Scanning-type observation device

Abstract

This scanning-type observation device comprises: a first emission optical system 10 for emitting first primary light including a spatially modulated component; a second emission optical system 20 for emitting coherent second primary light; a multiplexing unit 15 for coaxially multiplexing the first primary light and the second primary light; a scanning unit 30 for coaxially scanning the multiplexed first primary light and second primary light; a first objective lens 40 for condensing the scanned first primary light and second primary light; a mounting part 50 for mounting a sample at the condensing position of the first objective lens; a second objective lens 45 for collecting secondary light from the sample; a demultiplexing unit 75 for demultiplexing the secondary light into first secondary light emitted from the sample by being irradiated with the first primary light and second secondary light emitted from the sample by being irradiated with the second primary light; a first detection unit 70 for detecting the first secondary light; and a second detection unit 80 for detecting the second secondary light.

Description

Scanning Observation Device

[0001] The present invention relates to a scanning observation device.

[0002] In recent years, there has been growing expectation for methods to observe or evaluate living cells without staining in fields such as regenerative medicine, intraoperative rapid diagnosis, and bioproduction. Methods for imaging living cells without staining or contact include detecting the phase difference of light transmitted through the cells and detecting Raman scattered light caused by the vibration of molecules that make up the cells.

[0003] Japanese Patent Application Laid-Open Publication No. 2019-35859 discloses an apparatus capable of observing live cells using both a phase contrast imaging method that detects phase differences as light intensity, and a CARS imaging method that detects anti-Stokes Raman scattered light generated by nonlinear optical effects.

[0004] Japanese Patent Application Laid-Open No. 2019-35859

[0005] In JP 2019-35859 A, a phase contrast image is obtained by illuminating the entire observation area at once and capturing it with a CCD camera, whereas a CARS image is obtained by focusing irradiated light at a single point in the observation area and scanning it. In other words, different imaging methods use different image formation means. Therefore, the primary images obtained by each imaging method have different observation areas, different pixel counts, and other factors, resulting in inconsistent image quality. Therefore, to align the image quality, operators are required to perform cumbersome pre-calibration of the device and post-processing of the images. Thus, there is a need to reduce the burden on operators who wish to observe live cells using multiple methods.

[0006] An object of the present invention is to provide a scanning observation apparatus that can easily match the image quality of a phase contrast image and a Raman image.

[0007] a scanning observation device according to an embodiment of the present invention, comprising: a first emission optical system that emits first primary light including a spatially modulated component; a second emission optical system that emits coherent second primary light; a multiplexing unit that coaxially multiplexes the first primary light and the second primary light; a scanning unit that coaxially scans the combined first primary light and the second primary light; a first objective lens that focuses the scanned first primary light and the second primary light; a mounting unit that mounts a sample at a focusing position of the first objective lens; a second objective lens that collects secondary light from the sample; a demultiplexing unit that demultiplexes the secondary light into first secondary light emitted from the sample by irradiation with the first primary light and second secondary light emitted from the sample by irradiation with the second primary light; a first detection unit that detects the first secondary light;

[0008] According to the present invention, it is possible to provide an observation device that can easily match the image quality of a phase contrast image and a Raman image.

[0009] FIG. 1 is a block diagram illustrating an overview of the connection relationships of elements that configure an observation device according to a first embodiment of the present invention. FIG. 2 is a schematic diagram illustrating the configuration of the observation device according to the first embodiment. FIG. 3 is a schematic diagram illustrating phase-contrast observation by the observation device according to the first embodiment. FIG. 4 is a schematic diagram illustrating SRS observation by the observation device according to the first embodiment. FIG. 5 is a diagram illustrating a pre-observation flow of the observation device according to the first embodiment. FIG. 6 is a diagram illustrating the relationship between a region of interest and an observation field of view according to the first embodiment. FIG. 7 is a diagram illustrating a schematic configuration of a scanning condition determination unit according to the first embodiment. FIG. 8 is a schematic diagram illustrating the configuration of an observation device according to a second embodiment. FIG. 9 is a schematic diagram illustrating the relationship between a phase plate and a transmitted light beam in a phase-difference detection unit according to the second embodiment. FIG. 10 is a schematic diagram illustrating the relationship between a phase plate and a transmitted light beam in a phase-difference detection unit according to the second embodiment.

[0010] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0011] First Embodiment The configuration of an observation device according to a first embodiment will be described with reference to FIGS.

[0012] Fig. 1 is a block diagram illustrating an outline of the connection relationships between elements that make up the observation device 1. In Fig. 1, solid lines connecting blocks (rectangles) indicate optical connections between corresponding components, and dashed lines connecting blocks (rectangles) indicate connections that enable transmission of signals related to measurement or control between corresponding components.

[0013] The observation device 1 includes a first emission optical system 10 (phase difference light source unit), a second emission optical system 20 (SRS light source unit), a combining unit 15, a scanning unit 30, a first objective lens 40, and a mounting unit 50, which are optically connected. The observation device 1 further includes a second objective lens 45, a relay unit 60, a branching unit 75, a first detection unit 70 (phase difference detection unit), and a second detection unit 80 (SRS detection unit). In addition, the observation device 1 includes a control unit 90. The control unit 90 is electrically connected to the first emission optical system 10 (phase difference light source unit), the second emission optical system 20 (SRS light source unit), the scanning unit 30, the mounting unit 50, the relay unit 60, the first detection unit 70 (phase difference detection unit), and the second detection unit 80 (SRS detection unit).

[0014] In the observation device 1, the first emission optical system 10 (phase difference light source unit) forms the first emission optical system, and the second emission optical system 20 (SRS light source unit) forms the second emission optical system. Also, the first detection unit 70 (phase difference detection unit) forms the first detection unit, and the SRS detection unit forms the second detection unit.

[0015] FIG. 2 is a schematic diagram illustrating the configuration of the observation device 1.

[0016] (Placement Unit) The placement unit 50 includes a stage 511 and a stage scanner 512. The stage 511 supports a sample 501, which is an observation target of the observation device 1, so that at least a portion of the sample 501 overlaps with the focusing position of a first objective lens 40, which will be described later. The placement unit 50 may also be referred to as a support unit 50 for the sample 501 or a sample stage 50. The focusing position of the first objective lens 40 may also be referred to as the focal point of the first objective lens 40, the focusing point of the first objective lens, etc.

[0017] The stage 511 has an optically opened portion that allows the primary light to enter the sample 501 and the secondary light to exit the sample 501. The opening may be either closed or open in the circumferential direction.

[0018] As a result, the sample 501 placed on the mounting unit 50 is irradiated with light from the first objective lens 40 via the stage 511. The stage scanner 512 is coupled to the stage 511. The stage scanner 512 moves the stage 511 parallel to the surface on which the sample 501 is placed. As a result, after placing the sample 501, the operator can easily move the portion of the sample 501 that the operator wants to observe to the observation area of ​​the observation device 1. In addition, the stage scanner 512 moves the stage 511 perpendicular to the surface on which the sample 501 is placed. As a result, the operator can easily focus on the sample 501 and also perform three-dimensional observation of the sample 501.

[0019] (First Objective Lens and Second Objective Lens) The first objective lens 40 and the second objective lens 45 each include an objective lens 401, 451. The objective lenses 401, 451 are arranged on opposite sides of the stage 511. The objective lenses 401, 451 are arranged to share a focal plane P501. This focal plane P501 may also be referred to as the sample plane P501. In this case, the pupil planes P401 and P451 of the objective lenses 401 and 405 are conjugate. This prevents the phase contrast image from becoming unclear, artifacts from being superimposed on the SRS image and SRS spectrum, and reduced detection sensitivity. It is desirable that the axial and off-axial focal points of the objective lenses 401, 451 be the same for all wavelengths from the visible range to the near-infrared range. Therefore, it is desirable that axial and lateral chromatic aberrations are sufficiently corrected. This reduces misalignment between the SRS image and the phase-contrast image in the horizontal direction (i.e., the direction of the sample surface P501) and in the depth direction (i.e., the optical axis direction), improving observation accuracy. Furthermore, it is desirable for the objective lenses 401 and 451 to have the same numerical aperture. This reduces artifacts that may be superimposed on the acquired SRS image and SRS spectrum.

[0020] (Phase Difference Light Source Unit) The first emission optical system 10 (phase difference light source unit) includes an LED 101, a collimator 102, a ring slit 103, relay lenses 104 and 106, and a pinhole 105. The LED 101 emits incoherent visible light. It is desirable that the LED 101 be considered a point light source. The LED 101 may be optically coupled to one end of a multimode fiber, for example, and emit visible light from the other end of the multimode fiber. The ring slit 103 comprises a portion through which annular light passes and a light-blocking portion other than that. The ring slit 103 is positioned at a position P103 conjugate with a pupil plane P401 of the objective lens 401. The pinhole 105 is positioned at a position P105 conjugate with a focal plane P501 of the objective lens, which will be described later. The ring slit 103 may also be referred to as a ring diaphragm 103, an annular slit 103, a circular annular slit 103, etc.

[0021] The first emission optical system 10 (phase difference light source unit) is another way of saying an irradiation optical system configured to emit first primary light including a spatially modulated component.

[0022] (Phase Difference Detection Unit) The first detection unit 70 (phase difference detection unit) includes relay lenses 702 and 704, a pinhole 703, a phase plate 705, a tube lens 706, and a photodetector 701. The pinhole 703 is disposed at a position P703 conjugate with the focal plane P501 of the objective lens 451. The phase plate 705 consists of an annular portion corresponding to the ring slit 103 and the remaining portion. The annular portion is formed with a wave plate that shifts the phase of light by 1 / 4 relative to the remaining portion, and an ND filter that attenuates light. The phase plate 705 is disposed at a position P705 conjugate with the pupil plane P451 of the objective lens 451. The light receiving surface of the photodetector 701 is disposed at the focal plane P701 of the tube lens 706, and is disposed at a position conjugate with the focal plane P501 of the objective lens 451. The focal plane P701 may also be referred to as the light receiving surface P701. The photodetector 701 includes, for example, a photodiode or a photomultiplier tube. The intensity of the visible light received by the photodetector 701 is acquired as a phase difference signal by the control unit 90. The annular portion of the phase plate 705 in this embodiment may be referred to as a circular annular portion.

[0023] (Second Emission Optical System) The observation device 1 according to this embodiment includes pulsed lasers 201 and 211 as pulsed light sources that are optically coupled to the second emission optical system and emit two synchronized pulsed beams (two types of pulsed beams) with different oscillation wavelengths. These two pulsed beams include Stokes beams and pump beams that exhibit a nonlinear optical effect on the sample 501.

[0024] The second emission optical system 20 (SRS light source unit) includes pulse lasers 201 and 211 with different oscillation wavelengths, and corresponding relay lenses 202 and 203, a mirror 204, relay lenses 212 and 213, and a dichroic mirror 214. Furthermore, the second emission optical system 20 (SRS light source unit) includes a laser synchronization detection unit 221 that detects synchronization between the emission timings of the pulsed light beams from the pulse lasers 201 and 211. Therefore, the second emission optical system 20 (SRS light source unit) also includes beam splitters 222 and 223 that reflect a portion of the light emitted from the pulse lasers 201 and 211 and guide the reflected light to the pulse synchronization detection unit 221.

[0025] For example, a mode-locked picosecond titanium sapphire laser, a mode-locked picosecond neodymium laser, a mode-locked picosecond ytterbium laser, etc. can be used as the pulsed lasers 201 and 211. One of the pulsed lasers 201 and 211 may be replaced with an optical parametric oscillator that converts the wavelength of the pulsed laser generated by the other of the pulsed lasers 201 and 211.

[0026] Of the pulsed light beams emitted from the pulsed lasers 201 and 211, the light beam with a shorter wavelength is used as pump light in SRS observation, and the light beam with a longer wavelength is used as Stokes light in SRS observation.

[0027] The repetition frequency of the emission of pulsed light from the pulsed lasers 201, 211 is half that of the other. For example, the repetition frequency of the Stokes light is half that of the pump light. The synchronization of the pump light and the Stokes light is detected by a pulse synchronization detection unit 221. Based on the synchronization signal from the pulse synchronization detection unit 221, the control unit 90 controls, for example, the resonator length of one or both of the pulsed lasers 201, 211, to maintain a synchronized state of the pump light and the Stokes light for a period of time sufficiently long for SRS observation.

[0028] The dichroic mirror 214 has wavelength characteristics that allow it to transmit the pulsed light emitted from the pulsed laser 201, while reflecting the pulsed light emitted from the pulsed laser 211. The dichroic mirror 214 is disposed so that the reflected light and transmitted light overlap coaxially.

[0029] The pulse synchronization detection unit 221 includes a mirror 224, a dichroic mirror 225, a lens 226, and a two-photon detector 227. The dichroic mirror 225 has wavelength characteristics that transmit the pulsed light emitted from the pulsed laser 201 while reflecting the pulsed light emitted from the pulsed laser 211. The dichroic mirror 225 is arranged so that the transmitted light and reflected light overlap coaxially. The two-photon detector 227 detects two-photon absorption that occurs when both the pump light and the Stokes light pulses arrive at the same time.

[0030] The pulsed lasers 201 and 211 and the pulse synchronization detector 221 may include a delay optical path (not shown) as needed.

[0031] (Second Detector) The second detector 80 (SRS detector) includes a bandpass filter 802, a lens 803, and a photodetector 801. The bandpass filter 802 has wavelength characteristics that transmit only the pulsed light having the higher repetition frequency out of the pump light and the Stokes light. The lens 803 reduces the beam diameter of the pulsed light that has passed through the bandpass filter 802 so that it fits within the light receiving surface of the photodetector 801. The photodetector 801 includes, for example, a photodiode. The SRS signal is acquired by lock-in detection in the control unit 90 of the modulated component of the intensity of the pulsed light received by the photodetector 801.

[0032] (Scanning Unit) The scanning unit 30 includes a biaxial scanner 301 and relay lenses 302 and 303. The biaxial scanner 301 has two mirrors that oscillate around two orthogonal axes, two-dimensionally displacing the angle of incident light relative to the optical axis. The amount of displacement is controlled by the magnitude of the oscillation, thereby controlling the scanning range on the sample surface. The sample surface is scanned two-dimensionally by varying the oscillation frequencies of the two mirrors. It is desirable that the midpoint between the two mirrors be conjugate with the pupil plane P401 of the objective lens 401 via the relay lenses 302 and 303. The biaxial scanner 301 can be, for example, a combination of a single-axis resonant scanner and a single-axis galvanometer scanner. The relay lenses 302 and 303 allow light to enter the pupil of the objective lens at an appropriate beam diameter and maximum angle.

[0033] (Relay Unit) The relay unit 60 includes relay lenses 601 and 602 and a two-axis scanner 603. The relay lenses 601 and 602 allow light to be incident on the two-axis scanner 603 with an appropriate beam diameter and maximum angle. The two-axis scanner 603 has two mirrors that oscillate around two axes that are orthogonal to each other. These mirrors oscillate so as to cancel out the angular displacement of the incident light beam relative to the optical axis.

[0034] That is, one mirror of the biaxial scanner 603 oscillates with one mirror of the biaxial scanner 301, and the other mirror of the biaxial scanner 603 oscillates with the other mirror of the biaxial scanner 301 at the same frequency and in phase or in opposite phase (depending on the installation method). Therefore, the light beam emitted from the biaxial scanner 603 travels parallel to the optical axis, or ideally, on the optical axis. This operation of the biaxial scanner is sometimes called descanning. It is desirable to position the middle of the two mirrors of the biaxial scanner at a position conjugate with the pupil plane P451 of the objective lens 451 via the relay lenses 601 and 602.

[0035] In particular, when the objective lens 451 and the objective lens 401 are the same, the relay lenses 601 and 602 can be the same as the relay lenses 303 and 302, respectively, and the two-axis scanner 603 can be the same as the two-axis scanner 301. In this case, the relay lenses 601 and 602 and the two-axis scanner 603 can be arranged symmetrically with the relay lenses 303 and 302 and the two-axis scanner 301 with respect to the sample surface.

[0036] In terms of its function, the relay unit 60 of the first embodiment having the biaxial scanner 603 may be referred to as the reverse scanning unit 60. The reverse scanning unit 60 is configured to perform reverse scanning in synchronization with the scanning unit 30 on the optical path between the branching unit 75 and the second objective lens 45.

[0037] (Wave-combining unit and wave-demultiplexing unit) The wave-combining unit 15 and the wave-demultiplexing unit 75 each include a dichroic mirror 151, 751. The dichroic mirror 151 has wavelength characteristics that transmit the light emitted from the LED 101 and reflect the light emitted from the pulsed lasers 201, 211. The dichroic mirror 151 is disposed so that the reflected light and transmitted light overlap coaxially. The dichroic mirror 751 has wavelength characteristics that transmit the light emitted from the LED 101 and the light emitted from the sample 501, while reflecting the light emitted from the pulsed lasers 201, 211 and the light emitted from the sample 501.

[0038] (Control Unit) The control unit 90 includes a control device 901, a keyboard 911, a mouse 912, and a display 921. The control device 901 can be formed by implementing a program that executes the control flow on a computer. The control device 901 may also include an FPGA, a microcomputer, or an electrical circuit that executes part of the control flow, or part of the program may be implemented on these. The control device 901 also includes a lock-in amplifier (not shown) that detects the modulated component of the light intensity detected by the photodetector 801 for SRS observation. Note that this intensity modulation can be referred to as an SRS signal. The control device 901 may also include an electrical circuit that controls one or both of the pulse lasers 201 and 211 based on a synchronization signal from the pulse synchronization detection unit 221. The control device 901 may also include an electrical circuit that generates a drive signal for the two-axis scanners 301 and 603.

[0039] The control device 901 generates a phase contrast image and an SRS image based on the signals output by the photodetector 701 and the photodetector 801, respectively. At this time, the control device 901 reads out the phase contrast signal and the SRS signal at the same pixel frequency (pixel rate) and converts them into luminance, thereby generating images with the same number of pixels. The generated phase contrast image and SRS image are stored in a storage device (not shown) of the control device 901 or output to a display 921. The storage device is, for example, a solid state drive or a hard disk drive.

[0040] A keyboard 911 and a mouse 912 are connected to the control device 901, and an operator operates the keyboard 911 and the mouse 912 to input instructions to the control device 901. The control device 901 is connected to the LED 101, the pulse laser 201, the pulse laser 211, the two-axis scanners 301 and 603, the stage scanner 512, and the photodetectors 701 and 801, and controls the operations of these elements in accordance with instructions from the operator.

[0041] The display 921 provides visual feedback to the operator's operations, and also displays images and character strings output by the control device 901 .

[0042] 3 is a schematic diagram illustrating phase-contrast observation using the observation device 1. Phase-contrast observation using the observation device 1 uses a first emission optical system 10 (phase-contrast light source unit), a combining unit 15, a scanning unit 30, a first objective lens 40, a mounting unit 50, a second objective lens 45, a relay unit 60, a branching unit 75, a first detection unit 70 (phase-contrast detection unit), and a control unit 90.

[0043] As shown by the dotted line in FIG. 3 , light emitted from the LED 101 of the first emission optical system 10 (phase-contrast light source) is collimated by the collimator 102. The annular light beam that passes through the ring slit 103 is focused to a single point toward the pinhole 105 by the relay lens 104. At this time, first-order and higher-order diffracted light generated at the edge of the ring slit 103 widens the focused point. The pinhole 105 blocks light from the periphery of the focused point while allowing light closer to the center to pass, thereby increasing the proportion of light that travels straight through the ring slit 103 (in other words, zeroth-order diffracted light). This prevents the light beam, which has been refocused into an annular beam by the relay lens 106, from losing its shape as it propagates through the subsequent optical path. This reduces artifacts in the acquired phase-contrast image.

[0044] The parallel light beam, the diameter of which has been enlarged or reduced by the relay lens 106 , passes through the dichroic mirror 151 of the multiplexing unit 15 and enters the two-axis scanner 301 of the scanning unit 30 .

[0045] The annular light beam is reflected by each of the two mirrors of the biaxial scanner 301. The diameter of the annular light beam emitted from the biaxial scanner 301 parallel to the optical axis or at an angle to the optical axis is expanded or reduced by relay lenses 302 and 303, and the light beam is incident on the objective lens 401 of the first objective lens 40. The objective lens 401 focuses the light beam to a single point toward the sample 501 on the mounting unit 50. The position of this focusing point on the sample plane P501 corresponds to the angle of the light beam emitted from the biaxial scanner 301 with respect to the optical axis. At this time, in addition to the straight light that travels straight through the sample (in other words, the zeroth-order diffracted light), first-order or higher diffracted light is generated according to the refractive index distribution and shape of the sample.

[0046] The straight light and diffracted light emerging from the sample 501 are collected by the objective lens 451 and emerge as parallel beams. At this time, the straight light at the sample 501 forms a ring shape, and the inside and outside of the ring represent diffracted light generated at the sample 501. The emergence angle of this parallel beam corresponds to the position of the focal point on the sample surface P501, i.e., the angle relative to the optical axis of the beam emerging from the biaxial scanner 301. The beam diameter of the beam emerging from the objective lens 451 is expanded or reduced by relay lenses 601 and 602 of the relay unit 60, and the beam is incident on the biaxial scanner 603. This beam is reflected by each of the two mirrors of the biaxial scanner 603. Regardless of the incident angle, the beam emerging from the biaxial scanner 603 parallel to or aligned with the optical axis passes through the dichroic mirror 751 of the demultiplexing unit 75 and enters the relay lens 702 of the first detection unit 70 (phase difference detection unit).

[0047] This light beam is focused to a single point by a relay lens 702 toward a pinhole 703. The pinhole 703 blocks light from the periphery of the focused point, thereby blocking diffracted light and scattered light generated outside the focused point within the sample plane P501. This makes it possible to suppress artifacts in the phase-contrast image to be acquired.

[0048] The light beam that has passed through the pinhole 703 has its beam diameter expanded or reduced by the relay lens 704, becoming a parallel light beam again, and then passing through the phase plate 705. At this time, the straight light in the sample 501 passes through the annular portion and is phase-modulated and attenuated, while the diffracted light in the sample 501 passes through portions other than the annular portion. The light beam that has emerged from the phase plate 705 is focused by the tube lens 706 onto a single point toward the light-receiving surface P701 of the photodetector 701. At the light-receiving surface P701, the straight light and diffracted light in the sample 501 interfere with each other, and the photodetector 701 detects the light intensity.

[0049] 4 is a schematic diagram illustrating SRS observation using the observation device 1. SRS observation using the observation device 1 uses a second emission optical system 20 (SRS light source unit), a multiplexing unit 15, a scanning unit 30, a first objective lens 40, a mounting unit 50, a second objective lens 45, a relay unit 60, a branching unit 75, a second detection unit 80 (SRS detection unit), and a control unit 90.

[0050] 4, a part of the parallel light beam emitted by the pulsed laser 201 of the SRS light source unit is reflected by the beam splitter 222 and enters the pulse synchronization detection unit 221, while the majority of the parallel light beam is transmitted and enters the relay lens 202. The light beam, whose beam diameter has been expanded or reduced by the relay lenses 202 and 203, is reflected by the mirror 204 and then transmitted through the dichroic mirror 214.

[0051] A part of the parallel light beam emitted by the pulse laser 211 is reflected by the beam splitter 223 and enters the pulse synchronization detection unit 221, while the majority of the light beam is transmitted and enters the relay lens 212. The light beam, whose beam diameter has been expanded or reduced by the relay lenses 212 and 213, is reflected by the dichroic mirror 214. At this time, the light beam is superimposed coaxially on the light beam that has passed through the dichroic mirror 214.

[0052] The light beam from the pulsed laser 201 reflected by the beam splitter 222 is reflected by a mirror 224 of the pulse synchronization detection unit 221 and passes through a dichroic mirror 225. The light beam from the pulsed laser 211 reflected by the beam splitter 223 is reflected by the dichroic mirror 225. At this time, it is superimposed coaxially on the light beam that passed through the dichroic mirror 225. This superimposed coaxial light beam (in other words, both the pump light beam and the Stokes light beam) is focused by a lens 226 toward a two-photon detector 227.

[0053] The light beams coaxially superimposed by the dichroic mirror 214 (in other words, both the pump light and the Stokes light beams) are reflected by the dichroic mirror 151 of the multiplexing unit 15. At this time, the light beams are coaxially superimposed on the annular light beams that have been emitted from the first emission optical system 10 (phase difference light source unit) and transmitted through the dichroic mirror 151.

[0054] The light beam reflected by the dichroic mirror 151 enters the biaxial scanner of the scanning unit 30 and is reflected by each of the two mirrors. The beam diameter of the light beam emerging from the biaxial scanner 301 is expanded or reduced by relay lenses 302 and 303, and the light beam enters the objective lens 401 of the first objective lens 40. The objective lens 401 focuses the light beam to a single point toward the sample 501 on the mounting unit 50. The position of the focusing point within the sample plane P501 corresponds to the angle relative to the optical axis when the light beam emerges from the biaxial scanner 301. At this time, an SRS process occurs at the focusing point. That is, stimulated Raman loss occurs for the pump beam and stimulated Raman gain occurs for the Stokes beam in response to the vibrational levels of the molecules present at the focusing point. In addition to the pump beam and the Stokes beam, the light emerging from the first exit optical system 10 (phase-contrast light source) is also focused coaxially, so the focusing points coincide. Therefore, the SRS image and the phase contrast image are acquired simultaneously and have the same field of view.

[0055] The pump light and Stokes light emitted from the sample 501 are collected by the objective lens 451 and emitted as parallel beams. At this time, the emission angle with respect to the optical axis corresponds to the position of the focal point on the sample surface P501, in other words, the angle with respect to the optical axis of the beam emitted from the two-axis scanner 301. The beam diameter of the beam emitted from the objective lens 451 is expanded or reduced by relay lenses 601 and 602 of the relay unit 60, and the beam is incident on the two-axis scanner 603.

[0056] The light beams are reflected by each of the two mirrors of the two-dimensional scanner 603. Regardless of the angle of incidence, the pump light and Stokes light beams emitted from the two-axis scanner 603 parallel to or coincident with the optical axis are reflected by the dichroic mirror 751 of the demultiplexing unit 75 and enter the bandpass filter 802 of the second detection unit 80 (SRS detection unit). The bandpass filter 802 transmits only the pump light or the Stokes light with a higher pulse repetition frequency, and blocks the light with a lower repetition frequency (half of the higher one). The light beams transmitted through the bandpass filter 802 are focused by the lens 803 onto the light-receiving surface of the photodetector 801, and the photodetector 801 detects the light intensity.

[0057] (Observation Flow) Figures 5A to 5C are diagrams illustrating an observation flow of a sample 501 using the observation device 1. Figure 5A is a flowchart explaining the observation flow, and Figure 5B is a diagram explaining a low-magnification field of view R502, a high-magnification field of view R503, and a region of interest R504 in the flowchart. Figure 5C is a diagram showing the schematic configuration of a scanning condition determination unit. The region of interest R504 may also be referred to as a region of interest R504.

[0058] 5C , the control unit 90 includes an image generation unit 92 that generates a first image captured by the first objective lens 40 and the second objective lens 45 at a predetermined magnification and displays the first image on a display unit 921 (display). The control unit 90 further includes an input unit 910 that accepts input of information regarding a region of interest from an operator based on the displayed first image, and a scanning condition acquisition unit 94 that acquires scanning conditions SC0 for the scanning unit 30 based on the accepted information regarding the region of interest. The control unit 90 further includes a scanning condition determination unit 96 that determines scanning conditions SC1 for the scanning unit 30 during imaging based on the acquired scanning conditions SC0. The control unit 90 further includes an update unit 98 that displays the scanning conditions SC1 acquired by the scanning condition acquisition unit 96 on the display unit and accepts updates to the scanning conditions SC1.

[0059] First, the sample 501 is held on the stage 511. From this state, the observation flow starts. First, the sample 501 is observed with phase contrast over a wide field of view. Conditions for low-magnification phase contrast observation are input to the control device 901, and low-magnification phase contrast observation is started (steps S10 and S20).

[0060] The stage scanner 512 is driven to change the position of the stage 511, thereby moving the low-magnification field of view R502 shown in FIG. 5B. If the region of interest R504 of the sample 501 is not present within the low-magnification field of view R502, the low-magnification field of view R502 is moved to continue low-magnification phase-contrast observation. However, if the region of interest R504 is found, the low-magnification phase-contrast observation is completed (step S30). In this way, by first performing real-time phase-contrast observation of the sample 501 with a wide field of view, the time required to determine the position of the sample 501 relative to the sample surface P501 and to find the region of interest R504 can be reduced. This reduces damage to the sample 501.

[0061] Next, a determination is made as to whether or not to check the details of the phase-contrast image of the region of interest R504 from the low-magnification phase-contrast image. (Step S40) If checking the details, the field of view is narrowed and real-time phase-contrast observation is performed. In this case, high-magnification phase-contrast observation conditions are input into the control device 901, and high-magnification phase-contrast observation is initiated. (Steps S50 and S60) If necessary, the high-magnification field of view R503 shown in FIG. 5B is moved so that the region of interest R504 fits within the field of view, and the necessity of SRS observation is examined from the high-magnification phase-contrast image. On the other hand, if it is determined from these phase-contrast images that SRS observation is not necessary, the system returns to low-magnification phase-contrast observation and searches for another region of interest R504 on the sample 501. On the other hand, if it is determined that SRS observation is necessary, the system proceeds to SRS observation. (Step S70) In this way, smoothly enlarging the region of interest R504 on the sample 501 and performing phase-contrast observation allows the necessity of SRS observation to be quickly determined. Therefore, unnecessary SRS observations can be reduced, which not only improves measurement efficiency but also suppresses damage to the sample.

[0062] Next, before starting SRS observation, a decision is made as to whether to perform only SRS observation or both SRS observation and phase-contrast observation (step S80). If only SRS observation is to be performed, the SRS observation conditions are input into the control device 901, and SRS observation is initiated (steps S90 and S110). On the other hand, if both SRS observation and phase-contrast observation are to be performed, both the SRS observation conditions and the phase-contrast observation conditions are input into the control device 901, and simultaneous SRS observation and phase-contrast observation is initiated (steps S100 and S120). Once SRS observation or simultaneous SRS observation and phase-contrast observation is completed, the series of observation flows ends. In this way, when observing dynamic events in the sample 501 that require simultaneous SRS and phase-contrast images, for example, the observation accuracy can be improved by selecting simultaneous observation of both. Furthermore, when phase-contrast images are not required, unnecessary light is prevented from being irradiated onto the sample 501, thereby minimizing damage.

[0063] The low-magnification and high-magnification phase-contrast observation conditions are, for example, the intensity of the light output of the LED 101, the number of times of integration, and the size of the field of view. The SRS observation conditions are, for example, the intensity and wavelength of the light output of the pulse lasers 201 and 211, the number of times of integration, and the size of the field of view.

[0064] In the observation flow of FIG. 5A , when the region of interest R504 is found in step S30, the operator inputs each observation condition in the subsequent steps S50, S90, and S100. To improve operator convenience, step S35, in which the operator inputs position information of the region of interest R504, may be added between steps S30 and S40. The position information can be input, for example, by the operator tracing the outline of the region of interest R504 or drawing a mark using the mouse 912 on the low-magnification phase-contrast image displayed on the display 921 of the control unit 90. Then, based on the position information, the control device 901 may predict appropriate observation conditions and present them to the operator in steps S50, S90, and S100. Supporting the operator in inputting each observation condition in this way reduces the burden on the operator.

[0065] When step S35 is added, the control device 901 may determine appropriate observation conditions based on the position information and automatically input the observation conditions in steps S50, S90, and S100. This eliminates the need for the operator to input the observation conditions, further reducing the burden on the operator.

[0066] In the observation device 1 of FIG. 2, the ring slit 103 is arranged in the first emission optical system 10 (phase difference light source unit), and the phase plate 705 is arranged in the first detection unit 70 (phase difference detection unit), but a phase difference image can be obtained even if the arrangement of these two is reversed.

[0067] As described above, the observation device according to the first embodiment of the present invention simultaneously acquires a phase contrast signal and an SRS signal from the same point to generate a phase contrast image and an SRS image with the same number of pixels. In other words, it is possible to acquire primary images of phase contrast images and SRS images with matched image quality. This makes it possible to reduce the burden on the operator associated with matching image quality.

[0068] Second Embodiment The configuration of an observation device according to a second embodiment will be described with reference to Fig. 6. Fig. 6 is a schematic diagram illustrating the configuration of an observation device 2 according to this embodiment. The observation device 2 has the same configuration as the observation device 1 according to the first embodiment, except for the parts that will be described next. Therefore, common elements are assigned the same reference numbers, and duplicate descriptions will be omitted.

[0069] 6 , the biaxial scanner 603 of the relay unit 60 of the observation device 1 has been removed, and a dichroic mirror 751 of the branching unit 75 has been installed in its place. In addition, the pinhole 703 of the first detection unit 70 (phase difference detection unit) of the observation device 1 has been removed. Furthermore, the lens 803 of the second detection unit 80 (SRS detection unit) of the observation device 1 has been replaced with relay lenses 804 and 805. The relay unit 60 of the second embodiment differs from that of the first embodiment in that it is not electrically connected to the control unit 90.

[0070] (Relay Unit) The observation device 2 does not have a two-axis scanner that cancels out the angular displacement of the incident light beam relative to the optical axis of the relay unit 60. Therefore, the angle of the light beam emerging from the relay unit 60 of the observation device 2 relative to the optical axis corresponds to the position of the focal point on the sample surface P501, in other words, the angle of the light beam emerging from the two-axis scanner 301 of the scanning unit 30 relative to the optical axis.

[0071] (Branching Unit) The dichroic mirror 751 of the branching unit 75 of the observation device 2 has wavelength characteristics similar to those of the observation device 1. The dichroic mirror 751 of the observation device 2 is installed near a position conjugate with the pupil plane P451 of the objective lens 451 of the second objective lens 45 via the relay lenses 601 and 602 of the relay unit 60.

[0072] (Phase Difference Detection Unit) The first detection unit 70 (phase difference detection unit) of the observation device 2 does not have a pinhole 703 at a position conjugate to the focal plane P501 of the objective lens 451 (in other words, an intermediate image plane) P703. At the intermediate image plane P703, a point-like image moves in response to the focal point scanning the sample plane P501. Similarly, a point-like image also moves at the focal plane P701 of the tube lens 706, which is located at a position conjugate to the focal plane P501. Therefore, the photodetector 701 of the observation device 2 requires a larger light-receiving area than that of the observation device 1. For this reason, the photodetector 701 of the observation device 2 may use a photodiode array or an image sensor in which multiple photodiodes are arranged two-dimensionally.

[0073] The phase plate 705 of the first detection unit 70 (phase difference detection unit) of the observation device 2 is similar to the observation device 1 in that it is placed at a position conjugate with the pupil plane P451 of the objective lens 451. However, it differs from the observation device 1 in that the angle of the light beam passing through the phase plate 705 with respect to the optical axis changes according to the position of the focal point on the sample plane P501, in other words, the angle of the light beam emitted from the two-axis scanner 301 with respect to the optical axis. Taking this into consideration, it is necessary to design an annular portion that modulates the phase of the straight light (zeroth-order diffracted light) at the sample 501 and attenuates the light.

[0074] 7A and 7B are schematic diagrams illustrating the relationship between the phase plate 705 of the observation device 2 and the light beams passing through it. The phase plate 705 has two concentric cylinders 705_1 and 705_2 around a central axis 705_0 that coincides with the optical axis. A quarter-wave plate and an ND filter are formed in the region sandwiched between the inner cylinder 705_1 and the outer cylinder 705_2 (the shaded area in FIGS. 7A and 7B ). An annular parallel beam of light generated by the straight light at the sample 501 passes through this region. Of this annular beam of light, the innermost beam L103_1 and the outermost beam L103_2 form concentric cylinders around the chief ray of the parallel beam of light (including both the straight light and diffracted light at the sample 501) that passes through the phase plate 705. The cylindrical diameters of these light beams L103_1 and L103_2 correspond to the inner and outer diameters, respectively, of the ring slit 103 that exists at a position conjugate with the plane P705 on which the center of the thickness of the phase plate 705 is located. Specifically, by multiplying the inner radius I and the outer radius E of the ring slit 103 by the imaging magnification m, the radius of the light beam L103_1 becomes mI and the radius of the light beam L103_2 becomes mE.

[0075] 7A shows the case where the angle of the parallel light beam transmitted through the phase plate 705 with respect to the optical axis is maximum. In FIG. 7A, the chief ray L103_0 is at a maximum angle θ ０ and intersect on a plane P705 that coincides with the center of the thickness of the phase plate 705. At this time, all of the light beams between the light beams L103_1 and L103_2 pass through the region sandwiched between the cylinders 705_1 and 705_2 of the phase plate 705, so that the radii of the cylinders 705_1 and 705_2 satisfy the following formulas 1 and 2.

[0076]

[0077]

[0078] In addition, in Formula 1 and Formula 2, the incident angle of the light beam on the phase plate 705 is assumed to be at most 10°, and refraction occurring at a quarter-wave plate, an ND filter, etc. is not taken into consideration. More accurately, it is desirable to determine the above radius taking these refractions into consideration.

[0079] 7B shows the case where the parallel light beams passing through the phase plate 705 become parallel to the optical axis. When the formulas 1 and 2 are satisfied, all of the light beams between the light beams L103_1 and L103_2 pass through the region of the phase plate 705 sandwiched between the cylinders 705_1 and 705_2.

[0080] In this way, by designing the phase plate 705 so that all light traveling straight through the sample 501 undergoes phase modulation and dimming in the phase plate 705 regardless of the angle relative to the optical axis when passing through the phase plate 705, artifacts in the acquired phase contrast image can be suppressed.

[0081] (SRS Detector) The second detector 80 (SRS detector) of the observation device 2 includes relay lenses 804 and 805, a bandpass filter 802, and a photodetector 801. The relay lenses 804 and 805 expand or reduce the beam diameter of the incident light beam to suit the size of the light receiving surface of the photodetector 801. Between the relay lenses 804 and 805, there is a plane P804 conjugate to the focal plane P501 of the objective lens 451 of the second objective lens 45. The light receiving surface of the photodetector 801 of the observation device 2 is disposed at a position conjugate to the pupil plane P451 of the objective lens 451. The bandpass filter 802 of the observation device 2 has wavelength characteristics similar to those of the observation device 1 and is disposed between the relay lens 805 and the photodetector 801.

[0082] As described above, the observation apparatus according to the second embodiment of the present invention does not have a two-axis scanner in the relay unit 60, and therefore, in addition to simplifying control, it is possible to facilitate optical adjustment, thereby improving the stability of the observation apparatus.

[0083] In the above embodiment, in order to acquire a Raman image with high sensitivity and high speed, coherent pulsed light of different wavelengths is focused on the sample to generate the SRS process and perform imaging. However, a Raman image can also be acquired by focusing incoherent light of a predetermined wavelength on the sample to generate the spontaneous Raman scattering process and perform imaging. In this case, for example, a monochromator can be used as the detection unit. In this case, the same effect can be obtained.

[0084] Although the present invention has been described above using the embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be apparent to those skilled in the art that various modifications and improvements can be made to the above embodiments. It is clear from the claims that such modifications and improvements can also be included within the technical scope of the present invention.

[0085] As described above, the scanning observation apparatus according to the embodiment of the present invention includes the following configurations 1 to 14.

[0086] (Configuration 1) A first emission optical system that emits first primary light including a spatially modulated component; a second emission optical system that emits coherent second primary light; a multiplexing unit that coaxially multiplexes the first primary light and the second primary light; a scanning unit that coaxially scans the multiplexed first primary light and the second primary light; a first objective lens that collects the scanned first primary light and the second primary light; a mounting unit that mounts a sample at a focusing position of the first objective lens; a second objective lens that collects secondary light from the sample; a demultiplexing unit that demultiplexes the secondary light into first secondary light emitted from the sample by irradiation with the first primary light and second secondary light emitted from the sample by irradiation with the second primary light; a first detection unit that detects the first secondary light; and a second detection unit that detects the second secondary light. A scanning observation device having:

[0087] (Configuration 2) The scanning observation apparatus according to Configuration 1, further comprising a pulsed light source optically coupled to the second emission optical system and emitting two synchronized pulsed beams having different oscillation wavelengths.

[0088] (Configuration 3) The scanning observation apparatus according to Configuration 2, wherein the two pulsed beams include Stokes beam and pump beam that exhibit a nonlinear optical effect on the sample.

[0089] (Configuration 4) The scanning observation device according to any one of Configurations 1 to 3, wherein the second secondary light includes light emitted from the sample due to a nonlinear optical effect that occurs in the sample when irradiated with the second primary light.

[0090] (Configuration 5) The scanning observation apparatus according to any one of Configurations 1 to 4, wherein the first emission optical system includes a first light source that emits incoherent light of a predetermined wavelength.

[0091] (Configuration 6) In the scanning observation device according to any one of Configurations 1 to 5, the first emission optical system includes a modulation optical element that annularly modulates an intensity component of the first primary light, and the first detection unit includes a demodulation optical element having an annular portion that demodulates an intensity component and a phase component of the first secondary light.

[0092] (Configuration 7) The scanning observation apparatus according to any one of Configurations 1 to 6, wherein the first exit optical system includes a pinhole that blocks a part of the collected first primary light.

[0093] (Configuration 8) The scanning observation device according to any one of Configurations 1 to 7, wherein the first emission optical system includes a modulation optical element having an annular portion that modulates an intensity component and a phase component of the first primary light, and the first detection unit includes a demodulation optical element that annularly demodulates the intensity component of the first secondary light.

[0094] (Configuration 9) The scanning observation device according to any one of Configurations 1 to 8, further comprising a reverse scanning unit that performs reverse scanning in synchronization with the scanning unit, on an optical path between the branching unit and the second objective lens.

[0095] (Configuration 10) The scanning observation device according to Configuration 9, wherein the reverse scanning unit focuses the first secondary light toward the first detection unit, and the first detection unit includes a pinhole that blocks a portion of the focused first secondary light.

[0096] (Configuration 11) The scanning observation device according to any one of Configurations 1 to 10, further comprising an image generation unit that generates a first image captured by the first objective lens and the second objective lens at a predetermined magnification, and displays the first image on a display unit.

[0097] (Configuration 12) The scanning observation device according to Configuration 11, including: an input unit that receives input of information related to a region of interest from the operator based on the displayed first image; and a scanning condition acquisition unit that acquires scanning conditions for the scanning unit based on the received information related to the region of interest.

[0098] (Configuration 13) The scanning observation apparatus according to Configuration 12, further comprising: a scanning condition determination unit that determines scanning conditions for the scanning unit during imaging based on the acquired scanning conditions.

[0099] (Configuration 14) The scanning observation apparatus according to Configuration 12, further comprising an update unit that displays the scanning conditions acquired by the scanning condition acquisition unit on the display unit and accepts updates of the scanning conditions.

[0100] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0101] This application claims priority based on Japanese Patent Application No. 2023-204961, filed December 4, 2023, the entire contents of which are incorporated herein by reference.

[0102] REFERENCE SIGNS LIST 1, 2 Scanning observation device 10 First emission optical system (phase difference light source unit) 15 Combining unit 20 Second emission optical system (SRS light source unit) 30 Scanning unit 40 First objective lens (light collecting unit) 45 Second objective lens (light collecting unit) 50 Mounting unit 60 Reverse scanning unit (relay unit) 70 First detection unit (phase difference detection unit) 75 Branching unit 80 Second detection unit (SRS detection unit) 90 Control unit

Claims

1. A scanning observation device having: a first emission optical system that emits a first primary light including a spatially modulated component; a second emission optical system that emits a coherent second primary light; a combining unit that coaxially combines the first primary light and the second primary light; a scanning unit that coaxially scans the combined first primary light and the second primary light; a first objective lens that focuses the scanned first primary light and the second primary light; a mounting unit that mounts a sample at a focusing position of the first objective lens; a second objective lens that collects secondary light from the sample; a demultiplexing unit that demultiplexes from the secondary light into a first secondary light emitted from the sample by irradiation with the first primary light and a second secondary light emitted from the sample by irradiation with the second primary light; a first detection unit that detects the first secondary light; and a second detection unit that detects the second secondary light.

2. A scanning observation apparatus according to claim 1, further comprising a pulse light source optically coupled to said second emission optical system for emitting two pulsed beams having different oscillation wavelengths and synchronized with each other.

3. A scanning observation apparatus according to claim 2, wherein the two pulsed beams include Stokes beam and pump beam which produce a nonlinear optical effect on the sample.

4. A scanning observation apparatus according to claim 1 or 2, wherein the second secondary light includes light emitted from the sample due to a nonlinear optical effect produced in the sample by irradiation with the second primary light.

5. A scanning observation apparatus according to claim 1 or 2, wherein the first emission optical system comprises a first light source that emits incoherent light of a predetermined wavelength.

6. A scanning observation device as described in claim 1 or 2, wherein the first emission optical system is provided with a modulation optical element that annularly modulates the intensity component of the first primary light, and the first detection unit is provided with a demodulation optical element having an annular portion that demodulates the intensity component and phase component of the first secondary light.

7. A scanning observation apparatus according to claim 1 or 2, wherein the first exit optical system is provided with a pinhole that blocks a portion of the condensed first primary light.

8. A scanning observation device as described in claim 1 or 2, wherein the first emission optical system is provided with a modulation optical element having an annular portion that modulates the intensity component and phase component of the first primary light, and the first detection unit is provided with a demodulation optical element that annularly demodulates the intensity component of the first secondary light.

9. A scanning observation apparatus according to claim 1 or 2, further comprising a reverse scanning section which performs reverse scanning in synchronization with said scanning section, and which is provided on the optical path between said branching section and said second objective lens.

10. A scanning observation device as described in claim 9, wherein the reverse scanning unit focuses the first secondary light toward the first detection unit, and the first detection unit is provided with a pinhole that blocks a portion of the focused first secondary light.

11. A scanning observation device as described in claim 1 or 2, further comprising an image generating unit that generates a first image captured by the first objective lens of a predetermined magnification and the second objective lens, and displays the first image on a display unit.

12. A scanning observation device as described in claim 11, comprising: an input unit that accepts input of information regarding a region of interest from the operator based on the first image displayed on the display unit; and a scanning condition acquisition unit that acquires scanning conditions for the scanning unit based on the accepted information regarding the region of interest.

13. A scanning observation apparatus according to claim 12, further comprising a scanning condition determination unit that determines scanning conditions for said scanning unit during imaging based on said acquired scanning conditions.

14. The scanning observation apparatus according to claim 12, further comprising an update unit that displays the scanning conditions acquired by the scanning condition acquisition unit on the display unit and accepts updates to the scanning conditions.

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