the arrangement of the polarization conversion unit, phase conversion unit, and ring mask. [Figure 19] Figure 19 is a flowchart illustrating the operation of the microscope apparatus. [Figure 20] Sections (a) and (b) of Figure 20 show the shape of the focusing spot in a cross-section that includes the optical axis and is parallel to the optical axis. [Figure 21] Figure 21 is a graph showing the lateral resolution ratio for each aperture ratio when a ring mask is not used. [Figure 22] Figure 22 is a graph showing the lateral resolution ratio for each aperture ratio when using a ring mask. [Figure 23] Sections (a) to (d) of Figure 23 show the fluorescence detected at the second harmonic in a cross-section that includes the optical axis and is parallel to the optical axis direction. [Figure 24] Sections (a) to (d) of Figure 24 show the fluorescence detected at the third harmonic in a cross-section that includes the optical axis and is parallel to the optical axis direction. [Figure 25] Figure 25 is a schematic diagram showing the configuration of a light irradiation device according to the second modified example. [Figure 26] Figure 26 shows an example of a phase pattern that constitutes an amplitude-modulated ring mask. [Figure 27] Figure 27 shows an example of a phase pattern that constitutes an amplitude-modulated ring mask. [Modes for carrying out the invention]

[0023] Hereinafter, embodiments of the microscope apparatus and image acquisition method according to this disclosure will be described in detail with reference to the attached drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant descriptions are omitted. [First Embodiment]

[0024] Figure 1 shows the configuration of a microscope apparatus 1 according to the first embodiment of this disclosure. The microscope apparatus 1 is a device for acquiring an image of an object B to be observed, and comprises an excitation light output unit (excitation light output device) 10, an optical system 20, a harmonic detection unit 30, an image generation unit 40, and a signal generator (function generator) 50. The object B to be observed is, for example, a biological sample.

[0025] The excitation light output unit 10 outputs excitation light La to excite the object B to be observed. The excitation light La is either coherent or incoherent light. The wavelength of the excitation light La is, for example, included in the near-infrared region. Specifically, the wavelength of the excitation light La is in the range of 650 nm to 1800 nm. The excitation light La is, for example, laser light. The following equation (1) is an equation that shows the time waveform of the excitation light La. In equation (1), Ir is the light intensity of the excitation light La, t is time, f is frequency, and a and b are constants.

number

number

[0026] In one embodiment, the excitation light output unit 10 includes a light source 11 and an optical modulator 12. The light source 11 outputs pulsed light Lp. The light source 11 outputs pulsed light Lp having a time width, for example, on the order of picoseconds or femtoseconds. Here, the time width of the pulsed light Lp is, for example, the time during which the light intensity of the pulsed light Lp exceeds half of its peak value. Specifically, the time width of the pulsed light Lp is, for example, in the range of 10 femtoseconds to 50 picoseconds. The light source 11 is, for example, a laser light source, and in one example, a mode-locked laser light source. Alternatively, the light source 11 may be an incoherent light source such as a light-emitting diode. The preferred wavelength range of the pulsed light Lp is the same as the preferred wavelength range of the excitation light La described above.

[0027] The optical modulator 12 is an intensity-modulated optical modulator. The optical modulator 12 is optically coupled to the light source 11 via space or an optical waveguide. The optical modulator 12 is electrically connected to the signal generator 50. The optical modulator 12 modulates the pulsed light Lp output from the light source 11 to generate excitation light La. The modulation of the pulsed light Lp in the optical modulator 12 is synchronized with the output signal from the signal generator 50. The optical modulator 12 can be selected from various modulators, such as an electro-optical (EO) modulator, an acoustic-optical (AO) modulator, or a liquid crystal or ND (Neutral Density) filter whose transmittance or reflectance can be dynamically controlled. When the time width of the pulsed light Lp is on the order of femtoseconds, an AO modulator is particularly suitable as the optical modulator 12. Excitation light La may also be generated by controlling the magnitude of the drive current input to the light source 11, i.e., by a direct modulation method, instead of using the optical modulator 12. In that case, the optical modulator 12 is not necessary.

[0028] The optical system 20 is optically coupled to the excitation light output unit 10 via space. The optical system 20 irradiates the object B to be observed with excitation light La output from the excitation light output unit 10. In one embodiment, the optical system 20 includes a beam expander 21, an optical scanner 22, a relay lens system 23, a dichroic mirror 24, and an objective lens 25.

[0029] The beam expander 21 is optically coupled to the excitation light output unit 10 via space and expands the beam diameter of the excitation light La output from the excitation light output unit 10. The beam expander 21 includes, for example, a pair of lenses 211, 212 that are optically coupled to each other. One lens 211 is positioned in the front, i.e., closer to the excitation light output unit 10 than the other lens 212, and the other lens 212 is positioned in the rear, i.e., further away from the excitation light output unit 10 than the first lens 211. The front lens 211 diffuses the excitation light La, and the rear lens 212 parallelizes the excitation light La. The lenses 211, 212 are, for example, glass lenses.

[0030] The optical scanner 22 is optically coupled to the beam expander 21 via space. The optical scanner 22 scans the irradiation position of the excitation light La on the object B by moving the optical axis of the excitation light La in a plane perpendicular to the optical axis of the excitation light La. The optical scanner 22 can be composed of various optical scanners such as a galvanometer scanner, a resonant mirror, or a polygon mirror. In one example, the optical scanner 22 is a two-axis galvanometer scanner.

[0031] Figure 3 shows another example of the optical scanner 22. The optical scanner 22 shown in Figure 3 has two scanners 221 and 222. Both scanners 221 and 222 are uniaxial scanners. The scanning direction of scanner 221 and scanner 222 are orthogonal to each other. Scanners 221 and 222 are optically coupled to each other via an optical system 223 such as a relay lens. Scanners 221 and 222 are, for example, galvanometer scanners. In this way, the optical scanner 22 may be configured by combining multiple uniaxial scanners. Alternatively, the irradiation position of the excitation light La on the object to be observed B may be scanned by moving a stage on which the object to be observed B is placed in a plane perpendicular to the optical axis of the excitation light La. In that case, the optical scanner 22 may not be provided.

[0032] The relay lens system 23 is provided in the optical path between the optical scanner 22 and the objective lens 25, and optically couples the optical scanner 22 and the objective lens 25. The relay lens system 23 is, for example, a telecentric relay lens system. If the optical scanner 22 and the objective lens 25 are very close together, the relay lens system 23 can be omitted.

[0033] The dichroic mirror 24 transmits either the excitation light La from the optical scanner 22 or the fluorescence Lb from the object B being observed, and reflects the other. In the example shown in Figure 1, the dichroic mirror 24 transmits the excitation light La and reflects the fluorescence Lb. In the example shown in Figure 1, the dichroic mirror 24 is located in the optical path between the relay lens system 23 and the objective lens 25. The dichroic mirror 24 may also be located between the optical scanner 22 and the relay lens system 23, or between the beam expander 21 and the optical scanner 22.

[0034] The objective lens 25 is positioned opposite the object to be observed B and focuses the excitation light La into the interior of the object to be observed B. The relative distance between the objective lens 25 and the object to be observed B is variable. The objective lens 25 may be movable along the optical axis of the excitation light La, and the stage (not shown) on which the object to be observed B is placed may also be movable along the optical axis of the excitation light La. The mechanism for moving the objective lens 25 or the object to be observed B may be configured, for example, by a stepping motor or a piezo actuator. The arrangement of the objective lens 25 relative to the object to be observed B may be upright or inverted.

[0035] The objective lens 25 densely focuses the excitation light La at the object B, causing two-photon excitation at the object B and generating fluorescence Lb from it. The wavelength of fluorescence Lb is, for example, in the range of 350 nm to 900 nm. The objective lens 25 also has the function of collecting fluorescence Lb from the object B. In the illustrated example, the objective lens 25 serves as both the objective lens for the excitation light La and the lens for collecting fluorescence Lb. The objective lens for the excitation light La and the lens for collecting fluorescence Lb may be provided separately. For example, an objective lens with a high numerical aperture (NA) may be used for the excitation light La, and it may be locally focused by aberration correction. An objective lens with a large pupil may be used for fluorescence Lb to extract more light. The objective lens for the excitation light La and the lens for collecting fluorescence Lb may be arranged so that the object B is sandwiched between them, and fluorescence Lb emitted from the surface opposite to the incident surface of the excitation light La at the object B may be obtained. In that case, the dichroic mirror 24 will not be necessary.

[0036] The harmonic detection unit 30 detects the second harmonic (or the second and third harmonics) included in the time waveform of the light intensity of the fluorescent Lb. In one embodiment, the harmonic detection unit 30 includes a photodetector 31 and a lock-in amplifier 32. The photodetector 31 is optically coupled to the dichroic mirror 24. Alternatively, if separate objective lenses are provided for focusing the excitation light La and for collecting the fluorescent Lb, the photodetector 31 is optically coupled to the objective lens for collecting the fluorescent Lb. The photodetector 31 generates an electrical signal corresponding to the light intensity of the fluorescent Lb generated in the object B being observed. The photodetector 31 is sensitive to the wavelength of the fluorescent Lb and has a frequency range necessary to detect the second harmonic of the fluorescent Lb, or both the second and third harmonics. The photodetector 31 may be selected from one-dimensional photodetectors such as a photomultiplier tube or an avalanche photodiode. Alternatively, various two-dimensional photodetectors such as a multi-anode PMT (Photomultiplier Tube), a CCD image sensor, or a CMOS image sensor may be selected as the photodetector device 31. A filter may be provided in the optical path between the harmonic detection unit 30 and the dichroic mirror 24 (or a lens for collecting fluorescence Lb) to cut out the wavelength of the excitation light La and wavelengths that are not needed for observation.

[0037] The lock-in amplifier 32 is electrically connected to the photodetector 31 and the signal generator 50. The lock-in amplifier 32 receives an electrical signal from the photodetector 31 corresponding to the light intensity of the fluorescence Lb. In addition, the lock-in amplifier 32 receives a sinusoidal signal from the signal generator 50 that has the same period as the periodic signal provided to the excitation light output unit 10. Using the sinusoidal signal from the signal generator 50 as a reference, the lock-in amplifier 32 detects the second harmonic, or both the second and third harmonics, contained in the time waveform of the signal from the photodetector 31.

[0038] The image generation unit 40 is electrically connected to the lock-in amplifier 32. The image generation unit 40 receives a signal from the lock-in amplifier 32 regarding the magnitude of the second harmonic included in the time waveform of the light intensity of the fluorescent Lb, and generates an observation image of the object B based on this second harmonic. Alternatively, the image generation unit 40 receives signals from the lock-in amplifier 32 regarding the magnitudes of both the second and third harmonics included in the time waveform of the light intensity of the fluorescent Lb, and generates an observation image of the object B based on one or both of the second and third harmonics. The image generation unit 40 may be composed of, for example, a computer including a central processing unit (CPU) and memory. The image generation unit 40 may further have a monitor for displaying the generated image.

[0039] Figure 4 is a flowchart illustrating the operation of the microscope device 1 according to this embodiment. Referring to Figure 4, the operation of the microscope device 1 and the image acquisition method according to this embodiment will be explained.

[0040] First, the excitation light output step S1 is performed. In the excitation light output step S1, the excitation light output unit 10 outputs excitation light La. As mentioned above, the time waveform of the light intensity of the excitation light La includes the square root of a linear function of a sine wave (see equation (1)). The maximum value Imax of the light intensity of the excitation light La in each period exceeds the saturation excitation intensity at the object B being observed. This excitation light output step S1 may also include a step S11 in which pulsed light Lp is generated in the light source 11, and an intensity modulation step S12 in which pulsed light Lp is modulated in the optical modulator 12 to generate excitation light La.

[0041] Next, the excitation light irradiation step S2 is performed. In the excitation light irradiation step S2, the excitation light La output in the excitation light output step S1 is irradiated onto the object B via the beam expander 21, optical scanner 22, relay lens system 23, dichroic mirror 24, and objective lens 25. Two-photon excitation by irradiation with excitation light La generates fluorescence Lb in the object B.

[0042] Next, the harmonic detection step S3 is performed. In the harmonic detection step S3, the harmonic detection unit 30 detects the second harmonic, or both the second and third harmonics, included in the time waveform of the light intensity of the fluorescent Lb. The harmonic detection step S3 may also include steps S31 and S32. In step S31, the photodetection device 31 generates a signal corresponding to the light intensity of the fluorescent Lb generated in the object B being observed. In step S32, the lock-in amplifier 32 outputs the second harmonic, or both the second and third harmonics, included in the time waveform of the signal generated by the photodetection device 31.

[0043] The above steps of excitation light output step S1, excitation light irradiation step S2, and harmonic detection step S3 are repeated while scanning the irradiation position of the excitation light La on the object B using the optical scanner 22 (steps S4, S5). This provides data on the magnitude of the second harmonic, or both the second and third harmonics, at multiple locations on the object B.

[0044] After scanning by the optical scanner 22 is completed (step S4; YES), the image generation step S6 is performed. If a second harmonic is detected in the time waveform of the light intensity of the fluorescent Lb in the harmonic detection step S3, in the image generation step S6, the image generation unit 40 generates an observation image of the object B based on the magnitude of the second harmonic included in the time waveform of the light intensity of the fluorescent Lb at multiple irradiation positions of the excitation light La. If both the second harmonic and the third harmonic are detected in the time waveform of the light intensity of the fluorescent Lb in the harmonic detection step S3, in the image generation step S6, the image generation unit 40 generates an observation image of the object B based on the magnitude of one or both of the second harmonic and the third harmonic included in the time waveform of the light intensity of the fluorescent Lb at multiple irradiation positions of the excitation light La.

[0045] The effects of the microscope device 1 and image acquisition method of this embodiment, as described above, will be explained along with the problems of conventional microscope devices.

[0046] Part 5(a) of Figure 5 shows an example of the fluorescence intensity distribution in a SAX microscope. In a SAX microscope, the peak intensity of the excitation light is made greater than the saturation excitation intensity of the object being observed. As a result, the fluorescence intensity within a certain range from the center of the fluorescence intensity distribution saturates, and the shape of the fluorescence intensity distribution (see solid line F2 in the figure) changes from the theoretical value when there is no saturation (see dashed line F1 in the figure). Therefore, by taking the difference between the theoretical and measured values ​​of fluorescence intensity (see solid line F3 in part 5(b)), the full width at half maximum of the intensity distribution can be reduced, and the spatial resolution of the microscope can be improved. In the case of single-photon excitation, the difference between the theoretical and measured values ​​of fluorescence intensity, or a value approximating the difference, can be obtained by making the time waveform of the excitation light intensity sinusoidal and detecting the harmonics of the time waveform of the fluorescence intensity, for example, the second harmonic or the third harmonic.

[0047] In a two-photon excitation microscope, long-wavelength ultrashort pulsed light, such as near-infrared light, is used as excitation light to irradiate the object to be observed. This generates two-photon excitation in the object, and the resulting fluorescence is detected to create an observation image. Because this two-photon excitation microscope uses long-wavelength light with excellent object penetration properties, it becomes possible to non-invasively observe, for example, the deep parts of biological tissue.

[0048] If a SAX microscope, which possesses the advantages described above, and a two-photon excitation microscope can be combined, a microscope that combines these advantages can be realized. However, in two-photon excitation, fluorescence intensity is proportional to the square of the excitation light intensity. Therefore, when an object to be observed is irradiated with excitation light having a sinusoidal time waveform, and two-photon excitation occurs in the object, the time waveform of the excitation light output from the object will be proportional to the square of the sine wave. Figure 6 is a conceptual diagram showing the time waveform of the excitation light intensity (see part 6(a)) and the time waveform of the fluorescence intensity (see part 6(b)) in the case of two-photon excitation. If an observation image is to be obtained based on fluorescence intensity having a time waveform deformed from a sine wave, as shown in part 6(b), it becomes necessary to detect high-order harmonics, such as the third harmonic or the fifth harmonic, as described in Patent Document 2, for example. Since there is a limit to the maximum frequency range of the device that detects the excitation light, such as the lock-in amplifier 32, if we try to detect high-order harmonics, we have no choice but to lower the frequency of the excitation light, which increases the time required to create the observation image. Alternatively, we may have to introduce equipment with a higher upper limit on the frequency range, which increases costs.

[0049] In the microscope apparatus 1 and image acquisition method of this embodiment, the time waveform of the excitation light La's light intensity includes the square root of a linear sinusoidal function (see equation (1)). As described above, in two-photon excitation, the light intensity of fluorescence Lb is proportional to the square of the light intensity of the excitation light La. Therefore, when excitation light La having a time waveform including the square root of a linear sinusoidal function is irradiated onto the object B, and two-photon excitation occurs in the object B, the time waveform of the excitation light La output from the object B is proportional not to the square of the sinusoidal function, but to a linear sinusoidal function. Figure 7 is a conceptual diagram showing the time waveform of such excitation light intensity (see part 7(a)) and the time waveform of fluorescence intensity (see part 7(b)).

[0050] Therefore, according to the microscope apparatus 1 and image acquisition method of this embodiment, observation images can be obtained based on lower-order harmonics, such as the second or third harmonic, similar to a general SAX microscope. This eliminates the need to reduce the frequency of the excitation light La due to the frequency range constraints of the photodetector device 31, thus avoiding the need for longer image creation times. Alternatively, it allows the use of equipment with a lower upper limit on the frequency range, leading to cost reduction.

[0051] To verify the above effects, the inventors measured the light intensity of fluorescence Lb in the microscope apparatus 1 of this embodiment. For comparison, the inventors also measured the fluorescence intensity using a sine wave as the time waveform of the excitation light intensity. In these measurements, the excitation light intensity and excitation light wavelength were kept the same, and the same object B was used as the object of observation.

[0052] Part 8(a) of Figure 8 is a graph showing the result of a Fourier transform on the time waveform of the fluorescence Lb light intensity measured in the microscope apparatus 1 of this embodiment. Part 8(b) of Figure 8 is a graph showing the result of a Fourier transform on the time waveform of the fluorescence intensity measured when the time waveform of the excitation light intensity is a sine wave. The arrow U1 in parts 8(a) and (b) of Figure 8 points to the fundamental wave, and only this fundamental wave is generated when saturation is not allowed. When the excitation light intensity is set to a magnitude that causes saturation and its time waveform is a sine wave, as shown in part 8(b), in addition to the fundamental wave, the second harmonic (arrow U2), third harmonic (arrow U3), fourth harmonic (arrow U4), fifth harmonic (arrow U5), and sixth harmonic (arrow U6) are generated due to saturation. In contrast, in the microscope apparatus 1 of this embodiment, as shown in part 8(a), in addition to the fundamental wave, the second harmonic (arrow V2) and third harmonic (arrow V3) are generated due to saturation. In these measurement results, the fluorescence intensity of the second harmonic (arrow V2) in part 8(a) was approximately equal to the fluorescence intensity of the third harmonic (arrow U3) in part 8(b). Furthermore, the fluorescence intensity of the third harmonic (arrow V3) in part 8(a) was approximately equal to the fluorescence intensity of the fifth harmonic (arrow U5) in part 8(b).

[0053] Part 9(a) of Figure 9 is a graph showing the relationship between the light intensity (fluorescence intensity) of the fundamental wave and second harmonic of the fluorescence Lb measured in the microscope apparatus 1 of this embodiment, and the relative intensity (relative excitation intensity) of the excitation light La. In Part 9(a) of Figure 9, plot P11 shows the fundamental wave, and plot P12 shows the second harmonic. Part 9(b) of Figure 9 is a graph showing the relationship between the light intensity (fluorescence intensity) of the fundamental wave, second harmonic, and third harmonic of the fluorescence measured when the time waveform of the excitation light intensity is a sine wave, and the relative intensity (relative excitation intensity) of the excitation light. In Part 9(b) of Figure 9, plot P21 shows the fundamental wave, plot P22 shows the second harmonic, and plot P23 shows the third harmonic. The relative intensity of the excitation light is the intensity of the periodic excitation light La after it has been output from the optical modulator 12, and is the ratio when the maximum intensity that can be output by the optical modulator 12 is set to 1. Comparing parts 9(a) and 9(b) in Figure 9, it can be seen that the arrangement of plot P12 in part 9(a) is roughly similar to the arrangement of plot P23 in part 9(b). In other words, the arrangement of the plot of the second harmonic of fluorescence Lb measured in the microscope apparatus 1 of this embodiment is roughly similar to the arrangement of the plot of the third harmonic of fluorescence measured with the time waveform of the excitation light intensity being a sine wave. The fact that the arrangement of plot P12 does not approximate the arrangement of plot P23 in the region of low fluorescence intensity is thought to be due to measurement error.

[0054] From the measurement results shown above, it can be seen that in the microscope apparatus 1 of this embodiment, where the time waveform of the excitation light La contains the square root of a linear function of a sine wave, the detection of the second harmonic (or third harmonic) corresponds to the detection of the third harmonic (or fifth harmonic) when the time waveform of the excitation light intensity is a sine wave. In other words, with the microscope apparatus 1 of this embodiment, observation images can be obtained based on harmonics of a lower order compared to the case where the time waveform of the excitation light intensity is a sine wave. Generally, equipment with a lower upper limit of the frequency range is often less expensive than equipment with a higher upper limit, thus enabling cost reduction.

[0055] As in this embodiment, the excitation light output unit 10 may include a light source 11 that outputs pulsed light Lp, and an intensity-modulated optical modulator 12 that modulates the pulsed light Lp output from the light source 11 to generate excitation light La. Similarly, the excitation light output step S1 may include an intensity modulation step S12 that modulates the pulsed light Lp to generate excitation light La. This makes it easy to generate excitation light La whose time waveform of light intensity includes the square root of a linear function of a sinusoidal wave. The intensity-modulated optical modulator 12 may be an AO modulator. AO modulators are suitable for high-speed and non-sinusoidal optical modulation as in this embodiment.

[0056] As in this embodiment, the light source 11 may be a laser light source, and the pulsed light Lp may be laser light. This makes it possible to generate excitation light La, which has a high light intensity capable of causing two-photon excitation, with a simple configuration.

[0057] As in this embodiment, the harmonic detection unit 30 may include a photodetector 31 that generates a signal corresponding to the light intensity of the fluorescence Lb generated in the object B, and a lock-in amplifier 32 that receives the signal from the photodetector 31 and outputs the second harmonic, or both the second and third harmonics, included in the time waveform of the signal. Similarly, the harmonic detection step S3 may include a step S31 that generates a signal corresponding to the light intensity of the fluorescence Lb generated in the object B, and a step S32 that outputs the second harmonic, or both the second and third harmonics, included in the time waveform of the signal. This makes it possible to easily and accurately detect the second harmonic, or both the second and third harmonics.

[0058] As in this embodiment, the harmonic detection unit 30 may detect the second and third harmonics included in the time waveform of the light intensity of the fluorescent Lb generated in the object B. Similarly, in the harmonic detection step S3, the second and third harmonics included in the time waveform of the light intensity of the fluorescent Lb generated in the object B may be detected. Then, in the image generation step S6, an observation image of the object B may be generated based on one or both of the second and third harmonics. In this case, an observation image using the harmonics suitable for the object B among the second and third harmonics can be easily generated. [First variation]

[0059] The time waveform of the excitation light La shown in equation (1), that is, the time waveform containing the square root of a linear function of the sine wave, is not limited to the example shown in equation (2) and Figure 2. For example, in the example shown in Figure 2, the time waveform of the excitation light La, i.e., the minimum value Imin in each period of the envelope K, is 0, but the minimum value Imin in each period may be greater than 0. In other words, in equation (1), the constant b may be greater than a. Alternatively, the minimum value Imin in each period of the time waveform of the excitation light La may be greater than 0.1% or 5% and less than 20% of the maximum signal that the harmonic detection unit 30, specifically the lock-in amplifier 32, can receive. Figure 10 is a conceptual diagram showing such a time waveform of the excitation light La. In this modified example as well, the maximum value Imax of the light intensity of the excitation light La in each period is set to a magnitude exceeding the saturation excitation intensity at the object of observation B.

[0060] Equation (3) is an example of the time waveform of the excitation light La in this modified example. Here, α is a real number greater than 0 and less than 1.

number

[0061] At and around the minimum value Imin within each period of the time waveform of the excitation light La's light intensity, the intensity of the generated fluorescence Lb is negligible, and the detection result is greatly affected by noise. As in this modified example, by making the minimum value Imin within each period of the time waveform of the excitation light La's light intensity greater than 0, the effect of noise can be reduced, improving the detection accuracy of the second and third harmonics and making the observed image clearer. Alternatively, the minimum value Imin within each period of the time waveform of the excitation light La's light intensity may be greater than 0.1% and less than 20% of the maximum signal that can be received in the harmonic detection unit 30 or the harmonic detection step S3. This reduces the effect of noise, improving the detection accuracy of the second and third harmonics and making the observed image clearer.

[0062] When the time waveform of the excitation light La contains the square root of a linear function of a sine wave, the rate of change of light intensity near the minimum value Imin within each period is larger compared to when the excitation light intensity time waveform is a sine wave. In other words, the time waveform of light intensity near the minimum value Imin within each period becomes steeper. This rate of change is largest when the minimum value Imin within each period is 0 (see Figure 2). Here, Figure 11 is a graph showing a typical example of the relationship between the applied voltage and output light intensity of a general AO modulator. As shown in Figure 11, the output light intensity from the AO modulator changes nonlinearly with respect to the applied input voltage. And in the region of low applied voltage, the change in output light intensity is extremely gradual. For this reason, if the rate of change of light intensity is large during periods when the light intensity of the excitation light La is small, it becomes necessary to rapidly change the applied voltage to the AO modulator, making it difficult to control the applied voltage. To address these challenges, as shown in this modified example, by making the minimum value Imin within each period greater than 0, the rate of change of light intensity near the minimum value Imin within each period can be reduced, thereby mitigating the sharpness of the time waveform. Alternatively, by making the minimum value Imin within each period greater than 0.1% and less than 20% of the maximum signal that can be received in the harmonic detection unit 30 or the harmonic detection step S3, the rate of change of light intensity near the minimum value Imin within each period can be reduced, thereby mitigating the sharpness of the time waveform. Therefore, the shaping of the time waveform of the excitation light La in the excitation light output unit 10, which has an AO modulator as the optical modulator 12, becomes easier, particularly the shaping of the time waveform near the minimum value Imin within each period.

[0063] As described above, if the minimum value Imin within each period is made greater than 0, the minimum value within each period in the time waveform of the fluorescent Lb light intensity will also become greater than 0. Therefore, if the time waveform of the fluorescent Lb light intensity is used as is in the lock-in amplifier 32, the detection accuracy of the second and third harmonics may decrease. To avoid this risk, the time waveform may be adjusted in the lock-in amplifier 32 or in the stage preceding the lock-in amplifier 32 so that the minimum value within each period in the time waveform of the fluorescent Lb light intensity becomes zero. In other words, the fluctuation in the time waveform of the fluorescent Lb caused by the difference between the minimum value Imin of the excitation light La and zero may be canceled. [Second Embodiment]

[0064] Figure 12 shows the configuration of the microscope apparatus 2 according to the second embodiment. The microscope apparatus 2 is a device for acquiring an image of the object to be observed B. The microscope apparatus 2 comprises an excitation light output unit (excitation light output device) 10, an optical system 20A, a harmonic detection unit 30, an image generation unit 40, and a signal generator 50. The configurations of the excitation light output unit 10, the harmonic detection unit 30, the image generation unit 40, and the signal generator 50 are the same as in the first embodiment.

[0065] The optical system 20A is an optical system for irradiating the object to be observed B with excitation light La output from the excitation light output unit 10. The optical system 20A includes an optical path for the excitation light La from the excitation light output unit 10 to the object to be observed B. The optical system 20A includes a beam expander 21, a polarization conversion unit 61, a phase conversion unit 62, a ring mask 63, an optical scanner 22, a relay lens system 23, a dichroic mirror 24, and an objective lens 25. The configurations of the beam expander 21, optical scanner 22, relay lens system 23, dichroic mirror 24, and objective lens 25 are the same as in the first embodiment. In the illustrated example, the polarization conversion unit 61, the phase conversion unit 62, and the ring mask 63 are provided on the optical path between the beam expander 21 and the optical scanner 22. In this embodiment as well, the irradiation position of the excitation light La on the object B may be scanned by moving the stage on which the object B is placed in a plane perpendicular to the optical axis of the excitation light La. In that case, the optical scanner 22 may not be provided. If the optical scanner 22 is provided, the polarization conversion unit 61, the phase conversion unit 62, and the ring mask 63 may be provided in front of the optical scanner 22.

[0066] The polarization conversion unit 61 is optically coupled to the excitation light output unit 10 via the beam expander 21. The polarization conversion unit 61 receives excitation light La as input, converts the excitation light La to azimuth polarization, and outputs it. The polarization state of the excitation light La before it is input to the polarization conversion unit 61 is, for example, linear polarization. Figure 13 shows the polarization direction of azimuth polarization in a plane perpendicular to the optical axis. In Figure 13, arrow A indicates the polarization direction. Figure 14 shows a focused image of azimuth polarization. In Figure 14, light intensity is shown by the shade of color, with darker areas indicating lower light intensity and lighter areas indicating higher light intensity. In Figure 14, parts (a) to (d) show the light intensity distribution in a plane perpendicular to the optical axis, and parts (e) to (h) show the light intensity distribution in a plane parallel to the optical axis that includes the optical axis. The XY coordinate system shown in the figure applies to parts (a) to (d). The ZX coordinate system shown in the figure applies to parts (e) to (h). In Figure 14, parts (b) and (f) are the light intensity (|Ex|) due to the vibration component Ex in the X direction of the electric field, i.e., perpendicular to the optical axis. 2(c) and (g) show the optical intensity (|Ey|) due to the vibration component Ey in the Y direction of the electric field, i.e., perpendicular to the optical axis and the X direction. 2 (d) and (h) show the optical intensity (|Ez|) due to the vibration component Ez in the Z direction of the electric field, i.e., in the optical axis direction. 2 (a) and (e) show the light intensity (|Ex|) obtained by combining the vibration components of the electric field in all directions. 2 +|Ey| 2 +|Ez| 2 Figures 13 and 14 show that, in azimuth polarization, the direction of electric field oscillation is aligned with the tangential direction of the circumference centered on the optical axis, and the light intensity is small near the optical axis. Therefore, the light intensity distribution of azimuth polarization in a plane perpendicular to the optical axis is annular. The polarization conversion unit 61 converts the excitation light La input from the excitation light output unit 10 into such azimuth polarization and outputs it. The polarization conversion unit 61 can be composed of, for example, an azimuth polarizer or two spatial light modulators. The azimuth polarizer may be a fixed type made by processing a glass plate, or a variable type using liquid crystal.

[0067] The phase conversion unit 62 is optically coupled to the excitation light output unit 10 via the beam expander 21 and the polarization conversion unit 61. The phase conversion unit 62 receives the excitation light La as input and modulates the excitation light La with a helical phase pattern. Figure 15 shows a helical phase pattern. In Figure 15, the magnitude of the phase is shown by the intensity of the color, with lighter areas indicating a smaller phase and darker areas indicating a larger phase. As shown in Figure 15, in the helical phase pattern, the phase changes monotonically according to the angle around the optical axis Q. In one example, in this helical phase pattern, the phase changes from 0 (rad) to 2π (rad). That is, the width of the phase change in one rotation is 2π (rad). The phase conversion unit 62 can be configured, for example, by a helical phase plate or a phase-modulation type spatial light modulator. The phase plate may be, for example, a glass plate that has been processed for phase modulation.

[0068] FIG. 16 is a diagram showing a converging image of light that provides phase modulation by a helical phase pattern and azimuthal polarization. In FIG. 16, the light intensity is indicated by the shade of color, with darker portions indicating lower light intensity and lighter portions indicating higher light intensity. In FIG. 16, parts (a) to (d) show the light intensity distribution in a plane perpendicular to the optical axis, and parts (e) to (h) show the light intensity distribution in a plane containing the optical axis and parallel to the optical axis. The XY coordinate system shown in the figure is applied to parts (a) to (d). The ZX coordinate system shown in the figure is applied to parts (e) to (h). In FIG. 16, parts (b) and (f) show the light intensity (|Ex| 2 ) due to the vibration component Ex in the X direction of the electric field, i.e., the direction perpendicular to the optical axis. Parts (c) and (g) show the light intensity (|Ey| 2 ) due to the vibration component Ey in the Y direction of the electric field, i.e., the direction perpendicular to the optical axis and the X direction. Parts (d) and (h) show the light intensity (|Ez| 2 ) due to the vibration component Ez in the Z direction of the electric field, i.e., the direction of the optical axis. Parts (a) and (e) show the light intensity (|Ex| 2 + |Ey| 2 + |Ez| 2 ) obtained by synthesizing the vibration components in all directions of the electric field. As shown in FIG. 16, when a helical phase pattern is combined with azimuthal polarization, the light intensity near the optical axis increases, and the light intensity distribution in the plane perpendicular to the optical axis changes from an annular shape to a solid circular shape. The excitation light La passes through the polarization conversion unit 61 and the phase conversion unit 62, and thus has such a solid circular light intensity distribution.

[0069] The ring mask 63 is optically coupled to the excitation light output unit 10 via the beam expander 21, the polarization conversion unit 61, and the phase conversion unit 62. The ring mask 63 receives excitation light La as input, spatially modulates the intensity of the excitation light La in the beam cross-section of the excitation light La, and outputs the modulated excitation light La. The ring mask 63 has a ring-shaped light-shielding portion and a transmissive portion provided in contact with the inside and outside of the light-shielding portion, respectively. The ring mask 63 of this embodiment is a so-called multiple ring mask. The ring mask 63 can be composed of, for example, a plate-shaped member with a light-shielding portion and a transmissive portion formed thereon, or a phase-modulation type spatial light modulator. The plate-shaped member can be composed of, for example, a light-shielding film formed as a light-shielding portion on a light-transmitting plate material. Ring masks exist in amplitude-modulation type (in other words, intensity-modulation type), phase-modulation type, and combined types thereof. The ring mask 63 of this embodiment is of amplitude-modulation type.

[0070] Figure 17 shows an example of the configuration of a ring mask 63 as viewed from the optical axis direction. This ring mask 63 has a plurality of ring-shaped light-shielding sections provided around the center position. In the illustrated example, the ring mask 63 has three light-shielding sections D1, D2, and D3. Furthermore, the ring mask 63 has a transparent section E1, a transparent section E2, a transparent section E3, a transparent section E4, and a light-shielding section D4. The transparent section E1 is the innermost transparent section provided inside the light-shielding section D1. The transparent section E2 is a ring-shaped transparent section provided between the light-shielding sections D1 and D2. The transparent section E3 is a ring-shaped transparent section provided between the light-shielding sections D2 and D3. The transparent section E4 is the outermost ring-shaped transparent section provided outside the light-shielding section D3. The light-shielding section D4 is provided outside the transparent section E4.

[0071] The light transmittance in the transparent sections E1 to E4 is greater than the light transmittance in the light-blocking sections D1 to D4. The light transmittance in the transparent sections E1 to E4 may be 1 or less than 1. The light transmittance in the light-blocking sections D1 to D4 may be 0 or greater than 0. The boundaries between adjacent transparent and light-blocking sections among the transparent section E1, light-blocking section D1, transparent section E2, light-blocking section D2, transparent section E3, light-blocking section D3, transparent section E4 and light-blocking section D4 may be concentric circles or ellipses. In the following explanation, we will assume that the boundaries are circles. Let e1 be the radial width, i.e., radius, of the transparent section E1. Let d1 be the radial width of the light-blocking section D1. Let e2 be the radial width of the transparent section E2. Let d2 be the radial width of the light-blocking section D2. Let e3 be the radial width of the transparent section E3. Let d3 be the radial width of the light-blocking section D3. Let e4 be the radial width of the transparent section E4.

[0072] The radial width of two adjacent ring-shaped light-shielding portions D1, D2, and D3 is greater than the radial width of the transparent portion provided between these two light-shielding portions. That is, in the ring mask 63 of this embodiment, the radial widths of the light-shielding portions D1, D2 and the transparent portion E2 have a relationship expressed by equation (4) below, and the radial widths of the light-shielding portions D2, D3 and the transparent portion E3 have a relationship expressed by equation (5) below. Such a relationship may exist for all combinations of two adjacent ring-shaped light-shielding portions.

number

number

[0073] In this embodiment, the order in which the polarization conversion unit 61, the phase conversion unit 62, and the ring mask 63 are arranged is not limited to the example shown in Figure 12, but can be changed in various ways. That is, the polarization conversion unit 61, the phase conversion unit 62, and the ring mask 63 may be arranged in any of the order shown in parts (a) to (e) of Figure 18, as viewed from the excitation light output unit 10. Part (a) of Figure 18: From the perspective of the excitation light output unit 10, the order is polarization conversion unit 61, ring mask 63, and phase conversion unit 62. Part (b) of Figure 18: Viewed from the excitation light output unit 10, the order is phase conversion unit 62, polarization conversion unit 61, and ring mask 63. Part (c) of Figure 18: Viewed from the excitation light output unit 10, the order is phase conversion unit 62, ring mask 63, and polarization conversion unit 61. Part (d) of Figure 18: From the perspective of the excitation light output unit 10, the order is ring mask 63, polarization conversion unit 61, and phase conversion unit 62. Section (e) of Figure 18: From the perspective of the excitation light output unit 10, the order is ring mask 63, phase conversion unit 62, and polarization conversion unit 61.

[0074] Refer again to Figure 12. In this embodiment, the optical scanner 22 is optically coupled to the beam expander 21 via a polarization conversion unit 61, a phase conversion unit 62, and a ring mask 63.

[0075] In this embodiment, the objective lens 25 is, for example, a dry objective lens, a water immersion objective lens, an oil immersion objective lens, or a silicone immersion objective lens. The objective lens 25 may be an objective lens used for observing a cleared sample. If the objective lens 25 is a water immersion objective lens, its numerical aperture is, for example, 1.2 or greater. If the objective lens 25 is an oil immersion objective lens, its numerical aperture is, for example, 1.45 or greater. The ratio (NA / R) of the refractive index R in the medium between the objective lens 25 and the object B to the numerical aperture NA of the objective lens 25 is, for example, 0.75 or greater.

[0076] Figure 19 is a flowchart illustrating the operation of the microscope device 2 according to this embodiment. The image acquisition method according to this embodiment will be explained along with the operation of the microscope device 2 while referring to Figure 19.

[0077] First, the excitation light output step S1 is performed. The details of the excitation light output step S1 are the same as in the first embodiment.

[0078] Next, the excitation light irradiation step S2a is performed. In the excitation light irradiation step S2a, the excitation light La output in the excitation light output step S1 is irradiated onto the object B via the beam expander 21, polarization conversion unit 61, phase conversion unit 62, ring mask 63, optical scanner 22, relay lens system 23, dichroic mirror 24, and objective lens 25. That is, in the excitation light irradiation step S2a, the excitation light La output in the excitation light output step S1 is subjected to polarization conversion processing S21 by the polarization conversion unit 61, phase conversion processing S22 by the phase conversion unit 62, and ring mask processing S23 by the ring mask 63. Furthermore, in the excitation light irradiation step S2a, a focusing process S24 is performed to focus the excitation light La on the object B using the objective lens 25. Polarization conversion processing S21 is a process that converts the excitation light La into azimuth polarization. Phase conversion processing S22 is a process that applies phase modulation by a helical phase pattern to the excitation light La. In the focusing process S24 of the excitation light irradiation step S2a, the excitation light La may be focused using a water immersion objective lens with a numerical aperture of 1.2 or more. Alternatively, the excitation light La may be focused using an oil immersion objective lens with a numerical aperture of 1.45 or more. The phase conversion process S22 and the ring mask process S23 may be performed using a phase-modulation type spatial light modulator.

[0079] Next, the harmonic detection step S3 is performed. The details of the harmonic detection step S3 are the same as in the first embodiment.

[0080] The above steps of excitation light output step S1, excitation light irradiation step S2a, and harmonic detection step S3 are repeated while scanning the irradiation position of the excitation light La on the object B using the optical scanner 22 (steps S4, S5). This provides data on the magnitude of the second harmonic, or both the second and third harmonics, at multiple locations on the object B.

[0081] After scanning by the optical scanner 22 is completed (step S4; YES), the image generation step S6 is performed. The details of the image generation step S6 are the same as in the first embodiment.

[0082] The microscope device 2 and image acquisition method of this embodiment described above can provide the same effects and advantages as the first embodiment. In addition, the microscope device 2 and image acquisition method of this embodiment can also provide the effects and advantages described below.

[0083] In a microscope, light emitted from a light source is focused and illuminated onto the surface or interior of the object being observed via a focusing lens. When focusing light in this way, the beam waist diameter, which is an indicator of the size of the focused beam, can only be reduced to about half the wavelength of the light. This is called the diffraction limit.

[0084] To reduce the focal diameter beyond the diffraction limit, a ring mask is used. There are two types of ring masks: single-ring masks and multi-ring masks. A single-ring mask has a single ring-shaped light-shielding section and transparent sections provided both inside and outside of the light-shielding section. Light passing through the transparent sections on both the inside and outside of the light-shielding section reaches the focal point via a focusing lens. At the focal point, the interference of these two beams of light allows the light to be focused into a region smaller than the diffraction limit. A multi-ring mask has multiple ring-shaped light-shielding sections arranged concentrically and multiple transparent sections provided between these light-shielding sections. Light passing through each transparent section reaches the focal point via a focusing lens. Even when using such a multi-ring mask, it is possible to focus light into a smaller region beyond the diffraction limit.

[0085] Parts (a) and (b) of Figure 20 show the shape of the focused spot in a cross-section that includes the optical axis and is parallel to the optical axis. In these figures, the solid line Ha shows the shape of the focused spot when a ring mask is provided, and the dashed line Hb shows the shape of the focused spot when a ring mask is not provided. Part (a) of Figure 20 assumes the use of an immersion objective lens with a numerical aperture of 0.9. Part (b) of Figure 20 assumes the use of an immersion objective lens with a numerical aperture of 1.3. As shown in Figure 20, the size of the focused spot can be reduced by providing a ring mask.

[0086] As a reference example, consider the case where a multiple ring mask is applied to circularly polarized light. Tables 1 and 2 below show an example of the relationship between the numerical aperture of the objective lens and the degree of reduction in the diameter of the focused spot due to the provision of a ring mask in the light irradiation device related to the reference example. The volume improvement rate shown in these tables is the value obtained by dividing the volume A1 of the focused spot when no ring mask is provided by the volume A2 of the focused spot when a ring mask is provided (A1 / A2). The lateral improvement rate is the value obtained by dividing the diameter Wa1 of the focused spot in the direction perpendicular to the optical axis when no ring mask is provided, i.e., the diameter shown in Figure 20, by the diameter Wa2 of the focused spot in the direction perpendicular to the optical axis when a ring mask is provided, i.e., the width shown in Figure 20 (Wa1 / Wa2). The vertical improvement rate is the value obtained by dividing the length of the focused spot in the optical axis direction without a ring mask, i.e., the length Wb1 shown in Figure 20, by the length of the focused spot in the optical axis direction with a ring mask, i.e., the length Wb2 shown in Figure 20 (Wb1 / Wb2). The diameter of the focused spot and the length of the focused spot are the full width at half maximum (FWHM) of the light intensity distribution. In these examples, the ring mask is a multi-ring mask with four light-shielding sections, and the ring mask configuration that minimizes the volume of the focused spot was explored under the condition that the light intensity of the side lobes is 2.5% or less of the light intensity of the main lobe, i.e., the focused spot. Specifically, the configuration of the ring mask refers to the widths d1 to d3 of the light-shielding sections D1 to D3, and the widths e1 to e4 of the transmission sections E1 to E4. Table 1 assumes a water immersion objective lens. Table 2 assumes an oil immersion objective lens. Table 3 assumes a dry objective lens. [Table 1] [Table 2] [Table 3]

[0087] As shown in Tables 1 and 2, regardless of the type of objective lens and numerical aperture, providing a ring mask increases the volume improvement rate, lateral improvement rate, and vertical improvement rate to more than 1. However, the lateral improvement rate is smaller than the vertical improvement rate. In particular, when using objective lenses with large numerical apertures, such as water immersion objective lenses with an numerical aperture of 1.05 or higher, oil immersion objective lenses with an numerical aperture of 1.25 or higher, and dry objective lenses with an numerical aperture of 0.8 or higher, the lateral improvement rate becomes 1.08 or lower, and this tendency is particularly pronounced. Here, the refractive index of the medium between the water immersion objective lens and the object B, i.e., the immersion liquid, is assumed to be 1.333. The refractive index of the medium between the oil immersion objective lens and the object B, i.e., the immersion liquid, is assumed to be 1.518. The refractive index of the medium between the dry objective lens and the object B, i.e., air, is assumed to be 1. At this point, the ratio (NA / R) between the numerical aperture (NA) at which the lateral improvement rate begins to fall below 1.08 and the refractive index (R) is 0.75 or higher for water immersion objectives, oil immersion objectives, and dry objectives. A smaller lateral improvement rate results in a smaller reduction in the diameter of the focused spot, and consequently, a smaller improvement in resolution in a microscope, for example.

[0088] To address the above problem, the inventors have found that by providing a polarization conversion unit 61 that converts to azimuth polarization and a phase conversion unit 62 that applies phase modulation using a helical phase pattern, in addition to the ring mask 63, the degree to which the focusing diameter can be reduced can be increased. Similarly, the inventors have found that by performing a polarization conversion process S21 that converts to azimuth polarization and a phase conversion process S22 that applies phase modulation using a helical phase pattern, the degree to which the focusing diameter can be reduced can be increased.

[0089] Figure 21 is a graph showing the lateral resolution ratio for each aperture ratio when a ring mask is not used. In Figure 21, bar line G11 represents the case where the light illuminating object B is circularly polarized. Bar line G12 represents the case where the light illuminating object B is radially polarized. Bar line G13 represents the case where the light illuminating object B is a combination of azimuth polarization and helical phase modulation. The vertical axis represents the lateral resolution ratio. The lateral resolution ratio is the value obtained by dividing the lateral resolution when the numerical aperture of the oil immersion objective lens is 1.50 and the light illuminating object B is circularly polarized by the lateral resolution for each numerical aperture and each polarization. The horizontal axis represents the numerical aperture. Referring to Figure 21, it can be seen that even when a ring mask is not used, the lateral resolution ratio improves at all numerical apertures when the light illuminating object B is a combination of azimuth polarization and helical phase modulation compared to when the light illuminating object B is circularly polarized or radially polarized.

[0090] Figure 22 is a graph showing the lateral resolution ratio for each aperture ratio when using a ring mask. In Figure 22, bar line G21 represents the case where the light illuminating object B is circularly polarized. Bar line G22 represents the case where the light illuminating object B is radially polarized. Bar line G23 represents the case where the light illuminating object B is a combination of azimuth polarization and helical phase modulation. The vertical axis represents the lateral resolution ratio. The lateral resolution ratio is the value obtained by dividing the lateral resolution when the numerical aperture of the oil immersion objective lens is 1.50 and the light illuminating object B is circularly polarized, when a ring mask is not used, by the lateral resolution for each numerical aperture and polarization when a ring mask is used. The horizontal axis represents the numerical aperture. Referring to Figure 22, when a ring mask is used, the lateral resolution ratio is improved compared to when a ring mask is not used, regardless of the polarization state of the light illuminating object B. In particular, when the light irradiated onto object B is a combination of azimuth polarization and helical phase modulation, the lateral resolution ratio is dramatically improved.

[0091] Thus, by irradiating the object B with light combining azimuth polarization and helical phase modulation, in addition to the ring mask, the lateral resolution ratio, or lateral improvement rate, is dramatically improved. Therefore, according to this embodiment, the degree to which the focusing diameter can be reduced can be increased.

[0092] Figure 23 shows fluorescence detected at the second harmonic in a cross section containing the optical axis and parallel to the optical axis direction. Figure 24 shows fluorescence detected at the third harmonic in the same cross section. In these figures, light intensity is indicated by the shade of color, with darker areas indicating lower light intensity and lighter areas indicating higher light intensity. In Figures 23 and 24, parts (a) and (b) show fluorescence images when the excitation light La irradiated onto object B is azimuth-polarized light with helical phase modulation. Parts (c) and (d) show fluorescence images as reference examples when the excitation light La irradiated onto object B is circularly polarized. In Figures 23 and 24, parts (a) and (c) show fluorescence images when the ring mask 63 is provided. Parts (b) and (d) show fluorescence images when the ring mask 63 is not provided.

[0093] In Figures 23 and 24, comparing parts (a) and (c) with parts (b) and (d), it can be seen that when the ring mask 63 is provided, the horizontal and vertical dimensions of the fluorescence image are smaller than when the ring mask 63 is not provided. In Figures 23 and 24, comparing parts (a) and (b) with parts (c) and (d), it can be seen that when the excitation light La irradiated onto the object B is azimuth-polarized light with helical phase modulation, the horizontal dimensions of the fluorescence image are particularly smaller than when the excitation light La irradiated onto the object B is circularly polarized.

[0094] As mentioned above, the ring mask 63 and the ring mask processing S23 may be of amplitude modulation type. Ring masks exist in amplitude modulation type, phase modulation type, and hybrid types thereof. The light utilization efficiency of the phase modulation type is higher than that of the amplitude modulation type, so the phase modulation type can efficiently irradiate light while reducing losses. However, when using a phase modulation type ring mask, unwanted light-gathering portions called side lobes tend to be generated on both sides of the focusing spot in the optical axis direction. For example, Non-Patent Literature 3 discloses a method of reducing the size of the main lobe by interfering the side lobes with the main lobe. For example, in single-photon excitation type fluorescence microscopes, a confocal optical system is used, and the resolution can be increased by the confocal effect of the pinhole, so the generation of such unwanted light-gathering portions is to be tolerated to some extent. However, in two-photon excitation type fluorescence microscopes, for example, the excitation efficiency is lower than that of single-photon excitation type, and furthermore, when observing deep tissues, a large discrepancy occurs between the pinhole position and the focusing position due to the effect of aberrations, resulting in large light losses. By using an amplitude-modulated ring mask, the unwanted light-gathering side lobes are reduced compared to using a phase-modulated ring mask. This eliminates the need for a confocal optical system including a pinhole, contributing to miniaturization of the device while suppressing optical loss.

[0095] The ratio (NA / R) of the refractive index R in the medium between the objective lens 25 and the object B to the numerical aperture NA of the objective lens 25 may be 0.75 or greater. Similarly, in the focusing process S24 of the excitation light irradiation step S2, the excitation light La may be focused using an objective lens 25 in which the ratio (NA / R) of the refractive index R in the medium between the objective lens 25 and the object B to the numerical aperture NA of the objective lens 25 is 0.75 or greater. According to the microscope apparatus 2 and image acquisition method of this embodiment, when the numerical aperture of the objective lens is large in this way, the degree of reduction in the focusing diameter can be further increased. As the objective lens, a water immersion objective lens, an oil immersion objective lens, a dry objective lens, a silicone immersion objective lens, or an objective lens corresponding to a clearing solution can be used.

[0096] As described above, the phase conversion unit 62 and the ring mask 63 may each be configured with a phase-modulated spatial light modulator. Similarly, the phase conversion process S22 and the ring mask process S23 may each be performed using a phase-modulated spatial light modulator. In this case, the phase pattern in the phase conversion unit 62 or the phase conversion process S22 can be easily changed. Furthermore, the widths d1 to d3 of the light-shielding parts D1 to D3 and the widths e1 to e4 of the light-transmitting parts E1 to E4 in the ring mask 63 or the ring mask process S23 can be easily changed.

[0097] As described above, the ring mask 63 may have a plurality of ring-shaped light-shielding portions D1 to D3 provided around the central position, transparent portions E2 and E3 provided between two adjacent light-shielding portions D1 to D3, an innermost transparent portion E1 provided inside the innermost light-shielding portion D1 of the plurality of light-shielding portions D1 to D3, and an outermost transparent portion E4 provided outside the outermost light-shielding portion D3 of the plurality of light-shielding portions D1 to D3. By using such a multi-ring mask, the degree to which the light-gathering diameter can be reduced can be further increased. [Second variation]

[0098] Figure 25 shows a schematic diagram of the configuration of a microscope apparatus 2A according to one modified example of the second embodiment. Microscope apparatus 2A differs from the microscope apparatus 2 of the second embodiment in that it is equipped with a phase-modulated spatial light modulator 28 and an aperture optical system 29 instead of the phase conversion unit 62 and ring mask 63 of the microscope apparatus 2 described above. The other configurations of microscope apparatus 2A are the same as those of the microscope apparatus 2 of the second embodiment. Depending on the relative arrangement of the excitation light output unit 10 and the spatial light modulator 28, the excitation light output unit 10 and the spatial light modulator 28 may be optically coupled by an optical system such as a mirror 9.

[0099] The spatial light modulator 28 combines the functions of the phase conversion unit 62 and the ring mask 63. In other words, in this modified example, the spatial light modulator constituting the phase conversion unit 62 is the same as the spatial light modulator constituting the ring mask 63. The spatial light modulator 28 presents a phase pattern obtained by superimposing a phase pattern for constituting the phase conversion unit 62 and a phase pattern for constituting the ring mask 63. The phase pattern may also have a phase pattern superimposed to correct aberrations. In particular, aberrations possessed by the spatial light modulator 28 itself affect the accuracy of helical phase modulation, so they are corrected. Aberrations that occur during deep observation, such as spherical aberration arising from the refractive index difference between the object B and the immersion liquid, may also be corrected simultaneously by the spatial light modulator 28. The spatial light modulator 28 constitutes an amplitude-modulated ring mask 63 by its phase pattern. Figure 26 shows an example of a phase pattern constituting an amplitude-modulated ring mask 63. In Figure 26, the phase value of each pixel constituting the phase pattern is shown by the intensity of the color, with lighter colors indicating smaller phase values ​​and darker colors indicating larger phase values. In this example, the ring mask 63 is a triple ring mask. That is, this ring mask 63 has multiple ring-shaped light-shielding sections provided around the central position. In the illustrated example, the ring mask 63 has two light-shielding sections D5 and D6. Furthermore, the ring mask 63 has an innermost transparent section E5 provided inside the light-shielding section D5, a ring-shaped transparent section E6 provided between the light-shielding sections D5 and D6, a ring-shaped outermost transparent section E7 provided outside the light-shielding section D6, and a light-shielding section D7 provided outside the transparent section E7.

[0100] The light-shielding sections D5 to D7 are composed of gratings whose phase values ​​change periodically, while the phase values ​​of the light-transmitting sections E5 to E7 are kept constant. Specifically, in the light-shielding sections D5 to D7, a phase distribution that monotonically increases from 0 (rad) to 2π (rad) is periodically repeated in each period. Due to this phase pattern, the direction of emission of light from the light-shielding sections D5 to D7 is tilted relative to the direction of emission of light from the light-transmitting sections E5 to E7.

[0101] Refer again to Figure 25. The aperture optical system 29 is located downstream of the spatial light modulator 28 and is optically coupled to the spatial light modulator 28. The aperture optical system 29 has a pair of lenses 291 and 292 and an aperture 293 positioned between the lenses 291 and 292. Light emitted from the spatial light modulator 28 forms a beam waist between lenses 291 and 292. The aperture 293 is located in the beam waist and shields at least a portion of the light emitted from the light-shielding sections D5 to D7 of the spatial light modulator 28. As a result, the light emitted from the light-shielding sections D5 to D7 of the spatial light modulator 28 is attenuated or filtered out, and the light emitted from the light-transmitting sections E5 to E7 passes through the aperture 293. In this modified example as well, the light transmittance of the light-transmitting section is greater than that of the light-shielding section. The light transmittance of the transparent portion is defined as the ratio of the light intensity of the light that exits from the transparent portion E5 to E7 and passes through the aperture 293 to the light intensity of the light incident on the transparent portion E5 to E7. The light transmittance of the light-shielding portion is defined as the ratio of the light intensity of the light that exits from the light-shielding portion D5 to D7 and passes through the aperture 293 to the light intensity of the light-shielding portion D5 to D7 to the light intensity of the light-shielding portion D5 to D7. The light transmittance of the transparent portion may be 1 or less than 1. The light transmittance of the light-shielding portion may be 0 or greater than 0.

[0102] Alternatively, as shown in Figure 27, the transmission sections E5 to E7 may be composed of gratings with periodically changing phase values, while the phase values ​​of the light-shielding sections D5 to D7 may be constant. Specifically, in the transmission sections E5 to E7, a phase distribution that monotonically increases from 0 (rad) to 2π (rad) is periodically repeated in each period. Due to this phase pattern, the direction of emission of light from the transmission sections E5 to E7 is tilted relative to the direction of emission of light from the light-shielding sections D5 to D7. The position of the aperture 293 is then slightly shifted relative to the beam waist in a direction intersecting the optical axis. Even in this configuration, the aperture 293 can shield at least a portion of the light emitted from the light-shielding sections D5 to D7 of the spatial light modulator 28, while allowing the light emitted from the transmission sections E5 to E7 of the spatial light modulator 28 to pass through.

[0103] As shown in this modified example, the spatial light modulator constituting the phase conversion unit 62 may be the same as the spatial light modulator constituting the ring mask 63. The common spatial light modulator 28 may present a phase pattern in which the phase pattern for constituting the phase conversion unit 62 and the phase pattern for constituting the ring mask 63 are superimposed. Similarly, in the image acquisition method of the second embodiment (see Figure 19), the spatial light modulator that performs the phase conversion process S22 may be the same as the spatial light modulator that performs the ring mask process S23. The spatial light modulator may present a phase pattern in which the phase pattern for performing the phase conversion process S22 and the phase pattern for performing the ring mask process S23 are superimposed. In this case, the components constituting the phase conversion unit 62 and the components constituting the ring mask 63 can be combined into one, simplifying the configuration of the device. Alternatively, the components that perform the phase conversion process S22 and the components that perform the ring mask process S23 can be combined into one, simplifying the configuration of the device.

[0104] The microscope apparatus and image acquisition method according to this disclosure are not limited to the embodiments described above, and various other modifications are possible. For example, the above embodiments described the case in which two-photon excitation is generated in the object to be observed. The microscope apparatus and image acquisition method according to this disclosure are effective in the case in which n-photon excitation (n is an integer of 2 or more) is generated in the object to be observed. That is, in the intensity modulation step S12 of the excitation light output step S1, the optical modulator 12 of the excitation light output unit 10 outputs excitation light La whose time waveform of light intensity includes the nth root of a linear function of a sine wave. Therefore, all descriptions of "two-photon excitation" and "square root" in the above embodiments can be replaced with "n-photon excitation" and "nth root".

[0105] In the above embodiment, the time waveform of the excitation light La is the square root of a linear sinusoidal function for the entire duration of each period. However, the embodiment is not limited to this form, and the time waveform of the excitation light La may be the square root of a linear sinusoidal function only for a portion of the duration of each period, typically the duration containing the maximum value Imax.

[0106] In the second embodiment, an example is given in which the polarization conversion unit 61, the phase conversion unit 62, and the ring mask 63 are arranged in the optical path between the excitation light output unit 10 and the optical scanner 22. At least one of the polarization conversion unit 61, the phase conversion unit 62, and the ring mask 63 may be arranged in the optical path between the optical scanner 22 and the objective lens 25. At least one of the polarization conversion unit 61, the phase conversion unit 62, and the ring mask 63 may be arranged in the optical path between the objective lens 25 and the object to be observed B.

[0107] In the second embodiment, the case in which the ring mask 63 is amplitude-modulated is illustrated. Even if the ring mask 63 is phase-modulated, or a hybrid of amplitude-modulated and phase-modulated, the focusing diameter can be reduced by using light that combines azimuth polarization and helical phase, as in the second embodiment.

[0108] While the principles of the present invention have been illustrated and described in preferred embodiments, it will be recognized by those skilled in the art that the present invention can be modified in arrangement and detail without departing from such principles. The present invention is not limited to the specific configurations disclosed in these embodiments. Accordingly, all modifications and changes arising from the scope of the claims and their spirit are claimed. [Explanation of symbols]

[0109] 1,2,2A…Microscope apparatus, 9…Mirror, 10…Excitation light output unit, 11…Light source, 12…Optical modulator, 20,20A…Optical system, 21…Beam expander, 22…Optical scanner, 23…Relay lens system, 24…Dichroic mirror, 25…Objective lens, 28…Spatial light modulator, 29…Aperture optical system, 30…Harmonic detection unit, 40…Image generation unit, 50…Signal generator, 211,212…Lenses, 221,222…Scanner, 223…Optical system, 31…Optical detection device, 32…Lock-in amplifier, 61…Polarization conversion unit, 62…Phase conversion unit, 63…Ring mask, S1…Excitation light output S12...Intensity modulation step, S2, S2a...Excitation light irradiation step, S3...Harmonic detection step, S6...Image generation step, S4, S5, S11, S31, S32...Steps, S21...Polarization conversion process, S22...Phase conversion process, S23...Ring mask process, S24...Focusing process, B...Object to be observed, J...Pulse, K...Envelope, G11~G13, G21~G23...Bar line, Ha, Hb...Focused spot shape, La...Excitation light, Lb...Fluorescence, Lp...Pulsed light, P11, P12, P21~P23...Plot, Q...Optical axis, Wa1...Diameter, Wa2...Width, Wb1, Wb2...Length.

Claims

1. A multiphoton saturation excitation microscope, An excitation light output unit outputs excitation light in which the time waveform of the light intensity includes the nth root of a linear sinusoidal function (where n is an integer greater than or equal to 2), and the maximum value of the light intensity exceeds the saturation excitation intensity at the object being observed. An optical system that irradiates an object to be observed with the excitation light output from the excitation light output unit, A harmonic detection unit that detects the second harmonic included in the time waveform of the fluorescence light intensity generated in the object under observation by n-photon excitation caused by irradiation with the excitation light, An image generation unit that generates an observation image of the object to be observed based on the second harmonic, A microscope device equipped with the following features.

2. The excitation light output unit is, A light source that emits pulsed light, An intensity-modulated optical modulator that modulates the pulsed light output from the light source to generate the excitation light, A microscope apparatus according to claim 1, having the following features.

3. The microscope apparatus according to claim 2, wherein the optical modulator is an AO modulator.

4. The microscope apparatus according to claim 2, wherein the light source is a laser light source.

5. The microscope apparatus according to any one of claims 1 to 4, wherein the minimum value within each period in the time waveform of the excitation light intensity is greater than 0.

6. The microscope apparatus according to claim 5, wherein the minimum value within each period of the time waveform of the excitation light intensity is greater than 0.1% and less than 20% of the maximum signal that the harmonic detection unit can receive.

7. The harmonic detection unit is, A photodetector that generates a signal corresponding to the light intensity of the fluorescence generated in the object being observed, A lock-in amplifier that receives the signal from the aforementioned photodetector and outputs the second harmonic included in the time waveform of the signal, A microscope apparatus according to any one of claims 1 to 4, having the following features.

8. The microscope apparatus according to any one of claims 1 to 4, wherein the harmonic detection unit further detects a third harmonic included in the time waveform of the fluorescence light intensity generated in the object being observed.

9. The time waveform of the light intensity of the excitation light output from the excitation light output unit includes the square root of a linear function of a sine wave. The microscope apparatus according to any one of claims 1 to 4, wherein the harmonic detection unit detects the second harmonic contained in the time waveform of the fluorescence light intensity generated in the object to be observed by two-photon excitation by irradiation with the excitation light.

10. A method for acquiring an image by multiphoton saturation excitation, An excitation light output step in which the time waveform of the light intensity includes the nth root of a linear sinusoidal function (where n is an integer greater than or equal to 2), and the maximum value of the light intensity exceeds the saturation excitation intensity at the object being observed, An excitation light irradiation step is performed in which the excitation light output in the excitation light output step is irradiated onto the object to be observed, A harmonic detection step involves detecting a second harmonic included in the time waveform of the fluorescence light intensity generated in the object under observation by n-photon excitation caused by irradiation with the excitation light, An image generation step of generating an observation image of the object to be observed based on the second harmonic, Image acquisition methods, including those mentioned above.

11. The excitation light output step is, The image acquisition method according to claim 10, comprising an intensity modulation step of modulating pulsed light to generate the excitation light.

12. The image acquisition method according to claim 11, wherein the pulsed light is laser light.

13. The image acquisition method according to any one of claims 10 to 12, wherein the minimum value within each period in the time waveform of the light intensity of the excitation light is greater than 0.

14. The image acquisition method according to claim 13, wherein the minimum value within each period of the time waveform of the light intensity of the excitation light is greater than 0.1% and less than 20% of the maximum signal that can be received in the harmonic detection step.

15. The harmonic detection step is as follows: The steps include generating a signal corresponding to the light intensity of the fluorescence generated in the object being observed, The steps include outputting a second harmonic included in the time waveform of the aforementioned signal, The image acquisition method according to any one of claims 10 to 12, including the method described in any one of claims 10 to 12.

16. In the harmonic detection step, the third harmonic included in the time waveform of the fluorescence light intensity generated in the object being observed is further detected. The image acquisition method according to any one of claims 10 to 12, wherein the image generation step generates an observation image of the object to be observed based on one or both of the second harmonic and the third harmonic.

17. In the excitation light output step, the time waveform of the light intensity of the excitation light output includes the square root of a linear function of a sine wave. The image acquisition method according to any one of claims 10 to 12, wherein in the harmonic detection step, the second harmonic is detected, which is included in the time waveform of the fluorescence light intensity generated in the object to be observed by two-photon excitation by irradiation with the excitation light.

Images