Microscope device and image acquisition method

Abstract

To provide a light irradiation device, a microscope device, a light irradiation method, and an image acquisition method with which, when using a ring mask, it is possible to raise a level of reduction of a focus spot diameter.SOLUTION: A light irradiation device 1 comprises: a light output unit 10 for outputting coherent light; and an optical system 20 for irradiating an object B with light La that is outputted from the light output unit 10. The optical system 20 includes an objective lens 27 for focusing the light La outputted from the light output unit 10 on to the object B, and a polarization conversion unit 22, a phase conversion unit 23 and a ring mask 24 provided on an optical path between the light output unit 10 and the object B. The polarization conversion unit 22 is constituted to convert the light La having been inputted to the polarization conversion unit 22 into, and output as, azimuth polarization. The phase conversion unit 23 is constituted to apply phase modulation by a spiral phase pattern to the light La having been inputted to the phase conversion unit 23.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a light irradiation device, a microscope device, a light irradiation method, and an image acquisition method. [Background technology]

[0002] Non-Patent Document 1 discloses a two-photon microscope. In this two-photon microscope, excitation light is generated by combining azimuthally polarized light with phase modulation using a spiral phase pattern. Non-Patent Document 2 discloses a configuration that combines azimuthally polarized light, phase modulation using a spiral phase pattern, and amplitude modulation. Non-Patent Document 3 discloses a phase-modulated multi-ring mask. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Xiangping Li et al., "Super-resolved pure-transverse focalfields with an enhanced energy density through focus of an azimuthallypolarized first-order vortex beam", Optics Letters, Volume 39, No. 20, pp.5961-5964, October 15, 2014 [Non-patent document 2] GH Yuan et al., "Generation of nondiffracting quasi-circular polarization beams using an amplitude modulated phase hologram", Journal of the Optical Society of America A, Volume 28, No. 8, pp. 1716-1720, August2011 [Non-patent document 3] M. Martinez-corral et al., "Tailoring the axial shape of the point spread function using the Toraldo concept", Optics Express, Volume 10, Issue 1, pp. 98-103, February 2002 Summary of the Invention [Problem to be solved by the invention]

[0004] When observing an object to be observed or processing a workpiece, light output from a light source such as a laser light source is focused and irradiated onto the surface or interior of the object (object to be observed or processed) through a condenser lens. When focusing light in this way, the beam waist diameter, which is a measure of the size of the focused light diameter, can only be made small enough to about half the wavelength of the light. This is called the diffraction limit.

[0005] Ring masks are used to reduce the focused diameter beyond the diffraction limit. For example, a single ring mask has a single ring-shaped light-shielding portion and transparent portions located inside and outside the light-shielding portion. Light passing through the transparent portions inside and outside the light-shielding portion passes through a focusing lens and reaches a focusing position. At the focusing position, these two beams interfere with each other, allowing the light to be focused into an area smaller than the diffraction limit. A multi-ring mask also has multiple ring-shaped light-shielding portions arranged concentrically and multiple transparent portions located between these multiple light-shielding portions. Light passing through each transparent portion passes through a focusing lens and reaches a focusing position. Even when using such a multi-ring mask, it is possible to focus light into a smaller area beyond the diffraction limit.

[0006] When using a ring mask, it is advisable to adjust the widths of the light-shielding and transmitting portions according to the numerical aperture of the objective lens in order to minimize the light-focus diameter. However, even if the widths of the light-shielding and transmitting portions are adjusted to minimize the light-focus diameter, there are cases in which the degree of reduction in the diameter is small (or almost no reduction at all). This phenomenon becomes more pronounced as the numerical aperture of the objective lens increases.

[0007] The present disclosure aims to provide a light irradiation device, a microscope device, a light irradiation method, and an image acquisition method that can increase the degree of reduction in the focused light diameter when a ring mask is used. [Means for solving the problem]

[0008] In order to solve the above-mentioned problems, the present disclosure provides a light irradiation device comprising a light output unit that outputs coherent light and an optical system that irradiates an object with the light output from the light output unit. The optical system includes an objective lens that focuses the light output from the light output unit on the object, and a polarization conversion unit, a phase conversion unit, and a ring mask that are provided on an optical path between the light output unit and the object. The polarization conversion unit is configured to convert light input to the polarization conversion unit into azimuthally polarized light and output the light. The phase conversion unit is configured to impart phase modulation using a spiral phase pattern to the light input to the phase conversion unit. According to the inventor's findings, the degree of reduction in the focused light diameter can be increased by providing a polarization conversion unit that converts light into azimuthally polarized light and a phase conversion unit that imparts phase modulation using a spiral phase pattern in addition to the ring mask.

[0009] The light irradiation method according to the present disclosure includes a light output step of outputting coherent light, and a light irradiation step of performing polarization conversion processing, phase conversion processing, and ring mask processing on the light output in the light output step, and focusing the light on an object. The polarization conversion processing is a process of converting the light output in the light output step into azimuthally polarized light. The phase conversion processing is a process of imparting phase modulation using a spiral phase pattern to the light output in the light output step. According to the inventor's findings, the degree of reduction in the focused light diameter can be increased by performing the polarization conversion processing of converting to azimuthally polarized light and the phase conversion processing of imparting phase modulation using a spiral phase pattern in addition to the ring mask processing.

[0010] In the light irradiation device and light irradiation method described above, the ring mask and ring mask processing may be amplitude modulation type. Ring masks include amplitude modulation type, phase modulation type, and a combination of these. Because the light utilization efficiency of the phase modulation type is higher than that of the amplitude modulation type, the phase modulation type allows for efficient light irradiation while reducing loss. However, when using a phase modulation type ring mask, unwanted light-focused portions (side lobes) tend to occur on both sides of the focused spot in the optical axis direction. For example, single-photon excitation fluorescence microscopes use confocal optical systems and can enhance resolution through the confocal effect of pinholes, so the occurrence of such unwanted light-focused portions is tolerable to a certain extent. However, for example, two-photon excitation fluorescence microscopes have lower excitation efficiency than single-photon excitation microscopes, and during deep observation, aberrations cause a large misalignment between the pinhole position and the focused position, resulting in significant light loss. Using an amplitude modulation type ring mask reduces unwanted light-focused portions (side lobes) compared to a phase modulation type, eliminating the need for a confocal optical system including a pinhole, contributing to the miniaturization of the device while reducing light loss.

[0011] In the above light irradiation device, the ratio (NA / R) of the refractive index R of the medium between the objective lens and the object to the numerical aperture NA of the objective lens may be 0.75 or more. Similarly, in the light irradiation step of the above light irradiation method, light may be condensed using an objective lens in which the ratio (NA / R) of the refractive index R of the medium between the objective lens and the object to the numerical aperture NA of the objective lens is 0.75 or more. According to the above light irradiation device and light irradiation method, when the numerical aperture of the objective lens is large, the degree of reduction in the condensed light diameter can be further increased.

[0012] In the above-mentioned light irradiation device, at least one of the phase conversion unit and the ring mask may be configured by a phase modulation type spatial light modulator. Similarly, in the above-mentioned light irradiation method, at least one of the phase conversion process and the ring mask process may be performed using a phase modulation type spatial light modulator. In this case, it is possible to easily change the phase pattern in the phase conversion unit (phase conversion process) and / or change the widths of the light-shielding and light-transmitting parts in the ring mask (ring mask process).

[0013] In the above-mentioned light irradiation device, the spatial light modulator constituting the phase conversion unit may be common to the spatial light modulator constituting the ring mask, and the spatial light modulator may present a phase pattern in which a phase pattern for constituting the phase conversion unit and a phase pattern for constituting the ring mask are superimposed. Similarly, in the above-mentioned light irradiation method, the spatial light modulator performing the phase conversion process may be common to the spatial light modulator performing the ring mask process, and the spatial light modulator may present a phase pattern in which a phase pattern for constituting the phase conversion process and a phase pattern for constituting the ring mask are superimposed. In this case, the components constituting the phase conversion unit and the components constituting the ring mask (or the components performing the phase conversion process and the components performing the ring mask process) can be combined into one, thereby simplifying the configuration of the device.

[0014] In the light irradiation device, the ring mask may have a plurality of ring-shaped light-shielding portions arranged around a central position, a transmitting portion provided between two adjacent light-shielding portions of the plurality of light-shielding portions, an innermost transmitting portion provided inside the innermost light-shielding portion of the plurality of light-shielding portions, and an outermost transmitting portion provided outside the outermost light-shielding portion of the plurality of light-shielding portions. By using such a multiple ring mask, it is possible to further reduce the diameter of the focused light.

[0015] Furthermore, a microscope apparatus according to the present disclosure includes any one of the above light irradiation devices, a detection unit that detects light generated in an object by irradiation with light output from the light output unit, and an image generation unit that generates an observation image of the object based on the detection result of the detection unit. An image acquisition method according to the present disclosure includes any one of the above light irradiation methods, a detection step that detects light generated in the object by irradiation with light in the light irradiation step, and an image generation step that generates an observation image of the object based on the detection result in the detection step. These microscope apparatuses and image acquisition methods include any one of the above light irradiation devices or include any one of the above light irradiation methods, thereby making it possible to increase the degree to which the light collection diameter is reduced. Therefore, the resolution of the observation image can be increased.

[0016] In the above microscope device, the detection unit may detect fluorescence generated in the object by multiphoton excitation caused by irradiation with light output from the light output unit. Similarly, in the detection step of the image acquisition method, the fluorescence generated in the object by multiphoton excitation caused by irradiation with light in the light irradiation step may be detected. In such a multiphoton excitation type microscope device, the resolution of the observed image can also be improved.

[0017] In the above microscope device, the light output unit may output light whose time waveform of light intensity includes an nth root (n is an integer of 2 or more) of a linear function of a sine wave and whose maximum light intensity exceeds the saturation excitation intensity in the object, and the detection unit may detect a second harmonic contained in the time waveform of the light intensity of fluorescence generated in the object by n-photon excitation due to irradiation with light output from the light output unit. Similarly, in the light output step of the above image acquisition method, light whose time waveform of light intensity includes an nth root (n is an integer of 2 or more) of a linear function of a sine wave and whose maximum light intensity exceeds the saturation excitation intensity in the object may be output, and the detection step may detect a second harmonic contained in the time waveform of the light intensity of fluorescence generated in the object by n-photon excitation due to irradiation with light in the light irradiation step.

[0018] In these microscope devices and image acquisition methods, the time waveform of the light intensity of the light (excitation light) output from the light output unit (light output step) includes the nth root of a linear function of a sine wave. In n-photon excitation, the fluorescence intensity is proportional to the nth power of the excitation light intensity. Therefore, when excitation light having a time waveform including the nth root of a linear function of a sine wave is irradiated onto an object to cause n-photon excitation in the object, the time waveform of the fluorescence output from the object is proportional to a linear function of a sine wave. Therefore, similar to a typical saturated excitation (SAX) microscope, an observation image can be obtained based on a lower harmonic, such as the second or third harmonic. Therefore, with these microscope devices 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, thereby avoiding the long time required to create an observation image. [Effects of the Invention]

[0019] According to the present disclosure, it is possible to provide a light irradiation device, a microscope device, a light irradiation method, and an image acquisition method that can increase the degree of reduction in the focused light diameter when a ring mask is used. [Brief explanation of the drawings]

[0020] [Figure 1]1 is a diagram showing an outline of the configuration of a light irradiation device according to a first embodiment. [Figure 2] FIG. 2 is a diagram showing the polarization direction of azimuthally polarized light in a plane perpendicular to the optical axis. [Figure 3] FIG. 10 is a diagram showing a focused image of azimuthally polarized light. [Figure 4] FIG. 10 is a diagram showing a spiral phase pattern. [Figure 5] FIG. 10 is a diagram showing a focused image when phase modulation by a spiral phase pattern is applied to azimuthally polarized light. [Figure 6] FIG. 2 is a diagram showing an example of the configuration of a ring mask as viewed from the optical axis direction. [Figure 7] 10(a) to 10(e) are diagrams showing examples of the arrangement order of a polarization conversion unit, a phase conversion unit, and a ring mask. [Figure 8] FIG. 1 illustrates an example of an optical scanner. [Figure 9] 10 is a flowchart for explaining the operation of the microscope device. [Figure 10] 10A and 10B are diagrams showing the shape of a focused spot on a cross section that includes the optical axis and is parallel to the optical axis. [Figure 11] 10 is a graph showing the lateral resolution ratio for each aperture ratio when a ring mask is not used. [Figure 12] 10 is a graph showing a lateral resolution ratio for each aperture ratio when a ring mask is used. [Figure 13] FIG. 10 is a diagram showing a condensed image in a cross section that includes the optical axis and is parallel to the optical axis direction. [Figure 14] FIG. 10 is a diagram showing an outline of the configuration of a light irradiation device according to a first modified example. [Figure 15] 10A and 10B are diagrams illustrating examples of phase patterns constituting an amplitude modulation type ring mask. [Figure 16] FIG. 10 is a diagram showing another example of a phase pattern constituting an amplitude modulation type ring mask. [Figure 17] FIG. 10 is a diagram illustrating an outline of the configuration of a microscope apparatus according to a second embodiment. [Figure 18] 10 is a flowchart for explaining the operation of the microscope device. [Figure 19] FIG. 10 is a diagram showing an outline of the configuration of a microscope apparatus according to a second modified example. [Figure 20] FIG. 10 is a diagram illustrating an outline of the configuration of a microscope apparatus according to a third embodiment. [Figure 21] FIG. 2 is a diagram showing the time waveform of excitation light. [Figure 22] 10 is a flowchart for explaining the operation of the microscope device. [Figure 23] 1A is a diagram conceptually showing the intensity distribution of fluorescence in a SAX microscope, and FIG. 1B is a diagram conceptually showing the difference between the theoretical and measured values ​​of fluorescence intensity. [Figure 24] 1A is a diagram conceptually showing the time waveform of excitation light intensity, and FIG. 1B is a diagram conceptually showing the time waveform of fluorescence intensity. [Figure 25] 1A is a diagram conceptually showing the time waveform of excitation light intensity, and FIG. 1B is a diagram conceptually showing the time waveform of fluorescence intensity. [Figure 26] 10A is a graph showing the result of Fourier transform of the time waveform of the light intensity of the fluorescence measured in the microscope device of the third embodiment, and FIG. 10B is a graph showing the result of Fourier transform of the time waveform of the fluorescence intensity measured when the time waveform of the excitation light intensity is a sine wave. [Figure 27] 10A is a graph showing the relationship between the light intensity of the fundamental wave and second harmonic of the fluorescence measured in the microscope apparatus of the third embodiment and the relative intensity of the excitation light, and FIG. 10B is a graph showing the relationship between the light intensity of the fundamental wave, second harmonic, and third harmonic of the fluorescence measured in the case where the time waveform of the excitation light intensity is a sine wave, and the relative intensity of the excitation light. [Figure 28] FIG. 10 is a diagram showing a fluorescent image in which the fundamental wave is detected in a cross section that includes the optical axis and is parallel to the optical axis direction. [Figure 29] FIG. 10 is a diagram showing a fluorescent image obtained by detecting second harmonics in a cross section that includes the optical axis and is parallel to the optical axis direction. [Figure 30] FIG. 10 is a diagram showing a fluorescent image obtained by detecting the third harmonic in a cross section that includes the optical axis and is parallel to the optical axis direction. [Figure 31] FIG. 2 is a diagram conceptually showing the time waveform of excitation light. [Figure 32]1 is a graph conceptually showing the relationship between the applied voltage and the output light intensity of a typical AO modulator. [Figure 33] FIG. 10 is a diagram showing an outline of the configuration of a light irradiation device according to a fourth modified example. DETAILED DESCRIPTION OF THE INVENTION

[0021] Hereinafter, embodiments of a light irradiation device, a microscope device, a light irradiation method, and an image acquisition method according to the present disclosure will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are designated by the same reference numerals, and duplicated explanations will be omitted. [First embodiment]

[0022] 1 is a diagram showing an outline of the configuration of a light irradiation device 1 according to an embodiment of the present disclosure. The light irradiation device 1 is used, for example, in an optical microscope to irradiate an object to be observed with light. Alternatively, the light irradiation device 1 is used, for example, in a laser processing device to irradiate an object to be processed with laser light. The light irradiation device 1 includes a light output unit 10 and an optical system 20.

[0023] The light output unit 10 outputs coherent light La. The light output unit 10 is, for example, a pulsed light source, and outputs pulsed light La. In this case, the pulse width of the light La is, for example, on the order of picoseconds or femtoseconds. Here, the pulse width of the light La is, for example, the time during which the optical intensity of the light La exceeds half of the peak value of the pulse. Specifically, the pulse width of the light La is, for example, within a range of 10 femtoseconds to 50 picoseconds. Furthermore, the light output unit 10 is, for example, a laser light source, and in one example, a mode-locked laser light source. The wavelength of the light La is, for example, within the near-infrared range. Specifically, the wavelength of the light La is, for example, within a range of 650 nm to 1800 nm.

[0024] The optical system 20 is an optical system for irradiating the light La output from the light output unit 10 onto an object B, which is an object to be observed or processed. The optical system 20 includes an optical path for the light La from the light output unit 10 to reach the object B. The optical system 20 has a beam expander 21, a polarization conversion unit 22, a phase conversion unit 23, a ring mask 24, an optical scanner 25, a relay lens system 26, and an objective lens 27. The beam expander 21 and the optical scanner 25 are provided on the optical path between the light output unit 10 and the objective lens 27. In the illustrated example, the polarization conversion unit 22, the phase conversion unit 23, and the ring mask 24 are provided on the optical path between the beam expander 21 and the optical scanner 25.

[0025] The beam expander 21 is optically coupled to the light output unit 10 via a space, and expands the beam diameter of the light La output from the light output unit 10. The beam expander 21 includes, for example, a pair of lenses 211 and 212 that are optically coupled to each other. One lens 211 is provided in the front stage (on the light output unit 10 side), and the other lens 212 is provided in the rear stage (on the opposite side from the light output unit 10). The front lens 211 diffuses the light La, and the rear lens 212 collimates the light La. The lenses 211 and 212 are, for example, glass lenses.

[0026] The polarization conversion unit 22 is optically coupled to the optical output unit 10 via the beam expander 21. The polarization conversion unit 22 receives light La, converts it into azimuthally polarized light, and outputs it. The polarization state of the light La before it is input to the polarization conversion unit 22 is, for example, linear polarization. FIG. 2 is a diagram showing the polarization direction of azimuthally polarized light in a plane perpendicular to the optical axis. In FIG. 2, arrow A indicates the polarization direction. FIG. 3 is a diagram showing a focused image of azimuthally polarized light. In FIG. 3, the light intensity is indicated by shades of color, with darker areas indicating lower light intensity and lighter areas indicating higher light intensity. In FIG. 3, (a) to (d) show the light intensity distribution in a plane perpendicular to the optical axis, and (e) to (h) show the light intensity distribution in a plane that includes the optical axis and is parallel to the optical axis. In FIG. 3, (b) and (f) show the light intensity (|Ex|) due to the vibration component Ex of the electric field in the X direction (direction perpendicular to the optical axis). 2), and (c) and (g) show the light intensity (|Ey| 2 ), (d) and (h) show the light intensity (|Ez| 2 ), and (a) and (e) show the light intensity (|Ex| 2 +|Ey| 2 +|Ez| 2 2 and 3, in azimuthally polarized light, the vibration direction of the electric field is along the tangential direction of the circumference centered on the optical axis, and the light intensity decreases near the optical axis. Therefore, the light intensity distribution of azimuthally polarized light in a plane perpendicular to the optical axis is annular. The polarization conversion unit 22 converts the light La input from the light output unit 10 into such azimuthally polarized light and outputs it. The polarization conversion unit 22 can be configured, for example, by an azimuth polarizer (a fixed type made by processing a glass plate or a variable type using liquid crystal) or two spatial light modulators.

[0027] The phase converter 23 is optically coupled to the optical output unit 10 via the beam expander 21 and the polarization converter 22. The phase converter 23 inputs the light La and modulates the light La using a spiral phase pattern. FIG. 4 is a diagram illustrating the spiral phase pattern. In FIG. 4, the magnitude of the phase is indicated by the shade of color, with the lighter areas indicating a smaller phase and the darker areas indicating a larger phase. As shown in FIG. 4, in the spiral phase pattern, the phase changes monotonically depending on the angle around the optical axis Q. In one example, the phase in this spiral phase pattern changes from 0 (rad) to 2π (rad). That is, the range of phase change per revolution is 2π (rad). The phase converter 23 can be configured, for example, by a spiral phase plate (e.g., a glass plate processed for phase modulation) or a phase-modulating spatial light modulator.

[0028] Fig. 5 shows a focused image of light that has been phase-modulated by a spiral phase pattern and azimuthally polarized. In Fig. 5, the light intensity is indicated by shades of color, with darker areas indicating lower light intensity and lighter areas indicating higher light intensity. In Fig. 5, (a) to (d) show the light intensity distribution in a plane perpendicular to the optical axis, and (e) to (h) show the light intensity distribution in a plane along the optical axis. In Fig. 5, (b) and (f) show the light intensity (|Ex|) due to the vibration component Ex of the electric field in the X direction (direction perpendicular to the optical axis). 2 ), and (c) and (g) show the light intensity (|Ey| 2 ), (d) and (h) show the light intensity (|Ez| 2 ), and (a) and (e) show the light intensity (|Ex| 2 +|Ey| 2 +|Ez| 2 ) is shown. As shown in Fig. 5, when a spiral phase pattern is combined with azimuthally polarized light, 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 circle. Light La comes to have such a solid circular light intensity distribution by passing through the polarization conversion unit 22 and the phase conversion unit 23.

[0029] The ring mask 24 is optically coupled to the light output unit 10 via the beam expander 21, the polarization conversion unit 22, and the phase conversion unit 23. The ring mask 24 receives light La, spatially intensity-modulates the light La in the beam cross section of the light La, and outputs the modulated light La. The ring mask 24 has a ring-shaped light-shielding portion and transparent portions provided adjacent to the inside and outside of the light-shielding portion. The ring mask 24 of this embodiment is a so-called multi-ring mask. The ring mask 24 may be formed, for example, by a plate-like member having a light-shielding portion and a transparent portion formed thereon, or a phase-modulating spatial light modulator. The plate-like member may be formed, for example, by forming a light-shielding film as the light-shielding portion on a light-transmitting plate material. Ring masks include amplitude modulation (intensity modulation), phase modulation, and a combination thereof. The ring mask 24 of this embodiment is an amplitude modulation type.

[0030] 6 is a diagram showing an example of the configuration of ring mask 24 as viewed from the optical axis direction. This ring mask 24 has multiple (three in the illustrated example) ring-shaped light-shielding portions D1, D2, and D3 arranged around a central position. Furthermore, ring mask 24 has an innermost transmitting portion E1 arranged inside light-shielding portion D1, a ring-shaped transmitting portion E2 arranged between light-shielding portion D1 and light-shielding portion D2, a ring-shaped transmitting portion E3 arranged between light-shielding portion D2 and light-shielding portion D3, a ring-shaped outermost transmitting portion E4 arranged outside light-shielding portion D3, and a light-shielding portion D4 arranged outside light-shielding portion E4.

[0031] The light transmittance of the transmissive portions E1 to E4 is greater than the light transmittance of the light-shielding portions D1 to D4. The light transmittance of the transmissive portions E1 to E4 may be 1 or less than 1. The light transmittance of the light-shielding portions D1 to D4 may be 0 or greater than 0. The boundary between adjacent transmissive and light-shielding portions among the transmissive portion E1, light-shielding portion D1, transmissive portion E2, light-shielding portion D2, transmissive portion E3, light-shielding portion D3, transmissive portion E4, and light-shielding portion D4 may be concentric circles or ellipses. In the following description, the boundary is assumed to be a circle. The radial width (radius) of the transmitting portion E1 is e1, the radial width of the shading portion D1 is d1, the radial width of the transmitting portion E2 is e2, the radial width of the shading portion D2 is d2, the radial width of the transmitting portion E3 is e3, the radial width of the shading portion D3 is d3, and the radial width of the transmitting portion E4 is e4.

[0032] The radial width of each of two adjacent light-shielding portions among the ring-shaped light-shielding portions D1, D2, and D3 is greater than the radial width of the transmitting portion provided between these two light-shielding portions. That is, in the ring mask 24 of this embodiment, the radial widths of the light-shielding portions D1 and D2 and the transmitting portion E2 have a relationship expressed by the following formula (1), and the radial widths of the light-shielding portions D2 and D3 and the transmitting portion E3 have a relationship expressed by the following formula (2). It is preferable that such a relationship be satisfied for all combinations of two adjacent ring-shaped light-shielding portions.

number

number

[0033] In this embodiment, the arrangement order of the polarization conversion section 22, the phase conversion section 23, and the ring mask 24 is not limited to the example shown in Fig. 1, and can be changed in various ways. That is, they may be arranged in the order shown in any of Figs. 7(a) to 7(e) when viewed from the light output section 10 side. FIG. 7(a): When viewed from the optical output unit 10 side, the polarization conversion unit 22, the ring mask 24, and the phase conversion unit 23 are arranged in this order. FIG. 7(b): When viewed from the optical output unit 10 side, the phase conversion unit 23, the polarization conversion unit 22, and the ring mask 24 are arranged in this order. FIG. 7(c): When viewed from the optical output unit 10 side, the phase conversion unit 23, the ring mask 24, and the polarization conversion unit 22 are arranged in this order. FIG. 7(d): When viewed from the optical output section 10 side, the ring mask 24, the polarization conversion section 22, and the phase conversion section 23 are arranged in this order. FIG. 7(e): When viewed from the optical output unit 10 side, the ring mask 24, the phase conversion unit 23, and the polarization conversion unit 22 are arranged in this order.

[0034] The optical scanner 25 is optically coupled to the beam expander 21 via the polarization conversion unit 22, the phase conversion unit 23, and the ring mask 24. The optical scanner 25 scans the irradiation position of the light La on the object B by moving the optical axis of the light La in a plane perpendicular to the optical axis of the light La. The optical scanner 25 can be configured with various optical scanners such as a galvanometer scanner, a resonant mirror, or a polygon mirror. In one example, the optical scanner 25 is a two-axis galvanometer scanner.

[0035] FIG. 8 is a diagram showing another example of the optical scanner 25. The optical scanner 25 shown in FIG. 8 has two scanners 251 and 252. Both scanners 251 and 252 are uniaxial scanners. The scanning direction of scanner 251 and the scanning direction of scanner 252 are orthogonal to each other. Scanner 251 and scanner 252 are optically coupled to each other via an optical system 253 such as a relay lens. Scanners 251 and 252 are, for example, uniaxial galvanometer scanners. In this manner, the optical scanner 25 may be configured by combining uniaxial scanners. Note that the irradiation position of light La on object B may be scanned by moving a stage on which object B is placed in a plane perpendicular to the optical axis of light La. In this case, the optical scanner 25 may not be provided. Furthermore, when the optical scanner 25 is provided, it is preferable that the polarization conversion unit 22, the phase conversion unit 23, and the ring mask 24 be provided upstream of the optical scanner 25.

[0036] The relay lens system 26 is provided on the optical path between the optical scanner 25 and the objective lens 27, and optically couples the optical scanner 25 and the objective lens 27. The relay lens system 26 is, for example, a telecentric relay lens system. Note that if the optical scanner 25 and the objective lens 27 are extremely close to each other, the relay lens system 26 may be omitted. Furthermore, a relay lens system similar to the relay lens system 26 may be provided in at least one of the following locations: between the beam expander 21, the polarization conversion unit 22, and the phase conversion unit 23; between the beam expander 21, the polarization conversion unit 22, and the phase conversion unit 23 and the ring mask 24; and between the ring mask 24 and the optical scanner 25.

[0037] The objective lens 27 is disposed opposite the object B and focuses the light La onto the surface or interior of the object B. The objective lens 27 is, for example, a dry objective lens, a water immersion objective lens, an oil immersion objective lens, or a silicone immersion objective lens. An objective lens used for observing a transparentized sample may also be used. If the objective lens 27 is a water immersion objective lens, its numerical aperture is, for example, 1.2 or greater. If the objective lens 27 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 of the medium between the objective lens 27 and the object B to the numerical aperture NA of the objective lens 27 is, for example, 0.75 or greater. The relative distance between the objective lens 27 and the object B is variable. The objective lens 27 may be movable along the optical axis direction of the light La, or a stage (not shown) on which the object B is placed may be movable along the optical axis direction of the light La. The mechanism for moving the objective lens 27 or the object B may be configured by, for example, a stepping motor or a piezoelectric actuator. The position of the objective lens 27 relative to the object B may be either an upright type or an inverted type.

[0038] 9 is a flowchart for explaining the operation of the light irradiation device 1 according to this embodiment. With reference to FIG. 9, the operation of the light irradiation device 1 and the light irradiation method according to this embodiment will be described.

[0039] First, a light output step S1 is performed. In the light output step S1, the light output unit 10 outputs coherent light La. As described above, the light La is, for example, laser light, or pulsed light. The pulse width of the light La is, for example, on the order of picoseconds or femtoseconds.

[0040] Next, a light irradiation step S2 is performed. In the light irradiation step S2, the light La output in the light output step S1 is irradiated onto the object B via the beam expander 21, the polarization conversion unit 22, the phase conversion unit 23, the ring mask 24, the optical scanner 25, the relay lens system 26, and the objective lens 27. That is, in the light irradiation step S2, the light La output in the light output step S1 is subjected to a polarization conversion process S21 by the polarization conversion unit 22, a phase conversion process S22 by the phase conversion unit 23, and a ring mask process S23 by the ring mask 24, and a focusing process S24 is performed to focus the light La on the object B using the objective lens 27. The polarization conversion process S21 is a process of converting the light La into azimuthally polarized light. The phase conversion process S22 is a process of applying phase modulation to the light La using a spiral phase pattern. In the focusing process S24 of the light irradiation step S2, the light La may be focused using a water immersion objective lens having a numerical aperture of 1.2 or more. Alternatively, the light La may be focused using an oil immersion objective lens having a numerical aperture of 1.45 or more. Alternatively, the light La may be focused using an objective lens 27 having a ratio (NA / R) of 0.75 or more between the refractive index R of the medium between the objective lens 27 and the object B and the numerical aperture NA of the objective lens 27. The phase conversion process S22 and the ring mask process S23 may each be performed using a phase modulation type spatial light modulator.

[0041] The above-described light output step S1 and light irradiation step S2 are repeatedly performed while scanning the irradiation position of the light La on the object B with the optical scanner 25 (steps S3 and S4). This allows the light La to be continuously irradiated onto a plurality of positions on the object B.

[0042] The effects of the light irradiation device 1 and the light irradiation method of the present embodiment described above will be described together with the problems of the comparative example.

[0043] Figures 10(a) and 10(b) are diagrams showing the shape of the focused spot on 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. Figure 10(a) assumes the use of a water immersion objective lens with a numerical aperture of 0.9, and Figure 10(b) assumes the use of a water immersion objective lens with a numerical aperture of 1.3. As shown in Figure 10, the size of the focused spot can be reduced by providing a ring mask.

[0044] Tables 1, 2, and 3 below show examples 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 a light irradiation device according to a comparative example in which a multiple ring mask is applied to circularly polarized light. The volume improvement rate shown in these tables is the value obtained by dividing the volume of the focused spot when a ring mask is not provided by the volume of the focused spot when a ring mask is provided. The horizontal improvement rate is the value obtained by dividing the focused spot diameter in the direction perpendicular to the optical axis when a ring mask is not provided (diameter Wa1 shown in FIG. 10) by the focused spot diameter in the direction perpendicular to the optical axis when a ring mask is provided (width Wa2 shown in FIG. 10). The vertical improvement rate is the value obtained by dividing the focused spot length in the optical axis direction when a ring mask is not provided (length Wb1 shown in FIG. 10) by the focused spot length in the optical axis direction when a ring mask is provided (length Wb2 shown in FIG. 10). The diameter and 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 multiple ring mask with four light-shielding portions, and the ring mask configuration (widths d1 to d3 of the light-shielding portions D1 to D3 and widths e1 to e4 of the transparent portions E1 to E4) that minimizes the volume of the focused spot under the condition that the light intensity of the side lobes is 2.5% or less of the light intensity of the focused spot (main lobe) was searched for. Table 1 assumes that the objective lens is a water-immersion objective lens. Table 2 assumes that the objective lens is an oil-immersion objective lens. Table 3 assumes that the objective lens is a dry objective lens. [Table 1] [Table 2] [Table 3]

[0045] As shown in Tables 1 and 2, regardless of the type and numerical aperture of the objective lens, by providing a ring mask, the volumetric improvement rate, lateral improvement rate, and vertical improvement rate all exceed 1. However, the lateral improvement rate is smaller than the vertical improvement rate. In particular, when using an objective lens with a large numerical aperture, such as a water immersion objective lens with a numerical aperture of 1.05 or more, an oil immersion objective lens with a numerical aperture of 1.25 or more, or a dry objective lens with a numerical aperture of 0.8 or more, the lateral improvement rate becomes 1.08 or less, and this tendency is remarkable. If the refractive index of the medium (immersion liquid) between the water immersion objective and object B is 1.333, the refractive index of the medium (immersion liquid) between the oil immersion objective and object B is 1.518, and the refractive index of the medium (air) between the dry objective and object B is 1, the ratio (NA / R) of the numerical aperture NA at which the lateral improvement rate begins to drop below 1.08 to the refractive index R is 0.75 or greater for all of the water immersion objective, oil immersion objective, and dry objective. If the lateral improvement rate is small, the degree of reduction in the focused spot diameter will be small, and the degree of improvement in resolution in a microscope, for example, will also be small.

[0046] In response to the above problem, the inventors have discovered that the degree to which the focused light diameter can be reduced can be increased by providing, in addition to the ring mask 24, a polarization conversion section 22 that converts to azimuth polarization and a phase conversion section 23 that imparts phase modulation using a spiral phase pattern (or by performing a polarization conversion process S21 that converts to azimuth polarization and a phase conversion process S22 that imparts phase modulation using a spiral phase pattern).

[0047] FIG. 11 is a graph showing the lateral resolution ratio for each numerical aperture when a ring mask is not used. In FIG. 11, bars G11, G12, and G13 represent the cases where the light irradiated onto the object is circularly polarized, radially polarized, and a combination of azimuthally polarized light and spiral phase modulation, respectively. The vertical axis represents the lateral resolution ratio (the value obtained by dividing the lateral resolution for an oil immersion objective lens with a numerical aperture of 1.50 and circularly polarized light by the lateral resolution for each numerical aperture and each polarization). The horizontal axis represents the numerical aperture. Referring to FIG. 11, it can be seen that even without a ring mask, the lateral resolution ratio is improved at all numerical apertures when the light irradiated onto the object is a combination of azimuthally polarized light and spiral phase modulation, compared to when the light irradiated onto the object is circularly polarized or radially polarized.

[0048] FIG. 12 is a graph showing the lateral resolution ratio for each numerical aperture when a ring mask is used. In FIG. 12, bars G21, G22, and G23 represent the cases where the light irradiated onto the object is circularly polarized, radially polarized, and a combination of azimuthally polarized light and spiral phase modulation, respectively. The vertical axis represents the lateral resolution ratio (the value obtained by dividing the lateral resolution with an oil immersion objective lens numerical aperture of 1.50 and circularly polarized light without a ring mask by the lateral resolution with each numerical aperture and each polarization when a ring mask is used). The horizontal axis represents the numerical aperture. Referring to FIG. 12, it can be seen that when a ring mask is used, the lateral resolution ratio is improved regardless of the polarization state of the light irradiated onto the object, but the lateral resolution ratio is significantly improved when the light irradiated onto the object is a combination of azimuthally polarized light and spiral phase modulation.

[0049] In this way, by irradiating the target with light that combines azimuthally polarized light and spiral phase modulation in addition to the ring mask, the lateral resolution ratio (lateral improvement rate) is significantly improved. Therefore, according to this embodiment, the degree of reduction in the focused diameter can be increased.

[0050] FIG. 13 is a diagram showing a condensed image in a cross section that includes the optical axis and is parallel to the optical axis direction. In FIG. 13, light intensity is indicated by shades of color, with darker areas indicating lower light intensity and lighter areas indicating higher light intensity. In FIG. 13, (a) and (b) show condensed images when the light La irradiated onto the object B is azimuthally polarized light with spiral phase modulation, and (c) and (d) show condensed images when the light La irradiated onto the object B is circularly polarized light as a comparative example. Also, in FIG. 13, (a) and (c) show condensed images when the ring mask 24 is provided, and (b) and (d) show condensed images when the ring mask 24 is not provided.

[0051] 13(a) and 13(c) with (b) and (d), it can be seen that when the ring mask 24 is provided, the diameter and length of the focused spot are smaller than when the ring mask 24 is not provided. Also, when the light La irradiated onto the object B is azimuthally polarized light with spiral phase modulation, it can be seen that the diameter and length of the focused spot are smaller than when the light La irradiated onto the object B is circularly polarized light.

[0052] As mentioned above, the ring mask 24 and the ring mask process S23 may be amplitude modulation type. Ring masks include amplitude modulation type, phase modulation type, and a combination of these. Because the light utilization efficiency of the phase modulation type is higher than that of the amplitude modulation type, the phase modulation type allows for efficient light irradiation while reducing loss. However, when using a phase modulation type ring mask, unwanted light-focused portions (side lobes) tend to occur on both sides of the focused spot in the optical axis direction. For example, Non-Patent Document 3 discloses a method for reducing the main lobe by causing the side lobes to interfere with the main lobe. For example, single-photon excitation fluorescence microscopes use confocal optical systems and can enhance resolution through the confocal effect of a pinhole, so the occurrence of such unwanted light-focused portions is tolerable to a certain extent. However, for example, two-photon excitation fluorescence microscopes have lower excitation efficiency than single-photon excitation microscopes, and during deep observation, a large misalignment between the pinhole position and the focused position occurs due to the influence of aberrations, resulting in significant light loss. By using an amplitude modulation type ring mask, unnecessary light-condensing portions (side lobes) are reduced compared to a phase modulation type, so there is no need to install a confocal optical system including a pinhole, which contributes to miniaturizing the device while suppressing light loss.

[0053] The ratio (NA / R) of the refractive index R of the medium between the objective lens 27 and the object B to the numerical aperture NA of the objective lens 27 may be 0.75 or more. Similarly, in the light focusing process S24 of the light irradiation step S2, the light La may be focused using an objective lens 27 having a ratio (NA / R) of the refractive index R of the medium between the objective lens 27 and the object B to the numerical aperture NA of the objective lens 27 of 0.75 or more. According to the light irradiation device 1 and light irradiation method of this embodiment, when the numerical aperture of the objective lens is large, the degree of reduction in the focused light diameter can be further increased. Note that the objective lens may be a water immersion objective lens, an oil immersion objective lens, a dry objective lens, a silicone immersion objective lens, or an objective lens compatible with a clearing solution.

[0054] As described above, the phase conversion unit 23 and the ring mask 24 may each be configured with a phase modulation type spatial light modulator. Similarly, the phase conversion process S22 and the ring mask process S23 may each be performed using a phase modulation type spatial light modulator. In this case, it is easy to change the phase pattern in the phase conversion unit 23 (phase conversion process S22) and / or change the widths d1 to d3 of the light-shielding portions D1 to D3 and the widths e1 to e4 of the light-transmitting portions E1 to E4 in the ring mask 24 (ring mask process S23).

[0055] As described above, ring mask 24 may have a plurality of ring-shaped light-shielding portions D1-D3 arranged around the central position, transmitting portions E2 and E3 arranged between two adjacent light-shielding portions of the plurality of light-shielding portions D1-D3, an innermost transmitting portion E1 arranged inside light-shielding portion D1 of the plurality of light-shielding portions D1-D3, and an outermost transmitting portion E4 arranged outside light-shielding portion D3 of the plurality of light-shielding portions D1-D3. Use of such a multiple ring mask can further reduce the diameter of the focused light. [First Modification]

[0056] 14 is a diagram showing an outline of the configuration of a light irradiation device 1A according to a modified example of the first embodiment. The light irradiation device 1A differs from the light irradiation device 1 of the first embodiment in that it includes a phase-modulating spatial light modulator 28 and an aperture optical system 29 instead of the phase conversion unit 23 and ring mask 24 of the light irradiation device 1 described above. The other configuration of the light irradiation device 1A is the same as that of the light irradiation device 1 of the first embodiment. Note that, depending on the relative arrangement of the light output unit 10 and the spatial light modulator 28, the light output unit 10 and the spatial light modulator 28 may be optically coupled by an optical system such as a mirror 9.

[0057] The spatial light modulator 28 functions as both the phase conversion unit 23 and the ring mask 24. In other words, in this modification, the spatial light modulator constituting the phase conversion unit 23 is the same as the spatial light modulator constituting the ring mask 24. The spatial light modulator 28 presents a phase pattern in which a phase pattern for constituting the phase conversion unit 23 and a phase pattern for constituting the ring mask 24 are superimposed. A phase pattern for correcting aberrations may be further superimposed on the phase pattern. In particular, aberrations inherent in the spatial light modulator 28 itself affect the accuracy of the spiral phase modulation, so it is preferable to correct them. Furthermore, aberrations occurring during deep observation, such as spherical aberrations resulting from the refractive index difference between the object B and the immersion liquid, may be simultaneously corrected by the spatial light modulator 28. The spatial light modulator 28 constructs the amplitude modulation type ring mask 24 using the phase pattern. FIG. 15 shows an example of a phase pattern constituting the amplitude modulation type ring mask 24. In FIG. 15, the phase value of each pixel constituting the phase pattern is indicated by the shade of color, with the lighter the color, the smaller the phase value, and the darker the color, the larger the phase value. In this example, the ring mask 24 is a triple ring mask. That is, the ring mask 24 has multiple (two in the illustrated example) ring-shaped light-shielding portions D5 and D6 arranged around the center position. Furthermore, the ring mask 24 has an innermost transmitting portion E5 arranged inside the light-shielding portion D5, a ring-shaped transmitting portion E6 arranged between the light-shielding portions D5 and D6, a ring-shaped outermost transmitting portion E7 arranged outside the light-shielding portion D6, and a light-shielding portion D7 arranged outside the transmitting portion E7.

[0058] The light-shielding portions D5 to D7 are configured with gratings whose phase values ​​change periodically, while the phase values ​​of the light-transmitting portions E5 to E7 are constant. Specifically, in the light-shielding portions 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 emission direction of the light emitted from the light-shielding portions D5 to D7 is tilted relative to the emission direction of the light emitted from the light-transmitting portions E5 to E7.

[0059] Referring again to FIG. 14, the aperture optical system 29 is provided downstream of the spatial light modulator 28 and 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 disposed between the lenses 291 and 292. The light emitted from the spatial light modulator 28 forms a beam waist between the lenses 291 and 292. The aperture 293 is located at the beam waist and blocks at least a portion of the light emitted from the light-blocking portions D5 to D7 of the spatial light modulator 28. As a result, the light emitted from the light-blocking portions D5 to D7 of the spatial light modulator 28 is attenuated or eliminated, and the light emitted from the transmission portions E5 to E7 passes through the aperture 293. In this modification as well, the ratio of the light intensity of light that exits from the transmitting portions E5 to E7 and passes through aperture 293 to the light intensity of light that enters the transmitting portions E5 to E7 (i.e., the light transmittance of the transmitting portions) is greater than the ratio of the light intensity of light that exits from the light-shielding portions D5 to D7 and passes through aperture 293 to the light intensity of light that enters the light-shielding portions D5 to D7 (i.e., the light transmittance of the light-shielding portions). The light transmittance of the transmitting portions may be 1 or less than 1. The light transmittance of the light-shielding portions may be 0 or greater than 0.

[0060] Alternatively, as shown in FIG. 16, the transmissive portions E5 to E7 may be formed by gratings whose phase values ​​change periodically, while the phase values ​​of the shielding portions D5 to D7 may be constant. Specifically, the transmissive portions E5 to E7 periodically repeat a phase distribution that monotonically increases from 0 (rad) to 2π (rad) in each period. This phase pattern causes the direction of light emitted from the transmissive portions E5 to E7 to be tilted relative to the direction of light emitted from the shielding portions D5 to D7. The position of the aperture 293 is slightly shifted relative to the beam waist in a direction intersecting the optical axis. Even in this configuration, the aperture 293 can block at least a portion of the light emitted from the shielding portions D5 to D7 of the spatial light modulator 28 while allowing the light emitted from the transmissive portions E5 to E7 of the spatial light modulator 28 to pass through.

[0061] As in this modification, the spatial light modulator constituting the phase conversion unit 23 may be common to the spatial light modulator constituting the ring mask 24. The common spatial light modulator 28 may present a phase pattern in which a phase pattern for constituting the phase conversion unit 23 and a phase pattern for constituting the ring mask 24 are superimposed. Similarly, in the light irradiation method of the first embodiment (see FIG. 9 ), the spatial light modulator performing the phase conversion process S22 may be common to the spatial light modulator performing the ring mask process S23, and the spatial light modulator may present a phase pattern in which a phase pattern for constituting the phase conversion process S22 and a phase pattern for constituting the ring mask process S23 are superimposed. In this case, the components constituting the phase conversion unit 23 and the components constituting the ring mask 24 (or the components performing the phase conversion process S22 and the components performing the ring mask process S23) may be integrated into one device, thereby simplifying the configuration of the device. [Second embodiment]

[0062] FIG. 17 is a diagram illustrating an outline of the configuration of a microscope apparatus 2 according to an embodiment of the present disclosure. The microscope apparatus 2 is a fluorescence microscope that irradiates an object B with light La, which is excitation light, and detects fluorescence Lb obtained from the object B by this irradiation. The microscope apparatus 2 includes the light irradiation apparatus 1 of the first embodiment, a dichroic mirror 31, a detection unit 32, and an image generation unit 33. The wavelength of the excitation light is, for example, included in the near-infrared range. Specifically, the wavelength of the excitation light is within a range of 650 nm to 1800 nm. The excitation light is, for example, laser light.

[0063] The dichroic mirror 31 transmits one of the light La (excitation light) from the optical scanner 25 and the fluorescence Lb from the object B, and reflects the other. In the example shown in FIG. 17, the dichroic mirror 31 transmits the light La and reflects the fluorescence Lb. Note that, in the example shown in FIG. 17, the dichroic mirror 31 is provided on the optical path between the relay lens system 26 and the objective lens 27, but the dichroic mirror 31 may be provided between the optical scanner 25 and the relay lens system 26, or may be provided between the beam expander 21 and the optical scanner 25.

[0064] The objective lens 27 focuses the light La on the object B, generating fluorescence Lb from the object B. The objective lens 27 also functions to collect fluorescence Lb from the object B. In the illustrated example, the objective lens 27 serves as both an objective lens for light La and a lens for collecting fluorescence Lb. However, the objective lens for 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 light La, and local focusing may be performed using aberration correction. Alternatively, an objective lens with a large pupil may be used for fluorescence Lb, enabling more light to be extracted. The objective lens for light La and the lens for collecting fluorescence Lb may be positioned on either side of the object B to obtain fluorescence Lb emerging from the surface of the object B opposite the surface into which light La enters. In this case, the dichroic mirror 31 is not required.

[0065] The detection unit 32 detects fluorescence Lb generated in the object B by irradiation with light La. The detection unit 32 includes a photodetection device for detecting the fluorescence Lb. The detection unit 32 is optically coupled to the dichroic mirror 31. Alternatively, when an objective lens for collecting light La and an objective lens for collecting fluorescence Lb are provided separately, the detection unit 32 is optically coupled to the objective lens for collecting fluorescence Lb. The photodetection device of the detection unit 32 generates an electrical signal corresponding to the light intensity of the fluorescence Lb generated in the object B. The photodetection device of the detection unit 32 is sensitive to the wavelength of the fluorescence Lb. The photodetection device of the detection unit 32 may be selected from one-dimensional photodetection elements such as a photomultiplier tube or an avalanche photodiode. Alternatively, various two-dimensional photodetection elements such as a multi-anode PMT (photomultiplier tube), a CCD image sensor, or a CMOS image sensor may be selected as the photodetection device of the detection unit 32. A filter for cutting the wavelength of light La and wavelengths unnecessary for observation may be provided on the optical path between the detection unit 32 and the dichroic mirror 31 (or the lens for collecting the fluorescence Lb).

[0066] The image generation unit 33 is electrically connected to the detection unit 32. The image generation unit 33 receives a signal related to the light intensity of the fluorescence Lb, which is the detection result, from the detection unit 32, and generates an observation image of the object B based on the light intensity of the fluorescence Lb. The image generation unit 33 may be configured by, for example, a computer including a central processing unit (CPU) and a memory. The image generation unit 33 may further include a monitor for displaying the generated image.

[0067] 18 is a flowchart for explaining the operation of the microscope apparatus 2 according to this embodiment. With reference to FIG. 18, the operation of the microscope apparatus 2 and the image acquisition method according to this embodiment will be described.

[0068] First, a light output step S1 and a light irradiation step S2 are performed. The details of the light output step S1 and the light irradiation step S2 are the same as those in the first embodiment. However, in this embodiment, the light La output in the light output step S1 is excitation light for exciting the object B.

[0069] Next, a detection step S6 is performed. In the detection step S6, the detection unit 32 detects the light intensity of the fluorescence Lb generated in the object B by the irradiation of the light La. The detection step S6 may include a step in which the light detection device generates a signal corresponding to the light intensity of the fluorescence Lb generated in the object B.

[0070] The above-mentioned light output step S1, light irradiation step S2, and detection step S6 are repeatedly performed while scanning the irradiation position of the light La on the object B with the optical scanner 25 (steps S7 and S8). As a result, data on the light intensity of the fluorescence Lb at multiple positions on the object B is obtained.

[0071] After the scanning by the optical scanner 25 is completed (step S7; YES), an image generation step S9 is performed. In the image generation step S9, the image generation unit 33 generates an observation image of the object B based on the detection result in the detection step S6. In the image generation step S9, the image generation unit 33 may generate an observation image of the object B based on a signal generated by the optical detection device of the detection unit 32.

[0072] According to the microscope device 2 and image acquisition method of this embodiment described above, by including the light irradiation device 1 of the first embodiment or the light irradiation method of the first embodiment, it is possible to further reduce the diameter of the focused light, thereby increasing the resolution of the observed image. [Second Modification]

[0073] 19 is a diagram showing an outline of the configuration of a microscope apparatus 2A according to a modified example of the second embodiment. The microscope apparatus 2A differs from the microscope apparatus 2 of the second embodiment in that it includes a phase-modulation type spatial light modulator 28 instead of the phase conversion unit 23 and ring mask 24 of the microscope apparatus 2. The configurations of the spatial light modulator 28 and aperture optical system 29 are the same as those of the first modified example. The other configurations of the light irradiation device 1A are the same as those of the microscope apparatus 2 of the second embodiment. This modified example can also achieve the same effects as those of the second embodiment and the first modified example. [Third embodiment]

[0074] 20 is a diagram showing an outline of the configuration of a microscope apparatus 3 according to an embodiment of the present disclosure. The microscope apparatus 3 includes a light output unit 40, a detection unit 50, and an image generation unit 60 instead of the light output unit 10, the detection unit 32, and the image generation unit 33 of the microscope apparatus 2 according to the second embodiment described above. The microscope apparatus 3 also includes a signal generator (function generator) 70. The object B is, for example, a biological sample.

[0075] The light output unit 40 outputs light La, which is excitation light for exciting the object B. The light La is coherent light. The wavelength of the light La is, for example, included in the near-infrared region. Specifically, the wavelength of the light La is within the range of 650 nm to 1800 nm. The light La is, for example, laser light. The following formula (3) is a formula showing the time waveform of the light La output from the light output unit 40. In formula (3), Ir is the light intensity of the light La, t is time, f is frequency, and a and b are constants.

number

number

[0076] The light output unit 40 in one embodiment includes a light source 41 and an optical modulator 42. The light source 41 outputs pulsed light Lp. The light source 41 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 optical intensity of the pulsed light Lp exceeds half of its peak value. Specifically, the time width of the pulsed light Lp is, for example, within a range of 10 femtoseconds to 50 picoseconds. The light source 41 is, for example, a laser light source, and in one example, a mode-locked laser light source. The preferred wavelength range of the pulsed light Lp is the same as the preferred wavelength range of the light La described above.

[0077] The optical modulator 42 is an intensity-modulating optical modulator and is optically coupled to the light source 41 via space or an optical waveguide. The optical modulator 42 is electrically connected to the signal generator 70 and generates light La by modulating the pulsed light Lp output from the light source 41 in synchronization with the output signal from the signal generator 70. The optical modulator 42 can be selected from various modulators, such as an electro-optic (EO) modulator, an acousto-optic (AO) modulator, a liquid crystal or ND (neutral density) filter capable of dynamically controlling transmittance or reflectance. When the time width of the pulsed light Lp is on the order of femtoseconds, an AO modulator or an EO modulator is particularly suitable as the optical modulator 42. Note that light La may also be generated by controlling the magnitude of the drive current input to the light source 41 (direct modulation method) without using the optical modulator 42. In this case, the optical modulator 42 is not required.

[0078] The objective lens 27 focuses the light La at high density on the object B, causing two-photon excitation in the object B and generating fluorescence Lb from the object B. The wavelength of the fluorescence Lb is, for example, within the range of 300 nm to 900 nm.

[0079] The detection unit 50 detects the second harmonic (or second and third harmonics) contained in the time waveform of the light intensity of the fluorescence Lb. In one embodiment, the detection unit 50 includes a photodetection device 51 and a lock-in amplifier 52. The photodetection device 51 is optically coupled to the dichroic mirror 31. Alternatively, when an objective lens for collecting the light La and an objective lens for collecting the fluorescence Lb are provided separately, the photodetection device 51 is optically coupled to the objective lens for collecting the fluorescence Lb. The photodetection device 51 generates an electrical signal corresponding to the light intensity of the fluorescence Lb generated in the object B. The photodetection device 51 is sensitive to the wavelength of the fluorescence Lb and has a frequency range necessary to detect the second harmonic (or second and third harmonics) of the fluorescence Lb. The photodetection device 51 can be selected from one-dimensional photodetection elements such as a photomultiplier tube or an avalanche photodiode. Alternatively, various two-dimensional photodetection elements such as a multi-anode PMT, a CCD image sensor, or a CMOS image sensor may be selected as the photodetection device 51. A filter for cutting the wavelength of the light La and wavelengths unnecessary for observation may be provided on the optical path between the detection unit 50 and the dichroic mirror 31 (or a lens for collecting the fluorescence Lb).

[0080] The lock-in amplifier 52 is electrically connected to the photodetection device 51 and the signal generator 70. The lock-in amplifier 52 receives an electrical signal corresponding to the light intensity of the fluorescence Lb from the photodetection device 51, and also receives a sinusoidal signal from the signal generator 70 having the same period as the periodic signal provided to the light output unit 40. The lock-in amplifier 52 detects the second harmonic (or at least one of the second and third harmonic) contained in the time waveform of the signal from the photodetection device 51, using the signal from the signal generator 70 as a reference.

[0081] The image generating unit 60 is electrically connected to the lock-in amplifier 52. The image generating unit 60 receives a signal related to the magnitude of the second harmonic (or the second and third harmonics) contained in the time waveform of the light intensity of the fluorescence Lb from the lock-in amplifier 52, and generates an observation image of the object B based on this second harmonic (or at least one of the second and third harmonics). The image generating unit 60 may be configured by, for example, a computer including a central processing unit (CPU) and a memory. The image generating unit 60 may further include a monitor for displaying the generated image.

[0082] 22 is a flowchart for explaining the operation of the microscope apparatus 3 according to this embodiment. With reference to FIG. 22, the operation of the microscope apparatus 3 and the image acquisition method according to this embodiment will be described.

[0083] First, a light output step S1a is performed. In the light output step S1a, the light output unit 40 outputs light La, which is excitation light. As described above, the time waveform of the light intensity of light La includes the square root of a linear function of a sine wave (see equation (3)). Furthermore, the maximum value of the light intensity of light La in each period exceeds the saturated excitation intensity in the target B. This light output step S1a may include a step S11 in which pulsed light Lp is generated in the light source 41, and an intensity modulation step S12 in which the light modulator 42 modulates the pulsed light Lp to generate light La.

[0084] Next, the light irradiation step S2 is performed. The details of the light irradiation step S2 are the same as those in the first embodiment described above.

[0085] Subsequently, a detection step S6a is performed. In the detection step S6a, the detection unit 50 detects the second harmonic (or the second harmonic and the third harmonic) contained in the time waveform of the light intensity of the fluorescence Lb generated in the object B by two-photon excitation due to irradiation with the light La. The detection step S6a may include a step S61 in which the light detection device 51 generates a signal corresponding to the light intensity of the fluorescence Lb generated in the object B, and a step S62 in which the lock-in amplifier 52 outputs the second harmonic contained in the time waveform of this signal.

[0086] The above-described light output step S1a, light irradiation step S2, and detection step S6a are repeatedly performed (steps S7 and S8) while scanning the irradiation position of light La on object B using optical scanner 25. This allows data on the magnitude of the second harmonic (or third harmonic or higher harmonic) at multiple positions on object B to be obtained.

[0087] After the scanning by the optical scanner 25 is completed (step S7; YES), an image generation step S9a is performed. In the image generation step S9a, the image generation unit 60 generates an observation image of the object B based on the magnitude of the second harmonic (or at least one of the second harmonic and the third harmonic) contained in the time waveform of the light intensity of the fluorescence Lb at multiple irradiation positions of the light La.

[0088] The device and method of this embodiment described above can provide the same effects as those of Embodiment 2. In addition, the device and method of this embodiment can also provide the following effects.

[0089] Figure 23(a) shows an example of the fluorescence intensity distribution in a SAX microscope. In a SAX microscope, the peak intensity of the excitation light is set higher than the saturation excitation intensity of the object being observed. This causes the fluorescence intensity within a certain range from the center of the fluorescence intensity distribution to saturate, and the fluorescence intensity distribution shape (solid line F2 in the figure) changes from the theoretical value without saturation (dashed line F1 in the figure). Therefore, by taking the difference between the theoretical and measured fluorescence intensity values ​​(solid line F3 in Figure 23(b)), the full width at half maximum of the intensity distribution can be reduced, thereby improving the spatial resolution of the microscope. In the case of single-photon excitation, the difference between the theoretical and measured fluorescence intensity values, or a value approximating the difference, can be obtained by assuming the excitation light intensity time waveform to be sinusoidal and detecting harmonics (e.g., second or third harmonic) of the fluorescence intensity time waveform.

[0090] In two-photon excitation microscopes, an object to be observed is irradiated with long-wavelength ultrashort pulsed light, such as near-infrared light, as excitation light, causing two-photon excitation in the object, and the resulting fluorescence is detected to create an observation image. This two-photon excitation microscope uses long-wavelength light that has excellent object penetration, making it possible to observe, for example, deep inside living tissue non-invasively.

[0091] Combining a SAX microscope, which has the above advantages, with a two-photon excitation microscope would realize a microscope that combines these advantages. However, in two-photon excitation, the fluorescence intensity is proportional to the square of the excitation light intensity. Therefore, when an excitation light having a sinusoidal time waveform is irradiated onto an object to be observed and two-photon excitation occurs in the object, the time waveform of the excitation light output from the object to be observed is proportional to the square of the sine wave. Figure 24 conceptually illustrates the time waveform of the excitation light intensity (Figure 24(a)) and the time waveform of the fluorescence intensity (Figure 24(b)) in the case of two-photon excitation. To obtain an observation image based on the fluorescence intensity having a time waveform that is deformed from a sinusoidal wave, as shown in Figure 24(b), it becomes necessary to detect higher harmonics, such as the third and fifth harmonics, as described in Patent Document 2, for example. Since there is a limit to the maximum value of the frequency range of the device that detects the excitation light (for example, the maximum value of the frequency range of the lock-in amplifier 52), if one attempts to detect higher-order harmonics, the frequency of the excitation light must be reduced, which increases the time required to create an observation image.

[0092] In the microscope device 3 and image acquisition method of this embodiment, the time waveform of the light intensity of light La includes the square root of a linear function of a sine wave (see equation (3)). 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. Therefore, when light La having a time waveform including the square root of a linear function of a sine wave is irradiated onto object B to cause two-photon excitation in object B, the time waveform of light La output from object B will be proportional to a linear function of a sine wave, not the square of a sine wave. Figure 25 is a diagram conceptually showing such a time waveform of excitation light intensity (Figure 25(a)) and a time waveform of fluorescence intensity (Figure 25(b)).

[0093] Therefore, according to the microscope apparatus 3 and image acquisition method of this embodiment, like a general SAX microscope, an observation image can be obtained based on a lower harmonic, such as the second harmonic or the third harmonic. This eliminates the need to reduce the frequency of the light La due to the constraints of the frequency range of the light detection device 51, thereby preventing the time required to create an observation image from increasing.

[0094] To verify the above-mentioned effect, the inventors measured the light intensity of the fluorescence Lb in the microscope device 3 of this embodiment, and for comparison, measured the fluorescence intensity by setting the time waveform of the excitation light intensity to a sine wave. In these measurements, the excitation light intensity and wavelength were the same, and the same object B was used.

[0095] FIG. 26(a) is a graph showing the results of Fourier transforming the time waveform of the light intensity of fluorescence Lb measured in the microscope apparatus 3 of this embodiment. FIG. 26(b) is a graph showing the results of Fourier transforming the time waveform of fluorescence intensity measured when the time waveform of excitation light intensity is a sine wave. Arrow U1 in FIG. 26(b) indicates the fundamental wave, and only this fundamental wave is generated when saturation is not achieved. When the excitation light intensity is set to a magnitude that causes saturation and the time waveform is a sine wave, as shown in FIG. 26(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 3 of this embodiment, as shown in FIG. 26(a), in addition to the fundamental wave, the second harmonic (arrow V2) and third harmonic (arrow V3) are generated due to saturation. The fluorescence intensity of the second harmonic (arrow V2) in Figure 26(a) became approximately equal to the fluorescence intensity of the third harmonic (arrow U3) in Figure 26(b), and the fluorescence intensity of the third harmonic (arrow V3) in Figure 26(a) became approximately equal to the fluorescence intensity of the fifth harmonic (arrow U5) in Figure 26(b).

[0096] FIG. 27(a) is a graph showing the relationship between the light intensity of the fundamental wave and second harmonic of fluorescence Lb measured in the microscope apparatus 3 of this embodiment and the relative intensity of light La. In FIG. 27(a), plot P11 represents the fundamental wave, and plot P12 represents the second harmonic. FIG. 27(b) is a graph showing the relationship between the light intensity of the fundamental wave, second harmonic, and third harmonic of fluorescence measured when the time waveform of the excitation light intensity is a sine wave, and the relative intensity of the excitation light. In FIG. 27(b), plot P21 represents the fundamental wave, plot P22 represents the second harmonic, and plot P23 represents the third harmonic. The relative intensity of the excitation light is the intensity of the periodic excitation light (light La) after passing through the optical modulator 42, and is the ratio when the maximum intensity that can be output by the optical modulator 42 is set to 1. 27(a) and 27(b), it can be seen that the arrangement of plot P12 in Fig. 27(a) (the second harmonic of fluorescence Lb measured in the microscope apparatus 3 of this embodiment) and the arrangement of plot P23 in Fig. 27(b) (the third harmonic of fluorescence measured with the time waveform of the excitation light intensity as a sine wave) are roughly similar. Note that the discrepancy between the arrangement of plot P12 and the arrangement of plot P23 in areas where the fluorescence intensity is low is thought to be due to measurement error.

[0097] From the measurement results shown above, it can be seen that in the microscope device 3 of this embodiment, in which the time waveform of the light intensity of light La includes the square root of a linear function of a sine wave, detection of the second harmonic (or third harmonic) corresponds to 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 device 3 of this embodiment, it is possible to obtain an observation image based on a lower order harmonic compared to when the time waveform of the excitation light intensity is a sine wave. In general, devices with a lower upper limit in the frequency range are often cheaper than devices with a higher upper limit, and therefore costs can be reduced.

[0098] 28, 29, and 30 are diagrams showing fluorescence images in which the fundamental wave, second harmonic, and third harmonic are detected in a cross section that includes the optical axis and is parallel to the optical axis direction. In these diagrams, light intensity is indicated by shades of color, with darker areas indicating lower light intensity and lighter areas indicating higher light intensity. In FIGS. 28 to 30, (a) and (b) show fluorescence images in which the light La irradiated onto the object B is azimuthally polarized light with a spiral phase modulation, and (c) and (d) show fluorescence images in which the light La irradiated onto the object B is circularly polarized light, as a comparative example. In addition, in FIGS. 28 to 30, (a) and (c) show fluorescence images when the ring mask 24 is provided, and (b) and (d) show fluorescence images in which the ring mask 24 is not provided.

[0099] 29 and 30(a), (c) and (b), (d) show that when the ring mask 24 is provided, the horizontal and vertical dimensions of the fluorescence image are smaller than when the ring mask 24 is not provided. Furthermore, when (a), (b) and (c), (d) of FIG. 29 and 30(a), (b) and (c), (d) show that when the light La irradiating the object B is azimuthally polarized light with a spiral phase modulation, the diameter and length of the focused spot are smaller than when the light La irradiating the object B is circularly polarized. Furthermore, when (a), (b) and (c), (d) of FIG. 29 and 30(a), (b) and (c), (d) of FIG. 28 show that detecting the second or third harmonic reduces the horizontal dimensions of the fluorescence image, in particular, compared to when the fundamental wave is detected.

[0100] As in this embodiment, the light output unit 40 may include a light source 41 that outputs pulsed light Lp and an intensity-modulation-type optical modulator 42 that modulates the pulsed light Lp output from the light source 41 to generate light La. Similarly, the light output step S1a may include an intensity-modulation step S12 that modulates the pulsed light Lp to generate light La. This makes it possible to easily generate light La whose time waveform of light intensity includes the square root of a linear function of a sine wave. Furthermore, the intensity-modulation-type optical modulator 42 may be an AO modulator or an EO modulator. AO modulators and EO modulators are suitable for high-speed, non-sinusoidal optical modulation as in this embodiment.

[0101] As in the present embodiment, the light source 41 may be a laser light source, and the pulsed light Lp may be laser light, thereby making it possible to generate light La having a high optical intensity capable of causing two-photon excitation with a simple configuration.

[0102] As in the present embodiment, the detection unit 50 may include a photodetection device 51 that generates a signal corresponding to the light intensity of the fluorescence Lb generated in the object B, and a lock-in amplifier 52 that receives the signal from the photodetection device 51 and outputs the second harmonic (or the second and third harmonics) included in the time waveform of the signal. Similarly, the detection step S6a may include step S61 of generating a signal corresponding to the light intensity of the fluorescence Lb generated in the object B, and step S62 of outputting the second harmonic (or the second and third harmonics) included in the time waveform of the signal. This allows the second harmonic (or the second and third harmonics) to be detected easily and accurately.

[0103] As in the present embodiment, the detection unit 50 may detect the second harmonic and the third harmonic contained in the time waveform of the light intensity of the fluorescence Lb generated in the object B. Similarly, in the detection step S6a, the second harmonic and the third harmonic contained in the time waveform of the light intensity of the fluorescence Lb generated in the object B may be detected. Then, in the image generation step S9a, an observation image of the object B may be generated based on at least one of the second harmonic and the third harmonic. In this case, an observation image using the harmonic that is suitable for the object B out of the second harmonic and the third harmonic can be easily generated.

[0104] In this embodiment, similarly to the second modified example, a phase modulation type spatial light modulator 28 may be provided in place of the phase conversion unit 23 and the ring mask 24 of the microscope device 2, and an aperture optical system 29 may be provided in place of (or in addition to) the beam expander 21. In this case as well, the same effects as those of this embodiment and the second modified example can be achieved. [Third Modification]

[0105] The time waveform of light La shown in Equation (3), i.e., the time waveform including the square root of a linear function of a sine wave, is not limited to Equation (4) and the example shown in FIG. 21. For example, in the example shown in FIG. 21, the minimum value Imin in each period of the time waveform (envelope K) of light La is 0, but the minimum value Imin in each period may be greater than 0. In other words, in Equation (3), the constant b may be greater than a. Alternatively, the minimum value Imin in each period of the time waveform of light La may be greater than 0.1% (more preferably 5%) and less than 20% of the maximum signal that the detection unit 50 (specifically, the lock-in amplifier 52) can receive. FIG. 31 is a diagram conceptually illustrating such a time waveform of light La. Note that, in this modification as well, the maximum value of the light intensity of light La in each period is set to a magnitude that exceeds the saturation excitation intensity in the target B.

[0106] Equation (5) is an example of the time waveform of light La in this modification, where α is a real number greater than 0 and less than 1.

number

[0107] At and around the minimum value Imin in each period of the time waveform of the light intensity of light La, the intensity of the generated fluorescence Lb is small, and the detection result is significantly affected by noise. As in this modification, by making the minimum value Imin in each period of the time waveform of the light intensity of light La greater than 0 (more preferably greater than 0.1% and less than 20% of the maximum signal that the detection unit 50 (or in detection step S6a) can receive), the influence of noise can be reduced, the detection accuracy of the second harmonic can be improved, and the observed image can be made clearer.

[0108] Furthermore, when the time waveform of the light intensity of light La includes the square root of a linear function of a sine wave, the rate of change of the light intensity near the minimum value Imin in each period becomes larger (in other words, the time waveform becomes steeper) compared to when the time waveform of the excitation light intensity is a sine wave. This rate of change is greatest when the minimum value Imin in each period is 0 (see FIG. 21). Here, FIG. 32 is a graph showing a typical example of the relationship between the applied voltage and the output light intensity of a typical AO modulator. As shown in FIG. 32, the output light intensity of an AO modulator changes nonlinearly with respect to the applied voltage. Furthermore, in the region where the applied voltage is low, the change in the output light intensity is extremely gradual. Therefore, if the rate of change of the light intensity is large during periods when the light intensity of light La is low, it becomes necessary to suddenly change the applied voltage to the AO modulator, making it difficult to control the applied voltage. To address this issue, in this modified example, by making the minimum value Imin in each period greater than 0 (more preferably greater than 0.1% and less than 20% of the maximum signal that the detecting unit 50 (or in the detecting step S6a) can receive), the rate of change of the light intensity near the minimum value Imin in each period can be reduced, and the steepness of the time waveform can be alleviated. This makes it easier to shape the time waveform of the light intensity of light La (particularly, to shape the time waveform near the minimum value Imin in each period) in the light output unit 40, which has an AO modulator as the optical modulator 42.

[0109] Note that, when the minimum value Imin within each period is made greater than 0 as described above, the minimum value within each period in the time waveform of the light intensity of the fluorescence Lb also becomes greater than 0. Therefore, if the time waveform of the light intensity of the fluorescence Lb is used as is in the lock-in amplifier 52, there is a risk that the detection accuracy of the second and third harmonics will decrease. To avoid this risk, it is desirable to adjust the time waveform within the lock-in amplifier 52 or in a stage preceding the lock-in amplifier 52 so that the minimum value within each period in the time waveform of the light intensity of the fluorescence Lb becomes zero (in other words, to cancel the fluctuation in the time waveform of the fluorescence Lb caused by the difference between the minimum value Imin of the light La and zero). [Fourth Modification]

[0110] 33 is a diagram showing an outline of the configuration of a light irradiation device 1B according to another modified example of the first embodiment. The light irradiation device 1B differs from the light irradiation device 1 of the first embodiment in that, in addition to the configuration of the light irradiation device 1 described above, the light irradiation device 1B is provided with an optical axis adjustment mechanism 80 for adjusting the optical axis of the light La. Other configurations of the light irradiation device 1B are similar to those of the light irradiation device 1 of the first embodiment.

[0111] The optical axis adjustment mechanism 80 is provided on the optical path of the light La between the light output unit 10 and the optical system 20. The optical axis adjustment mechanism 80 includes a pair of variable-angle mirrors 81 and 82, a pair of beam samplers 83 and 84, a pair of cameras 85 and 86, and a control unit 89. The variable-angle mirror 81 is optically coupled to the light output unit 10 and reflects the light La received from the light output unit 10 toward the variable-angle mirror 82. The variable-angle mirror 82 is optically coupled to the light output unit 10 via the variable-angle mirror 81. It receives the light La reflected by the variable-angle mirror 82 and reflects the light La toward the optical system 20. Each of the variable-angle mirrors 81 and 82 has two degrees of freedom, allowing the reflection direction of the light La to be tilted at any angle and in any direction. The variable-angle mirrors 81 and 82 operate based on electrical drive signals input from the control unit 89.

[0112] The beam samplers 83 and 84 are provided at a distance from each other on the optical path of the light La between the angle variable mirror 82 and the optical system 20. The beam samplers 83 and 84 split a portion of the light La. In one example, the beam samplers 83 and 84 reflect a portion of the light La and transmit the remainder.

[0113] Camera 85 is optically coupled to beam sampler 83 via lens 87 and receives light (a part of light La) branched by beam sampler 83. Camera 86 is optically coupled to beam sampler 84 via lens 88 and receives light (another part of light La) branched by beam sampler 84. Cameras 85 and 86 capture images of the incident light and generate data corresponding to the light intensity distribution. Controller 89 detects the deviation and tilt of the optical axis of light La based on this data. Controller 89 provides drive signals to variable-angle mirrors 81 and 82 so that the deviation and tilt of the optical axis of light La approach zero.

[0114] The misalignment and tilt of the optical axis of light La occur, for example, due to wavelength change by a wavelength-tunable laser constituting the light output unit 10. Alternatively, the misalignment and tilt of the optical axis of light La occur due to fluctuations over time in the output offset and tilt of a piezo mirror included in the light output unit 10 for output stability. If the misalignment and / or tilt of the optical axis of light La occurs, the optical axis of light La will no longer coincide with the central positions of the polarization conversion unit 22, phase conversion unit 23, and ring mask 24. Therefore, in this modification, the optical axis adjustment mechanism 80 suppresses the misalignment and tilt of the optical axis of light La. This allows the central positions of the polarization conversion unit 22, phase conversion unit 23, and ring mask 24 to more accurately coincide with the optical axis of light La.

[0115] The light irradiation device, microscope device, light irradiation method, and image acquisition method according to the present disclosure are not limited to the above-described embodiments, and various other modifications are possible. For example, in the above-described embodiments, the polarization conversion unit 22, the phase conversion unit 23, and the ring mask 24 are arranged on the optical path between the light output unit 10 and the optical scanner 25. However, at least one of the polarization conversion unit 22, the phase conversion unit 23, and the ring mask 24 may be arranged on the optical path between the optical scanner 25 and the objective lens 27. Furthermore, at least one of the polarization conversion unit 22, the phase conversion unit 23, and the ring mask 24 may be arranged on the optical path between the objective lens 27 and the object B.

[0116] Furthermore, although the above embodiments have exemplified cases in which the ring mask 24 is of the amplitude modulation type, even if the ring mask 24 is of the phase modulation type or a combination of the amplitude modulation type and the phase modulation type, the focused light diameter can be reduced by using light that combines azimuthally polarized light and a spiral phase, as in the above embodiments.

[0117] Furthermore, although the third embodiment has been described as a case where two-photon excitation is generated in the object B, the microscope apparatus and image acquisition method according to the present disclosure are effective when multi-photon excitation, i.e., n-photon excitation (n is an integer equal to or greater than 2), is generated in the object B. That is, in the intensity modulation step S12 of the light output step S1a, the optical modulator 42 of the light output unit 40 outputs light La whose time waveform of light intensity includes the n-th root of a linear function of a sine wave. Therefore, the terms "two-photon excitation" and "square root" in the above-described embodiments can all be replaced with "n-photon excitation" and "n-th root."

[0118] Furthermore, in the third embodiment, the time waveform of the light intensity of light La is the square root of a linear function of a sine wave over the entire period of each cycle. However, the present invention is not limited to this, and the time waveform of the light intensity of light La may be the square root of a linear function of a sine wave over only a portion of each cycle (typically, the period including the maximum value Imax).

[0119] Furthermore, in the second and third embodiments, configurations in which the light irradiation device of the present disclosure is applied to a microscope device are exemplified, but the light irradiation device of the present disclosure is not limited to this and can be used for various light irradiation applications such as laser processing and light stimulation devices. [Explanation of symbols]

[0120] 1, 1A... light irradiation device, 2, 2A... microscope device, 3... microscope device, 9... mirror, 10... light output unit, 20... optical system, 21... beam expander, 22... polarization conversion unit, 23... phase conversion unit, 24... ring mask, 25... optical scanner, 26... relay lens system, 27... objective lens, 28... spatial light modulator, 29... aperture optical system, 31... dichroic mirror, 32... detection unit, 33... image generation unit, 40... light output unit, 41... light source, 42... optical modulator, 50... detection unit, 51... light detection device, 52... lock-in amplifier, 60... image generation unit, 70... signal generator, 211, 212... lens, 251, 252... scanner, 253... optical system, 291, 292... lens, 293... aperture , D1 to D7...light-shielding area, d1 to d3...width, B...object, J...pulse, K...envelope, E1 to E7...transmitting area, e1 to e4...width, G11 to G13, G21 to G23...bars, Ha, Hb...focused spot shape, La...light, Lb...fluorescence, Lp...pulsed light, S1, S1a...light output step, S2...light irradiation step, S3, S4, S7, S8...steps, S6, S6a...detection step, S9, S9a...image generation step, S12...intensity modulation step, S21...polarization conversion processing, S22...phase conversion processing, S23...ring mask processing, S24...light-focusing processing, S61, S62...steps, P11, P12, P21 to P23...plot, Q...optical axis, Wa1, Wa2...width, Wb1, Wb2...length.

Claims

1. an optical output unit that outputs coherent light; an optical system that irradiates an object with the light output from the light output unit; a detection unit that detects light generated in the object by irradiation with light output from the light output unit; an image generating unit that generates an observation image of the object based on the detection result of the detecting unit; Equipped with The optical system comprises: an objective lens that focuses the light output from the light output unit on the object; a polarization conversion unit, a phase conversion unit, and a ring mask provided on an optical path between the optical output unit and the object; and the polarization conversion unit is configured to convert the light input to the polarization conversion unit into azimuthally polarized light and output the converted light, the phase change unit is configured to impart phase modulation by a spiral phase pattern to light input to the phase change unit, the light output unit outputs light whose time waveform of light intensity includes an nth root (n is an integer of 2 or more) of a linear function of a sine wave and whose maximum value of light intensity exceeds a saturation excitation intensity in the object; The detection unit detects second harmonics included in a time waveform of the light intensity of fluorescence generated in the object by n-photon excitation caused by irradiation with light output from the light output unit.

2. 2. The microscope apparatus according to claim 1, wherein the ring mask is an amplitude modulation type.

3. 3. The microscope apparatus according to claim 1, wherein a ratio (NA / R) of a refractive index R of a medium between the objective lens and the object to a numerical aperture NA of the objective lens is 0.75 or more.

4. 4. The microscope apparatus according to claim 1, wherein at least one of the phase conversion unit and the ring mask is configured by a phase modulation type spatial light modulator.

5. the spatial light modulator constituting the phase conversion unit is common to the spatial light modulator constituting the ring mask, 5. The microscope apparatus according to claim 4, wherein the spatial light modulator presents a phase pattern in which a phase pattern for configuring the phase conversion unit and a phase pattern for configuring the ring mask are superimposed.

6. The ring mask is a plurality of ring-shaped light-blocking portions provided around a central position; a transmission portion provided between two adjacent light-shielding portions of the plurality of light-shielding portions; an innermost light-transmitting portion provided inside the innermost light-shielding portion of the plurality of light-shielding portions; an outermost light-transmitting portion provided outside the outermost light-shielding portion of the plurality of light-shielding portions; The microscope apparatus according to any one of claims 1 to 5, comprising:

7. an optical output step of outputting coherent light; a light irradiation step in which the light outputted in the light output step is subjected to polarization conversion processing, phase conversion processing, and ring mask processing, and is condensed on an object; a detection step of detecting light generated in the object by the irradiation of light in the light irradiation step; an image generation step of generating an observation image of the object based on the detection result in the detection step; Including, the polarization conversion process is a process of converting the light output in the light output step into azimuthally polarized light, the phase conversion process is a process of applying phase modulation by a spiral phase pattern to the light outputted in the light output step, In the light output step, light is output whose time waveform of light intensity includes an nth root (n is an integer of 2 or more) of a linear function of a sine wave and whose maximum value of light intensity exceeds a saturation excitation intensity in the object; An image acquisition method, wherein the detecting step detects a second harmonic contained in a time waveform of the light intensity of the fluorescence generated in the object by n-photon excitation due to the irradiation with light in the light irradiation step.

8. The image acquisition method according to claim 7 , wherein the ring masking is of the amplitude modulation type.

9. 9. The image acquisition method according to claim 7, wherein in the light irradiation step, the light is focused using an objective lens having a ratio (NA / R) of a refractive index R of a medium between the objective lens and the object to a numerical aperture NA of the objective lens of 0.75 or more.

10. 10. The image acquisition method according to claim 7, wherein at least one of the phase conversion process and the ring mask process is performed using a phase modulation type spatial light modulator.

11. the spatial light modulator that performs the phase conversion processing is common to the spatial light modulator that performs the ring mask processing, The image acquisition method according to claim 10 , wherein the spatial light modulator presents a phase pattern in which a phase pattern for performing the phase conversion processing and a phase pattern for performing the ring mask processing are superimposed.

Images