Scanning observation device

Abstract

To provide an observation device that can easily match the image qualities of a phase difference image and a Raman image.SOLUTION: A scanning observation device has: a first emission optical system 10 that emits first primary light including a spatially modulated component; a second emission optical system 20 that emits coherent second primary light; a multiplexing unit 15 that coaxially multiplexes the first primary light and the second primary light; a scanning unit 30 that coaxially scans the multiplexed first primary light and second primary light; a first objective lens 40 that condenses the scanned first primary light and second primary light; a mounting part 50 that mounts a sample at a condensing position of the first objective lens; a second objective lens 45 that collects secondary light from the sample; a demultiplexing unit 75 that demultiplexes 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 that detects the first secondary light; and a second detection unit 80 that detects the second secondary light.SELECTED DRAWING: Figure 2

Description

Technical Field

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

Background Art

[0002] In recent years, in the fields of regenerative medicine, intraoperative rapid diagnosis, bioproduction, etc., there has been an increasing expectation for methods of observing or evaluating living cells without staining them. As methods for imaging living cells without staining and non-contact, there are a method of detecting the phase difference of light transmitted through the cells, and a method of detecting Raman scattered light generated due to the vibration of molecules constituting the cells.

[0003] Patent Document 1 discloses an apparatus capable of observing living cells using both a phase contrast imaging method for detecting a phase difference as the intensity of light, and a CARS imaging method for detecting anti-Stokes Raman scattered light generated by a non-linear optical effect.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In Patent Document 1, the phase contrast image is obtained by illuminating the entire observation region at once and photographing it with a CCD camera, whereas the CARS image is obtained by condensing the irradiation light on a single point in the observation region and scanning it. That is, different imaging methods were used with different image forming means. Therefore, the primary images obtained by each imaging method had inconsistent image qualities, such as different observation regions or different numbers of pixels. Therefore, in order to match the image qualities, complicated pre-calibration of the apparatus and post-processing of the images were required for the operator. Thus, it has been demanded to reduce the burden on the operator who wants to observe living cells by multiple methods.

[0006] An object of the present invention is to provide a scanning type observation apparatus in which the image qualities of a phase difference image and a Raman image can be easily matched.

Means for Solving the Problems

[0007] The scanning type observation apparatus according to an embodiment of the present invention includes a first emission optical system that emits first primary light including spatially modulated components, a second emission optical system that emits coherent second primary light, a multiplexing unit that multiplexes the first primary light and the second primary light coaxially, a scanning unit that scans the multiplexed first primary light and second primary light coaxially, a first objective lens that condenses the scanned first primary light and second primary light, a placement unit that places a sample at the condensing position of the first objective lens, a second objective lens that collects secondary light from the sample, a demultiplexing unit that demultiplexes the first secondary light emitted from the sample by irradiation with the first primary light and the second secondary light emitted from the sample by irradiation with the second primary light from the secondary light, a first detection unit that detects the first secondary light, and a second detection unit that detects the second secondary light.

Effects of the Invention

[0008] According to the present invention, it becomes possible to provide an observation apparatus in which the image qualities of a phase difference image and a Raman image can be easily matched.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Embodiments for Carrying Out the Invention

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

[0011] <First Embodiment> The configuration of the observation device according to the first embodiment will be described with reference to FIGS. 1 to 5.

[0012] FIG. 1 is a block diagram for explaining the schematic connection relationship of the elements constituting the observation device 1. In FIG. 1, the solid lines connecting between the blocks (rectangles) represent the optical connections of the corresponding components, and the broken lines connecting between the blocks (rectangles) represent that there are connections through which signals related to measurement or control can be transmitted between the corresponding components.

[0013] The observation device 1 includes an optically connected first emission optical system 10 (phase difference light source unit), a second emission optical system 20 (SRS light source unit), a multiplexer 15, a scanning unit 30, a first objective lens 40, and a placement unit 50. The observation device 1 further includes a second objective lens 45, a relay unit 60, a demultiplexer 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 placement 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] Figure 2 is a schematic diagram for explaining 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, such that at least a part of the sample 501 overlaps with the light collection position of the first objective lens 40 described later. The placement unit 50 may be alternatively referred to as a support unit 50 for the sample 501 or a sample stage 50. Also, the light collection position of the first objective lens 40 may be alternatively referred to as the focal point of the first objective lens 40, the light collection point of the first objective lens, etc.

[0017] The stage 511 has a portion that is optically open so that primary light can enter and secondary light can be emitted with respect to the sample 501. The opening can adopt either a form that is closed in the circumferential direction or a form that is not closed.

[0018] Thus, the sample 501 placed on the placement unit 50 via the stage 511 is configured to be irradiated with light from the first objective lens 40. 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. Thereby, after the operator places the sample 501, the operator can easily move the portion of the sample 501 to be observed to the observation region of the observation device 1. Further, the stage scanner 512 moves the stage 511 perpendicular to the surface on which the sample 501 is placed. Thereby, the operator can easily focus on the sample 501 and can 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 objective lenses 401 and 451. The objective lenses 401 and 451 are arranged on opposite sides of the stage 511. The objective lenses 401 and 451 are arranged to share the focal plane P501. In some cases, this focal plane P501 may be referred to as the sample surface P501. At this time, the pupil planes P401 and P451 of the objective lenses 401 and 405 have a conjugate positional relationship. Thereby, it is possible to suppress the phase difference image obtained from becoming unclear, artifacts from being superimposed on the obtained SRS image and SRS spectrum, and the detection sensitivity from decreasing. It is desirable that the on-axis and off-axis foci of the objective lenses 401 and 451 be the same at any wavelength from the visible region to the near-infrared region. Therefore, it is desirable that the on-axis and magnification chromatic aberrations be sufficiently corrected. Thereby, since the deviation in the horizontal direction (in other words, the sample surface P501 direction) and the depth direction (in other words, the optical axis direction) of the SRS image and the phase difference image can be suppressed, the observation accuracy can be improved. Further, it is desirable that the objective lenses 401 and 451 have the same numerical aperture as each other. Thereby, it is possible to suppress the artifacts superimposed on the obtained SRS image and SRS spectrum.

[0020] (Phase difference light source unit) The first projection 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 can be regarded as a point light source. For example, the LED 101 may be optically coupled to one end of a multimode fiber and emit visible light from the other end of the multimode fiber. The ring slit 103 includes a portion through which annular light passes and a light-shielding portion other than that. The ring slit 103 is disposed at a position P103 conjugate to the pupil plane P401 of the objective lens 401. The pinhole 105 is disposed at a position P105 conjugate to the focal plane P501 of the objective lens described later. The ring slit 103 may be alternatively referred to as a ring aperture 103, an annular slit 103, a circular annular slit 103, or the like.

[0021] The first projection optical system 10 (phase difference light source unit) can be alternatively referred to as an illumination optical system configured to project first primary light including spatially modulated components.

[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 to the focal plane P501 of the objective lens 451. The phase plate 705 includes an annular portion corresponding to the ring slit 103 and other portions. In the annular portion, a wave plate that shifts the phase of light by 1 / 4 and a neutral density (ND) filter that attenuates light are formed with respect to the other portions. The phase plate 705 is disposed at a position P705 conjugate to 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 at a position conjugate to the focal plane P501 of the objective lens 451. The focal plane P701 may be alternatively referred to as the light receiving surface P701. The photodetector 701 has, for example, a photodiode or a photomultiplier tube. The intensity of the visible light received by the photodetector 701 is acquired by the control unit 90 as a phase difference signal. The annular portion of the phase plate 705 in the present embodiment may be alternatively referred to as a circular annular portion.

[0023] (Second emission optical system) The observation apparatus 1 according to the present embodiment includes pulse lasers 201 and 211 as a pulse light source that is optically coupled to a second emission optical system and emits two pulse lights (two types of pulse lights) having different oscillation wavelengths and synchronized with each other. Such two pulse lights include Stokes light and pump light that exhibit a non-linear optical effect on the sample 501.

[0024] The second emission optical system 20 (SRS light source unit) includes pulse lasers 201 and 211 having different oscillation wavelengths, relay lenses 202 and 203, mirrors 204 corresponding to each of them, relay lenses 212 and 213, and a dichroic mirror 214. Further, the second emission optical system 20 (SRS light source unit) includes a laser synchronization detection unit 221 that detects the synchronization of the emission timings of the pulse lights of 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 respectively reflect a part of the light emitted from the pulse lasers 201 and 211 and guide it to the pulse synchronization detection unit 221.

[0025] As the pulse lasers 201 and 211, 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. One of the pulse lasers 201 and 211 may be replaced with an optical parametric oscillator that converts the wavelength of the pulse laser generated by the other of the pulse lasers 201 and 211.

[0026] Among the pulse lights emitted from the pulse lasers 201 and 211, the light with the shorter wavelength is used as the pump light in SRS observation, and the light with the longer wavelength is used as the Stokes light in SRS observation.

[0027] The repetition frequencies of the pulsed light emitted from the pulsed lasers 201 and 211 are such that one is 1 / 2 of the other. For example, the repetition frequency of the Stokes light is 1 / 2 of that of the pump light. The synchronization of the pump light and the Stokes light is detected by the pulse synchronization detection unit 221. Based on the synchronization signal of the pulse synchronization detection unit 221, the controller 90 controls, for example, the resonator length of one or both of the pulsed lasers 201 and 211 to maintain a state where the pump light and the Stokes light are synchronized for a sufficiently long time for SRS observation.

[0028] The dichroic mirror 214 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 214 is arranged such that these 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 such that these 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 simultaneously.

[0030] Note that the pulsed lasers 201 and 211 and the pulse synchronization detection unit 221 may be provided with a delay optical path (not shown) as necessary.

[0031] (Second detection unit) The second detection unit 80 (SRS detection unit) includes a band-pass filter 802, a lens 803, and a photodetector 801. The band-pass filter 802 has wavelength characteristics that transmit only the pulsed light with the higher repetition frequency among the pump light and the Stokes light. The lens 803 reduces the beam diameter of the pulsed light transmitted through the band-pass filter 802 so that it fits within the light-receiving surface of the photodetector 801. The photodetector 801 has, for example, a photodiode. An SRS signal is acquired by performing lock-in detection on the modulation component of the intensity of the pulsed light received by the photodetector 801 in the control unit 90.

[0032] (Scanning unit) The scanning unit 30 includes a two-axis scanner 301 and relay lenses 302 and 303. The two-axis scanner 301 has two mirrors that swing around two axes orthogonal to each other, and two-dimensionally displace the angle of the incident light with respect to the optical axis. The displacement amount is controlled by the magnitude of the swing, thereby controlling the scanning range on the sample surface. By making the swing frequencies of the two mirrors different from each other, the sample surface is two-dimensionally scanned. It is desirable that the midpoint between the two mirrors be in a position conjugate to the pupil plane P401 of the objective lens 401 via the relay lenses 302 and 303. The two-axis scanner 301 can be formed by combining, for example, a resonant scanner and a galvanometer scanner for each axis. The relay lenses 302 and 303 cause light to enter the pupil of the objective lens at an appropriate beam diameter and maximum angle.

[0033] (Relay section) The relay unit 60 includes relay lenses 601 and 602 and a two-axis scanner 603. The relay lenses 601 and 602 cause 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 swing around two axes orthogonal to each other. These mirrors swing so as to cancel out the angular displacement of the incident light beam with respect to the optical axis. That is, one mirror of the two-axis scanner 603 swings at the same frequency as one mirror of the two-axis scanner 301, and the other mirror of the two-axis scanner 603 swings at the same frequency as the other mirror of the two-axis scanner 301, and in phase or out of phase (depending on the installation method). Therefore, the light beam emitted from the two-axis scanner 603 travels parallel to the optical axis or, ideally, on the optical axis. The operation of this two-axis scanner is sometimes called reverse scanning (descanning). It is desirable that the middle of the two mirrors of the two-axis scanner be arranged at a position conjugate to the pupil plane P451 of the objective lens 451 via the relay lenses 601 and 602.

[0034] Particularly 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 that case, the relay lenses 601 and 602 and the two-axis scanner 603 can be arranged symmetrically with respect to the relay lenses 303 and 302 and the two-axis scanner 301 with respect to the sample surface.

[0035] The relay unit 60 of the first embodiment having the two-axis scanner 603 may be equivalently referred to as a reverse scanning unit 60 from its function. The reverse scanning unit 60 is configured to perform reverse scanning in synchronization with the scanning unit 30 on the optical path between the wavelength division unit 75 and the second objective lens 45.

[0036] (Wavelength multiplexing unit and wavelength division unit) The multiplexing unit 15 and the 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 arranged such that these 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.

[0037] (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 for executing a control flow on a computer. Further, the control device 901 may include an FPGA, a microcomputer, and an electric circuit that execute a part of the control flow, or a part of the program may be implemented in these. A lock-in amplifier (not shown) that detects the modulation component of the light intensity detected by the photodetector 801 for SRS observation is also included in the control device 901. Note that this intensity modulation can be paraphrased as an SRS signal. Also, for example, an electric circuit that controls one or both of the pulsed lasers 201, 211 based on the synchronization signal of the pulse synchronization detection unit 221 can be included in the control 901. Also, for example, an electric circuit that generates drive signals for the two-axis scanners 301, 603 can be included in the control device 901.

[0038] Based on the signals output by the photodetector 701 and the photodetector 801, the control device 901 generates a phase difference image and an SRS image, respectively. At this time, the phase difference signal and the SRS signal are read out at the same pixel frequency (pixel rate) and converted into luminance respectively, and images with the same number of pixels are generated. The generated phase difference image and SRS image are stored in a storage device (not shown) of the control device 901 or output toward the display 921. The storage device is, for example, a solid state drive or a hard disk drive.

[0039] The keyboard 911 and the mouse 912 are connected to the control device 901, and the operator inputs instructions to the control device 901 by operating the keyboard 911 and the mouse 912. The control device 901 is connected to the LED 101, the pulsed laser 201, the pulsed lasers 211, the two-axis scanners 301, 603, the stage scanner 512, and the photodetectors 701, 801, and controls these operations according to the instructions from the operator.

[0040] The display 921 visually returns feedback to the operator's operation and displays the images and character strings output by the control device 901.

[0041] (Phase difference observation) FIG. 3 is a schematic diagram for explaining the phase difference observation by the observation apparatus 1. In the phase difference observation in the observation apparatus 1, the first emission optical system 10 (phase difference light source unit), the multiplexing unit 15, the scanning unit 30, the first objective lens 40, the placement unit 50, the second objective lens 45, the relay unit 60, the demultiplexing unit 75, the first detection unit 70 (phase difference detection unit), and the control unit 90 are used.

[0042] As shown by the dotted line in FIG. 3, the light emitted from the LED 101 of the first emission optical system 10 (phase difference light source unit) becomes a parallel light beam by the collimator 102. The annular light beam that has passed through the ring slit 103 is focused on the pinhole 105 by the relay lens 104. At this time, the diffracted light of the first order or higher generated at the edge of the ring slit 103 spreads the focal point. The pinhole 105 blocks the light at the peripheral part of this focal point while allowing the light closer to the center to pass through, thereby increasing the ratio of the light that has traveled straight through the ring slit 103 (in other words, the zero-order diffracted light). As a result, it is possible to suppress the shape of the light beam that has become annular again by the relay lens 106 from being distorted as it propagates through the subsequent optical path. Therefore, artifacts in the acquired phase difference image can be suppressed.

[0043] The parallel light beam whose annular diameter is 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. The annular light beam is reflected by each of the two mirrors of the two-axis scanner 301. The annular light beam that is emitted from the two-axis scanner 301 parallel to the optical axis or inclined with respect to the optical axis has its annular diameter enlarged or reduced by the relay lenses 302 and 303 and enters the objective lens 401 of the first objective lens 40. By the objective lens 401, the light beam is focused onto one point toward the sample 501 on the placement unit 50. The position of this focal point within the sample surface P501 corresponds to the angle with respect to the optical axis of the light beam emitted from the two-axis scanner 301. At this time, in addition to the direct light (in other words, the zero-order diffracted light) that travels straight through the sample, diffracted light of the first order or higher corresponding to the refractive index distribution and shape of the sample is generated.

[0044] The direct light and diffracted light emitted from the sample 501 are collected by the objective lens 451 and emitted as a parallel light beam. At this time, the direct light in the sample 501 becomes annular, and the inside and outside of the annulus become the diffracted light generated in the sample 501. The emission angle of this parallel light beam corresponds to the position of the focal point within the sample surface P501, that is, the angle with respect to the optical axis of the light beam emitted from the two-axis scanner 301. The light beam emitted from the objective lens 451 has its beam diameter enlarged or reduced by the relay lenses 601 and 602 of the relay unit 60 and enters the two-axis scanner 603. This light beam is reflected by each of the two mirrors of the two-axis scanner 603. The light beam that is emitted from the two-axis scanner 603 parallel or coincident with the optical axis regardless of the incident angle 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).

[0045] This light beam is focused onto one point toward the pinhole 703 by the relay lens 702. The pinhole 703 blocks the light at the peripheral portion of this focal point, thereby blocking the diffracted light and scattered light generated outside the focal point within the sample surface P501. Thereby, artifacts in the acquired phase difference image can be suppressed.

[0046] The light beam passing through the pinhole 703 has its beam diameter enlarged or reduced by the relay lens 704 and becomes a parallel light beam again, and then passes through the phase plate 705. At this time, the direct light in the sample 501 passes through the annular portion and undergoes phase modulation and light attenuation, while the diffracted light in the sample 501 passes through the portion other than the annulus. The light beam emitted from the phase plate 705 is focused at a single point toward the light receiving surface P701 of the photodetector 701 by the tube lens 706. On the light receiving surface P701, the direct light and the diffracted light in the sample 501 interfere, and the photodetector 701 detects the light intensity thereof.

[0047] (SRS Observation) FIG. 4 is a schematic diagram for explaining the SRS observation by the observation apparatus 1. In the SRS observation in the observation apparatus 1, the second emission optical system 20 (SRS light source unit), the multiplexing unit 15, the scanning unit 30, the first objective lens 40, the placement unit 50, the second objective lens 45, the relay unit 60, the demultiplexing unit 75, the second detection unit 80 (SRS detection unit), and the control unit 90 are used.

[0048] As shown by the dotted line in FIG. 4, 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 synchronous detection unit 221, while most of it passes through and enters the relay lens 202. The light beam whose beam diameter is enlarged or reduced by the relay lenses 202 and 203 is reflected by the mirror 204 and then passes through the dichroic mirror 214.

[0049] Part of the parallel light beam emitted by the pulsed laser 211 is reflected by the beam splitter 223 and enters the pulse synchronous detection unit 221, while most of it passes through and enters the relay lens 212. The light beam whose beam diameter is enlarged or reduced by the relay lenses 212 and 213 is reflected by the dichroic mirror 214. At this time, it is overlapped coaxially with the light beam that has passed through the aforementioned dichroic mirror 214.

[0050] The light beam from the pulsed laser 201 reflected by the beam splitter 222 is reflected by the mirror 224 of the pulse synchronization detection unit 221 and transmitted through the 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 superposed coaxially with the light beam transmitted through the dichroic mirror 225. The coaxially superposed light beams (in other words, both the pump light beam and the Stokes light beam) are condensed by the lens 226 toward the two-photon detector 227.

[0051] The light beams superposed coaxially by the dichroic mirror 214 (in other words, both the pump light beam and the Stokes light beam) are reflected by the dichroic mirror 151 of the multiplexing unit 15. At this time, it is emitted from the first emission optical system 10 (phase difference light source unit) described above. It is superposed coaxially with the annular light beam transmitted through the dichroic mirror 151.

[0052] The light beam reflected by the dichroic mirror 151 enters the two-axis scanner of the scanning unit 30 and is reflected by each of the two mirrors. The light beam emitted from the two-axis scanner 301 has its beam diameter enlarged or reduced by the relay lenses 302 and 303 and enters the objective lens 401 of the first objective lens 40. By the objective lens 401, the light beam is condensed into a single point toward the sample 501 on the placement unit 50. The position of the condensing point within the sample surface P501 corresponds to the angle with respect to the optical axis when the light beam is emitted from the two-axis scanner 301. At this time, the SRS process occurs at the condensing point. That is, corresponding to the vibration level of the molecules present at the condensing point, an induced Raman loss occurs with respect to the pump light, and an induced Raman gain occurs with respect to the Stokes light. In addition to the pump light and the Stokes light, the light emitted from the first emission optical system 10 (phase difference light source unit) is also coaxially condensed, so the condensing points coincide. Therefore, the SRS image and the phase difference image are acquired simultaneously, and the field of view is the same.

[0053] The pump light and the Stokes light injected with the sample 501 are collected by the objective lens 451 and emitted as parallel light beams. At this time, the emission angle with respect to the optical axis corresponds to the position of the condensing point in the sample surface P501, in other words, the angle with respect to the optical axis of the light beam injected by the two-axis scanner 301. The light beam emitted from the objective lens 451 has its beam diameter enlarged or reduced by the relay lenses 601 and 602 of the relay unit 60 and is incident on the two-axis scanner 603. The light beam is reflected by each of the two mirrors of the two-dimensional scanner 603. The light beams of the pump light and the Stokes light emitted from the two-axis scanner 603 parallel to or coinciding with the optical axis regardless of the incident angle are reflected by the dichroic mirror 751 of the wavelength division unit 75 and are incident on the band-pass filter 802 of the second detection unit 80 (SRS detection unit). Only the light with the higher pulse repetition frequency among the pump light or the Stokes light is transmitted by the band-pass filter 802, and the light with the lower repetition frequency (1 / 2 of the higher one) is blocked. The light beam transmitted through the band-pass filter 802 is condensed by the lens 803 toward the light receiving surface of the photodetector 801, and the photodetector 801 detects the light intensity thereof.

[0054] (Observation flow) FIG. 5 is a diagram illustrating the observation flow of the sample 501 using the observation apparatus 1. FIG. 5(a) is a flowchart for explaining the observation flow, and FIG. 5(b) is a diagram for explaining the low magnification field of view R502, the high magnification field of view R503, and the region of interest R504 of the flowchart. Further, FIG. 5(c) is a diagram showing the schematic configuration of the scanning condition determination unit. The region of interest R504 may be referred to as the region of interest R504.

[0055] As shown in FIG. 5(c), 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 the display unit 921 (display). The control unit 90 further includes an input unit 910 that receives an input of information regarding the region of interest from the operator based on the displayed first image, and a scanning condition acquisition unit 94 that acquires the scanning condition SC0 for the scanning unit 30 based on the received information regarding the region of interest. The control unit 90 further includes a scanning condition determination unit 96 that determines the scanning condition SC1 during imaging for the scanning unit 30 based on the acquired scanning condition SC0. The control unit 90 further includes an update unit 98 that displays the scanning condition SC1 acquired by the scanning condition acquisition unit 96 on the display unit and accepts an update of the scanning condition SC1.

[0056] First, the sample 501 is held on the stage 511. From this state, the observation flow starts. First, the sample 501 is observed in phase contrast with a wide field of view. The conditions for low-magnification phase contrast observation are input to the control device 901, and low-magnification phase contrast observation is started. (Steps S10, S20.) By driving the stage scanner 512 to change the position of the stage 511, the low-magnification field of view R502 shown in FIG. 5(b) is moved. If the region of interest R504 of the sample 501 does not exist within the low-magnification field of view R502, the low-magnification field of view R502 is moved and the low-magnification phase contrast observation is continued. On the other hand, 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 position of the sample 501 with respect to the sample surface P501 and the time required to discover the region of interest R504 can be shortened. Thereby, damage to the sample 501 can be suppressed.

[0057] Subsequently, check or determine the details of the phase difference image of the target region R504 from the low-magnification phase difference image. (Step S40.) When checking the details, narrow the field of view and perform real-time phase difference observation. In that case, input the high-magnification phase difference observation conditions into the control device 901 and start the high-magnification phase difference observation. (Steps S50, S60.) If necessary, move the high-magnification field of view R503 shown in Fig. 5(b) so that the target region R504 is within it, and consider the necessity of SRS observation from the high-magnification phase difference image. On the other hand, when not checking the details of the phase difference image of the target region R504, consider the necessity of SRS observation from the low-magnification phase difference image. If it is determined from these phase difference images that SRS observation is unnecessary, return to the low-magnification phase difference observation and search for another target region R504 of the sample 501. On the other hand, if it is determined that SRS observation is necessary, proceed to the SRS observation. (Step S70.) In this way, by smoothly expanding the target region R504 of the sample 501 and performing phase difference observation, the necessity of SRS observation can be quickly determined. Therefore, unnecessary SRS observations can be reduced, and in addition to improving the measurement efficiency, damage to the sample can also be suppressed.

[0058] Subsequently, before starting the SRS observation, determine whether to perform only the SRS observation or both the SRS observation and the phase difference observation. (Step S80.) When performing only the SRS observation, input the SRS observation conditions into the control device 901 and start the SRS observation. (Steps S90, S110.) On the other hand, when performing both the SRS observation and the phase difference observation, input both the SRS observation conditions and the phase difference observation conditions into the control device 901 and start the simultaneous observation of the SRS observation and the phase difference observation. (Steps S100, S120.) When the SRS observation or the simultaneous observation of the SRS observation and the phase difference observation is completed, end the series of observation flows. In this way, when observing, for example, the dynamic events of the sample 501 that require the SRS image and the phase difference image at the same time, the observation accuracy can be improved by selecting the simultaneous observation of both. Also, when the phase difference image is not required, unnecessary light irradiation of the sample 501 can be prevented, and damage can be suppressed.

[0059] Note that the low-magnification and high-magnification phase-contrast observation conditions are, for example, the intensity of the light output of LED 101, the number of integrations, and the size of the field of view. The SRS observation conditions are, for example, the intensity and wavelength of the light output of pulse lasers 201 and 211, the number of integrations, and the size of the field of view.

[0060] In the observation flow of FIG. 5(a), when the target region R504 is discovered in step S30, the operator inputs the respective observation conditions in subsequent steps S50, S90, and S100. In order to improve the convenience for the operator, a step S35 for the operator to input the position information of the target region R504 may be added between steps S30 and S40. The position information can be input, for example, by the operator tracing the contour of the target region R504 or drawing a mark using the mouse 912 with respect to the low-magnification phase-contrast image displayed on the display 921 of the control unit 90. Then, based on the above position information, the control device 901 may predict the appropriate respective observation conditions and present them to the operator in steps S50, S90, and S100. In this way, by supporting the operator's input of the respective observation conditions, the burden on the operator can be reduced.

[0061] When adding the above step S35, based on the above position information, the control device 901 may determine the appropriate observation conditions and automatically input the respective observation conditions in steps S50, S90, and S100. Thereby, the labor for the operator to input the respective observation conditions can be saved, and the burden can be further reduced.

[0062] Note that in the observation device 1 of FIG. 2, although the ring slit 103 is arranged in the first emission optical system 10 (phase-contrast light source unit) and the phase plate 705 is arranged in the first detection unit 70 (phase-contrast detection unit), even if both are arranged in an exchanged manner, a phase-contrast image can be acquired.

[0063] As described above, the observation device according to the first embodiment of the present invention simultaneously acquires the phase-contrast signal and the SRS signal from the same point and generates a phase-contrast image and an SRS image with the same number of pixels. That is, a primary image of a phase-contrast image and an SRS image with matched image quality can be acquired. Thereby, it becomes possible to reduce the burden on the operator regarding the matching of image quality.

[0064] <Second Embodiment> The configuration of the observation apparatus according to the second embodiment will be described with reference to FIG. 6. FIG. 6 is a schematic diagram for explaining the configuration of the observation apparatus 2 of the present embodiment. The observation apparatus 2 has the same configuration as the observation apparatus 1 of the first embodiment, except for the parts described below. Therefore, the same reference numerals are assigned to the common elements and the overlapping explanations are omitted.

[0065] In the observation apparatus 2 of FIG. 6, the two-axis scanner 603 of the relay unit 60 of the observation apparatus 1 is removed, and the dichroic mirror 751 of the beam splitter 75 is installed at that position. In addition, the pinhole 703 of the first detection unit 70 (phase difference detection unit) of the observation apparatus 1 is removed. Further, the lens 803 of the second detection unit 80 (SRS detection unit) of the observation apparatus 1 is replaced with relay lenses 804 and 805. The relay unit 60 of the second embodiment is different from that of the first embodiment in that it is not electrically connected to the control unit 90.

[0066] (Relay Unit) The observation apparatus 2 does not have a two-axis scanner that cancels the angular displacement of the incident light beam with respect to the optical axis of the relay unit 60. Therefore, the angle of the light beam emitted from the relay unit 60 of the observation apparatus 2 with respect to the optical axis corresponds to the position of the condensing point on the sample surface P501, in other words, the angle of the light beam emitted from the two-axis scanner 301 of the scanning unit 30 with respect to the optical axis.

[0067] (Beam Splitter) The dichroic mirror 751 of the beam splitter 75 of the observation apparatus 2 has the same wavelength characteristics as those of the observation apparatus 1. The dichroic mirror 751 of the observation apparatus 2 is installed near a position conjugate with the pupil surface P451 of the objective lens 451 of the second objective lens 45 via the relay lenses 601 and 602 of the relay unit 60.

[0068] (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, the intermediate imaging plane) P703. At the intermediate imaging plane P703, a dot-like image moves corresponding to the condensing point that scans the sample surface P501. Similarly, at the focal plane P701 of the tube lens 706 at a position conjugate to the focal plane P501, the dot-like image also moves. Therefore, the photodetector 701 of the observation device 2 requires a larger light-receiving area compared to the observation device 1. For this reason, the photodetector 701 of the observation device 2 may utilize a photodiode array or an image sensor in which a plurality of photodiodes are two-dimensionally arranged.

[0069] The phase plate 705 of the first detection unit 70 (phase difference detection unit) of the observation device 2 is arranged at a position conjugate to the pupil plane P451 of the objective lens 451, which is the same as that of the observation device 1. However, it is different from the observation device 1 in that the angle of the light beam transmitted through the phase plate 705 with respect to the optical axis changes corresponding to the position of the condensing point on the sample surface P501, in other words, the angle of the optical axis of the emitted light beam of the two-axis scanner 301. Considering this, it is necessary to design an annular portion that modulates the phase of the direct light (zero-order diffracted light) at the sample 501 and reduces the light intensity.

[0070] FIG. 7 is a schematic diagram for explaining the relationship between the phase plate 705 of the observation device 2 and the light beam transmitted through it. The phase plate 705 has two concentric cylinders 705_1 and 705_2 around the central axis 705_0 that coincides with the optical axis. In the region sandwiched between the inner cylinder 705_1 and the outer cylinder 705_2 (the shaded portion in FIG. 7), a quarter-wave plate and an ND filter are formed. The annular parallel light beam created by the direct light at the sample 501 passes through this region. Among this annular light beam, the innermost light beam L103_1 and the outermost light beam L103_2 draw concentric cylinders around the principal ray of the parallel light beam (including both the direct light and the diffracted light at the sample 501) transmitted through the phase plate 705. The diameters of the cylinders of these light beams L103_1 and L103_2 respectively correspond to the inner diameter and the outer diameter of the ring slit 103 that exists at a position conjugate to the plane P705 where 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.

[0071] FIG. 7(a) shows the situation when the angle of the parallel light beam transmitted through the phase plate 705 with respect to the optical axis is maximum. In FIG. 7(a), the principal ray L103_0 forms the maximum angle θ0 with respect to the optical axis 705_0 and intersects on the plane P705 that coincides with the center of the thickness of the phase plate 705. At this time, since all 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, the radii of the cylinders 705_1 and 705_2 satisfy the following equations (1) and (2).

[0072]

Equation

[0073]

Equation

[0074] In addition, in Equations (1) and (2), it is assumed that the incident angle of the light beam on the phase plate 705 is at most 10°, and the refraction caused by a quarter-wave plate, an ND filter, etc. is not considered. More precisely, it is desirable to determine the above radius in consideration of such refraction.

[0075] FIG. 7(b) shows the case where the parallel light beam transmitted through the phase plate 705 becomes parallel to the optical axis. When Equations (1) and (2) are satisfied, all 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.

[0076] In this way, by designing so that all the direct light at the sample 501 undergoes phase modulation and light attenuation at the phase plate 705 regardless of the angle with respect to the optical axis when passing through the phase plate 705, artifacts in the acquired phase difference image can be suppressed.

[0077] (SRS detection unit) The second detection unit 80 (SRS detection unit) of the observation apparatus 2 includes relay lenses 804 and 805, a band-pass filter 802, and a photodetector 801. The relay lenses 804 and 805 enlarge or reduce the beam diameter of the incident light beam so as to be suitable for 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 apparatus 2 is arranged at a position conjugate to the pupil plane P451 of the objective lens 451. The band-pass filter 802 of the observation apparatus 2 has the same wavelength characteristics as those of the observation apparatus 1 and is arranged between the relay lens 805 and the photodetector 801.

[0078] As described above, since the observation apparatus according to the second embodiment of the present invention does not have a two-axis scanner in the relay unit 60, in addition to simplifying the control, the optical adjustment can be facilitated. Thereby, the stability of the observation apparatus can be improved.

[0079] In the above embodiments, in order to acquire a Raman image with high sensitivity and high speed, coherent pulsed light of different wavelengths was condensed on a sample to generate an SRS process for imaging. However, a Raman image can also be acquired by condensing incoherent light of a predetermined wavelength on a sample to generate a spontaneous Raman scattering process for imaging. As a detection unit in this case, for example, a monochromator can be used. Even in this case, it is possible to obtain a similar effect.

[0080] As described above, the present invention has been described using embodiments. However, the technical scope of the present invention is not limited to the scope described in the above embodiments. It is obvious to those skilled in the art that various changes or improvements can be made to the above embodiments. It is clear from the description of the claims that forms with such changes or improvements can also be included in the technical scope of the present invention.

[0081] As described above, the scanning observation device according to the embodiment of the present invention includes the following configurations 1 to 14.

[0082] (Configuration 1) A first emission optical system that emits a first primary light including a spatially modulated component, A second emission optical system that emits coherent second primary light, A multiplexing unit that multiplexes the first primary light and the second primary light coaxially, A scanning unit that scans the multiplexed first primary light and second primary light coaxially, A first objective lens that condenses the scanned first primary light and second primary light, A placement unit that places a sample at the condensing position of the first objective lens, A second objective lens that collects secondary light from the sample, A demultiplexing unit that demultiplexes the first secondary light emitted from the sample by the irradiation of the first primary light and the second secondary light emitted from the sample by the irradiation of the second primary light from the secondary light, A first detection unit that detects the first secondary light, a second detection unit that detects the second second-order light; A scanning observation apparatus having the same.

[0083] (Configuration 2) The scanning observation apparatus according to Configuration 1, further comprising a pulse light source that is optically coupled to the second emission optical system and emits two pulse lights having different oscillation wavelengths and being synchronized.

[0084] (Configuration 3) The scanning observation apparatus according to Configuration 2, wherein the two pulse lights include Stokes light and pump light that exhibit a non-linear optical effect on the sample.

[0085] (Configuration 4) The scanning observation apparatus according to any one of Configurations 1 to 3, wherein the second second-order light includes light emitted from the sample due to a non-linear optical effect generated in the sample by irradiation with the second first-order light.

[0086] (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 light having a predetermined incoherent wavelength.

[0087] (Configuration 6) The first emission optical system includes a modulation optical element that modulates the intensity component of the first first-order light in a circular shape. Further, the first detection unit includes a demodulation optical element having a circular portion that demodulates the intensity component and the phase component of the first second-order light. The scanning observation apparatus according to any one of Configurations 1 to 5.

[0088] (Configuration 7) The scanning observation apparatus according to any one of Configurations 1 to 6, wherein the first emission optical system includes a pinhole that shields a part of the condensed first first-order light.

[0089] (Configuration 8) The first injection optical system includes a modulation optical element having an annular portion that modulates the intensity component and the phase component of the first primary light. Further, the first detection unit includes a demodulation optical element that demodulates the intensity component of the first secondary light in an annular shape. The scanning type observation apparatus according to any one of configurations 1 to 7.

[0090] (Configuration 9) The scanning type observation apparatus 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 the optical path between the wavelength division unit and the second objective lens.

[0091] (Configuration 10) The reverse scanning unit condenses the first secondary light toward the first detection unit, The first detection unit includes a pinhole that shields a part of the condensed first secondary light. The scanning type observation apparatus according to Configuration 9.

[0092] (Configuration 11) The scanning type observation apparatus 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.

[0093] (Configuration 12) The scanning type observation apparatus according to Configuration 11, further comprising an input unit that receives input of information regarding 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 regarding the region of interest.

[0094] (Configuration 13) The scanning type observation apparatus according to Configuration 12, further comprising a scanning condition determination unit that determines scanning conditions during imaging for the scanning unit based on the acquired scanning conditions.

[0095] (Configuration 14) The scanning type observation device 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 to the scanning conditions.

Explanation of Signs

[0096] 1, 2 Scanning type observation device 10 First injection optical system (phase difference light source unit) 15 Beam combining unit 20 Second injection optical system (SRS light source unit) 30 Scanning unit 40 First objective lens (condensing unit) 45 Second objective lens (light collection unit) 50 Mounting unit 60 Reverse scanning unit (relay unit) 70 First detection unit (phase difference detection unit) 75 Beam splitting unit 80 Second detection unit (SRS detection unit) 90 Control unit

Claims

1. A first emission optical system that emits a first primary light beam including a spatially modulated component; A second emission optical system that emits a coherent second primary light beam; A multiplexing unit that multiplexes the first primary light beam and the second primary light beam coaxially; A scanning unit that scans the multiplexed first primary light beam and second primary light beam coaxially; A first objective lens that condenses the scanned first primary light beam and second primary light beam; A placement unit that places a sample at the condensing 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, a first secondary light beam emitted from the sample by irradiation with the first primary light beam and a second secondary light beam emitted from the sample by irradiation with the second primary light beam; A first detection unit that detects the first secondary light beam; A second detection unit that detects the second secondary light beam; And a scanning type observation apparatus having the same.

2. The scanning type observation apparatus according to claim 1, further comprising a pulse light source that is optically coupled to the second emission optical system and emits two pulse light beams having different oscillation wavelengths and being synchronized.

3. The scanning type observation apparatus according to claim 2, wherein the two pulse light beams include Stokes light and pump light that exhibit a non-linear optical effect on the sample.

4. The scanning type observation apparatus according to claim 1 or 2, wherein the second secondary light beam includes light emitted from the sample due to a non-linear optical effect generated in the sample by irradiation with the second primary light beam.

5. The scanning type observation apparatus according to claim 1 or 2, wherein the first emission optical system includes a first light source that emits incoherent light having a predetermined wavelength.

6. The first projection optical system includes a modulation optical element that modulates the intensity component of the first primary light in a ring shape, and the first detection unit includes a demodulation optical element having an annular portion that demodulates the intensity component and the phase component of the first secondary light. The scanning type observation apparatus according to claim 1 or 2.

7. The first projection optical system includes a pinhole that shields a part of the condensed first primary light. The scanning type observation apparatus according to claim 1 or 2.

8. The first projection optical system includes a modulation optical element having an annular portion that modulates the intensity component and the phase component of the first primary light, and the first detection unit includes a demodulation optical element that demodulates the intensity component of the first secondary light in a ring shape. The scanning type observation apparatus according to claim 1 or 2.

9. On the optical path between the beam splitter and the second objective lens, a reverse scanning unit that performs reverse scanning in synchronization with the scanning unit is provided. The scanning type observation apparatus according to claim 1 or 2.

10. The reverse scanning unit condenses the first secondary light toward the first detection unit, The first detection unit includes a pinhole that shields a part of the condensed first secondary light. The scanning type observation apparatus according to claim 9.

11. The scanning type observation apparatus according to claim 1 or 2, 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.

12. An input unit that receives input of information regarding a region of interest from an 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 received information regarding the region of interest. The scanning type observation apparatus according to claim 11.

13. The scanning type observation apparatus according to claim 12, further comprising a scanning condition determination unit that determines scanning conditions during imaging for the scanning unit based on the acquired scanning conditions. Claim 14 The scanning type 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 an update of the scanning conditions.

Images