Scanning observation device

Abstract

The present invention provides a scanning observation device that enables reproducible adjustment of the phase difference detection region and the photothermal signal detection region. [Solution] The scanning observation device comprises: an emission optical system that emits a first light containing spatially modulated components; a first scanning unit that scans the first light; a focusing lens that focuses the scanned first light; a scanning range acquisition unit that receives the setting of the scanning area of ​​the first light and commands the first scanning unit; a placement unit on which a sample is placed at the focusing position of the focusing lens; a light-collecting lens located on the opposite side of the placement unit from the focusing lens and for collecting secondary light from the sample; an illumination optical system that irradiates the sample with a second light having a longer wavelength than the first light so that at least a part of the irradiation area of ​​the sample by the focusing lens overlaps; a detection unit that detects the secondary light through the light-collecting lens; and a demodulation unit that demodulates the signal from the detection unit to acquire the intensity modulated component of the secondary light synchronized with the emission of the second light.

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, and bioproduction, 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 without contact, there are a method of detecting the phase difference of light transmitted through the cells, and a method of detecting the absorption of mid-infrared light generated due to the vibration of molecules constituting the cells.

[0003] Patent Document 1 discloses an apparatus that combines a phase contrast imaging method for detecting a phase difference as the intensity of light, and an imaging method for detecting a temperature change caused by mid-infrared light absorption as a refractive index change. That is, a photo-thermal imaging apparatus is disclosed that observes the spatial distribution of infrared absorption amounts by detecting a refractive index change as a phase contrast change.

[0004] The photo-thermal imaging apparatus described in Patent Document 1 employs a wide-field simultaneous irradiation system that irradiates the irradiation surface of a subject all at once without serially dividing the irradiation spot with respect to the irradiation optical system for obtaining a phase contrast image.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] When comparing morphological images (phase contrast images) and functional images (mid-infrared photothermal images) using a common field of view, it is necessary to perform a procedure to overlap the region of interest with the field of view of the phase contrast image and the mid-infrared light irradiation area, and then capture the phase contrast image and the mid-infrared light absorption image. In this configuration, where a simultaneous irradiation system such as that described in Patent Document 1 is used as the irradiation system for phase contrast imaging, in addition to the objective lens and XY stage, a means for switching the objective lens and a field diaphragm are required, and the shooting sequence requires the mechanical operation of the objective lens and field diaphragm. Normally, the mechanical operation of the objective lens and field diaphragm, which are mounted on the mirror base, can cause vibrations that affect the positional relationship between the sample and the XY stage and the state of the sample, raising concerns about reduced reproducibility of the image. Furthermore, other means to reduce vibrations and waiting time required for vibration damping may be necessary, raising concerns about the size of the device and reduced usability of the image.

[0007] There was a need for a multimodal observation system in which the adjustment of the visible light illumination area for phase contrast imaging did not affect the optical system's configuration, relative to the infrared light illumination area for photothermal imaging.

[0008] The object of the present invention is to provide a scanning observation device that can reproducibly adjust the phase difference detection region and the photothermal signal detection region to match each other. [Means for solving the problem]

[0009] A scanning observation apparatus according to an embodiment of the present invention is characterized by comprising: an emission optical system that emits a first light containing a spatially modulated component; a first scanning unit that scans the first light; a scanning range acquisition unit that receives the setting of the scanning area of ​​the first light and commands the first scanning unit; a focusing lens that focuses the scanned first light; a placement unit on which a sample is placed at the focusing position of the focusing lens; a light-collecting lens located on the opposite side of the placement unit from the focusing lens and for collecting secondary light from the sample; an illumination optical system that irradiates the sample with a second light having a longer wavelength than the first light such that at least a portion of the irradiation area of ​​the sample by the focusing lens overlaps; a detection unit that detects the secondary light through the light-collecting lens; and a demodulation unit that demodulates the signal from the detection unit to acquire the intensity modulated component of the secondary light synchronized with the emission of the second light. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a scanning observation device that can reproducibly adjust the phase difference detection region and the photothermal signal detection region to match each other. [Brief explanation of the drawing]

[0011] [Figure 1] This is a block diagram illustrating the schematic connection relationships of the elements constituting the observation device according to the first embodiment of the present invention. [Figure 2] This is a schematic diagram illustrating the configuration of the observation device according to the first embodiment. [Figure 3] This is a schematic diagram illustrating the phase detection system of the observation device according to the first embodiment. [Figure 4] This is a schematic diagram illustrating the relationship between the phase plate and the transmitted light beam in the phase difference detection unit according to the first embodiment. [Figure 5] This is a schematic diagram illustrating the photothermal induction system of the observation device according to the first embodiment. [Figure 6] This is a schematic diagram illustrating the region and timing of light irradiated onto the sample in the observation apparatus according to the first embodiment. [Figure 7]This is a schematic diagram illustrating the method for generating infrared absorption images and phase contrast images in the observation apparatus according to the first embodiment. [Figure 8] This is a schematic diagram illustrating the method for identifying the infrared light irradiation area and setting the photothermal signal detection area in the observation device according to the first embodiment. [Figure 9] This figure illustrates the observation flow (A), the relationship between the region of interest and the observation field (B), and the schematic configuration of the scanning range acquisition unit (C) using the observation device according to the first embodiment. [Figure 10] This is a schematic diagram illustrating the configuration of the observation device according to the second embodiment. [Figure 11] This is a schematic diagram illustrating the region and timing of light irradiated onto the sample in the observation apparatus according to the second embodiment. [Figure 12] This is a schematic diagram illustrating the configuration of the observation device according to the third embodiment. [Modes for carrying out the invention]

[0012] Embodiments of the present invention will be described below with reference to the drawings.

[0013] <First Embodiment> The configuration of the observation apparatus according to the first embodiment will be explained using Figures 1 to 5. This observation apparatus 1 acquires an infrared absorption signal by detecting the change in refractive index of the medium (photothermal effect) caused by the heat generated by the absorption of mid-infrared light by molecules, using a phase difference. This infrared absorption signal may also be referred to as a photothermal signal or PT signal. A plot showing the wavenumber dependence of the PT signal intensity (sometimes simply called PT intensity) is called a PT spectrum, and a mapping of the two-dimensional or three-dimensional spatial distribution of the PT signal intensity is called a PT image. The PT spectrum and PT image may also be referred to as an infrared absorption spectrum and an infrared absorption image, respectively.

[0014] FIG. 1 is a block diagram for explaining the schematic connection relationship of the elements constituting the observation apparatus 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 connections enabling the transmission of signals related to measurement or control are made between the corresponding components.

[0015] The observation apparatus 1 optically includes an injection optical system 10 (phase difference light source unit), an irradiation optical system 20 (mid-infrared light source unit), a scanning unit 30, a first objective lens 40 (condensing lens), and a mounting unit 50. The observation apparatus 1 further includes a second objective lens 45 (light collection lens), a relay unit 60, and a detection unit 70 (phase difference detection unit). In addition, the observation apparatus 1 includes a control unit 90. The control unit 90 is electrically connected to the injection optical system 10, the irradiation optical system 20, the scanning unit 30, the mounting unit 50, and the detection unit 70.

[0016] FIG. 2 is a schematic diagram for explaining the configuration of the observation apparatus 1.

[0017] (Mounting unit) The mounting unit 50 includes a stage 511 and a stage scanner 512. The stage 511 supports the sample 501, which is the observation object of the observation apparatus 1, such that at least a part of the sample 501 overlaps with the condensing position of the first objective lens 40 described later. The mounting unit 50 may be alternatively referred to as a support unit 50 for the sample 501 or a sample stage 50. Also, the condensing position of the first objective lens 40 may be alternatively referred to as the focal point of the first objective lens 40, the condensing point of the first objective lens, etc.

[0018] The stage 511 has a portion that is optically opened so that primary light can enter and secondary light can exit with respect to the sample 501. The opening can adopt either a form closed in the circumferential direction or a form not closed.

[0019] This configuration ensures that light from the first objective lens 40 illuminates the sample 501 placed on 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. This allows the operator to easily move the part of the sample 501 they wish to observe to the observation area of ​​the observation device 1 after placing the sample 501. The stage scanner 512 also moves the stage 511 perpendicular to the surface on which the sample 501 is placed. This allows the operator to easily focus on the sample 501 and also observe the sample 501 in three dimensions.

[0020] (First objective lens and second objective lens) The first objective lens 40 and the second objective lens 45 are each equipped with objective lenses 401 and 451. The objective lenses 401 and 451 are positioned on opposite sides of the stage 511. The objective lenses 401 and 451 are positioned 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 objective lenses 401 and 405 are in a conjugate positional relationship. This suppresses the superposition of artifacts on the acquired phase difference signal, PT image, and PT spectrum, as well as a decrease in detection sensitivity. It is desirable that the on-axis and off-axis focal points of the objective lenses 401 and 451 are the same at any wavelength in the visible range. Therefore, it is desirable that axial and lateral chromatic aberrations are sufficiently corrected. This suppresses a decrease in the detection sensitivity of the PT signal.

[0021] (Phase difference light source) The emission optical system 10 (phase difference light source section) includes an LED 101, a collimator 102, a ring slit 103, relay lenses 104 and 106, and a pinhole 105. The LED 101 emits visible light including a predetermined central wavelength of incoherent light. The LED 101 emits light of a predetermined wavelength in the visible light range. It is desirable that the LED 101 be considered as a point light source. The LED 101 may, for example, be optically coupled to one end of a multimode fiber and emit visible light from the other end of the multimode fiber. The LED 101 may generate light pulses on the order of picoseconds or nanoseconds, or it may generate continuous light. In addition, various lasers such as LDs can be used instead of the LED 101. In this case, in order to suppress noise components in the acquired image due to high coherence, such as speckle noise, optical elements that make the light as close to incoherent as possible may be inserted into the optical path after emission. The ring slit 103 comprises a transmissive portion through which light passes as an annular light beam, and a light-shielding portion that shields the periphery of the transmissive portion and the center of the ring. The ring slit 103 is positioned at a position P103 conjugate to the pupil plane P401 of the objective lens 401. The pinhole 105 is positioned at a position P105 conjugate to the focal plane P501 of the objective lens, which will be described later. The ring slit 103 may also be referred to as a ring aperture 103, annular slit 103, or circular annular slit 103. The ring slit 103 can be described as a modulation optical element having an annular portion that modulates the incoherent intensity component of the first primary light. Furthermore, the exit optical system 10 (phase difference light source) can be described as comprising the ring slit 103 as a modulation optical element having an annular portion that modulates the incoherent intensity component of the first primary light.

[0022] The emission optical system 10 (phase difference light source) can be described as an illumination optical system configured to emit a first primary light containing spatially modulated components.

[0023] (Phase difference detection unit) The detection unit 70 (phase difference detection unit) comprises relay lenses 702 and 704, a phase plate 705, a tube lens 706, and a photodetector 701. Between the relay lenses 702 and 704, there is an intermediate imaging plane P703 that is conjugate to the focal plane P501 of the objective lens 451 (light-gathering lens 451). The phase plate 705 consists of an annular portion corresponding to the ring slit 103 and the remaining portion. The annular portion has a waveplate 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 positioned on a plane P705 that is conjugate to the pupil plane P451 of the objective lens 451 (light-gathering lens 451). In other words, the phase plate 705 is a demodulating optical element that separates and demodulates the intensity component and phase difference component of the secondary light from the sample 501, respectively. Furthermore, the detection unit 70 (phase difference detection unit) is said to include a phase plate 705 as a demodulating optical element that separates and demodulates the intensity component and phase difference component of the secondary light from the sample 501, respectively. The light-receiving surface of the photodetector 701 is positioned on the focal plane P701 of the tube lens 706 and on a plane conjugate to the focal plane P501 of the objective lens 451 (light-gathering lens 451). The focal plane P701 may be referred to as the light-receiving surface P701. The photodetector 701 may have, for example, a photodiode or a photomultiplier tube. Alternatively, a photodiode array in which multiple photodiodes are arranged in two dimensions may be used. The intensity of the visible light received by the photodetector 701 is acquired by the control unit 90 as a phase difference signal.

[0024] (irradiation optical system) The observation apparatus 1 according to this embodiment includes a laser 201 as a light source that is optically coupled to an irradiation optical system 20 and emits light with an oscillation wavelength in the mid-infrared region. This light includes light that exhibits infrared absorption with respect to the sample 501. The irradiation optical system 20 is a light source that irradiates the sample 501 with a second light having a longer wavelength than the first light emitted by the emission optical system 10, heating the irradiated area as a photothermal phenomenon and causing an immediate change in refractive index.

[0025] The irradiation optical system 20 (mid-infrared light source) includes a laser 201, cylindrical mirrors 202 and 203, a mirror 204, and a lens 205. The wavelength of the light output by the laser 201 is from 2 μm to 30 μm and may be continuous or pulsed light. For example, a quantum cascade laser can be used as the laser 201. Alternatively, a tunable laser system having an optical parametric oscillator or optical parametric amplifier can be used. The laser 201 has two states: one in which it emits mid-infrared light to induce a photothermal effect (PT-on), and another in which it does not emit mid-infrared light and does not induce a photothermal effect (PT-off). Therefore, the laser 201 may be equipped with an element that acts as an optical switch to turn the emission of mid-infrared light on and off, such as a photoacoustic modulator or an optical chopper.

[0026] Cylindrical mirrors 202 and 203 each have curvature in planes perpendicular to each other. For example, in Figure 2, cylindrical mirror 202 appears curved in a plane parallel to the plane of paper, but flat in a plane perpendicular to the plane of paper. On the other hand, cylindrical mirror 203 appears curved in a plane perpendicular to the plane of paper, but flat in a plane parallel to the plane of paper. By considering cylindrical mirrors 202 and 203 in combination with lens 205, they can be considered as a relay optical system with different magnification (or reduction) ratios in planes parallel to and perpendicular to the plane of paper. When a relay optical system is desired that magnifies (or reduces) at the same magnification in planes parallel to and perpendicular to the plane of paper, cylindrical mirrors 202 and 203 can be replaced with a single curved mirror, such as a parabolic mirror or a concave mirror. Alternatively, a cylindrical lens may be used instead of cylindrical mirrors 202 and 203.

[0027] (Scanning Department) The scanning unit 30 comprises a two-axis scanner 301 and relay lenses 302 and 303. The two-axis scanner 301 has two mirrors that oscillate around two mutually orthogonal axes, and emits incident light with a two-dimensional displacement relative to the optical axis. This displacement is controlled by the angles of the two mirrors, thereby controlling the scanning point and scanning range on the sample surface P501. By making the oscillation frequencies of the two mirrors different, the sample surface P501 is scanned in two dimensions. It is desirable that the midpoint between the two mirrors is conjugate to the pupil plane P401 of the objective lens 401 via the relay lenses 302 and 303. The two-axis scanner 301 can use a two-axis galvanometer scanner. It is also possible to configure the two-axis scanner 301 by combining a one-axis resonant scanner and a galvanometer scanner. The relay lenses 302 and 303 ensure that light is incident on the pupil of the objective lens 401 with an appropriate beam diameter and maximum angle.

[0028] (Relay team) The relay unit 60 includes relay lenses 601 and 602 and a mirror 603. 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. In this case, the relay lenses 601 and 602 can be arranged symmetrically with respect to the sample surface P501 with respect to the relay lenses 303 and 302. Alternatively, the mirror 603 may be arranged symmetrically with respect to the sample surface P501 at an intermediate position between the two mirrors of the two-axis scanner 301.

[0029] (Control Unit) The control unit 90 comprises a control device 901, a keyboard 911, a mouse 912, a display 921, a scanning range acquisition unit 94, and a demodulation unit 80. 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 a waveform or signal generator, measuring instruments, an FPGA, a microcontroller, an electrical circuit, or a server that executes part of the control flow, and part of the program may be implemented on these. For example, a waveform generator that controls the output of the LED 101 can be included in the control device 901. Also, for example, an electrical circuit or waveform generator that controls the output of the laser 201 can be included in the control device 901. Also, for example, an electrical circuit that generates a drive signal for the 2-axis scanner 301 can be included in the control device 901. Also, for example, a digitizer that converts the analog signal output from the photodetector 701 to digital, or an analyzer that analyzes the signal waveform can be included in the control device 901. Also, for example, an electrical circuit that acquires desired data at a predetermined pixel rate or frame rate can be included in the control device 901. Furthermore, the control device 901 may also include a server for saving the generated phase-contrast images and PT images, and for performing image processing and analysis, as well as network equipment required for communication with this server.

[0030] The control device 901 generates a phase contrast image and a PT image based on data acquired by analyzing the signal output by the photodetector 701. During image generation, the control device 901 identifies the position of the focal point (measurement point) on the sample surface P501 from the control signal of the two-axis scanner 301 and generates data for each pixel based on that information. The control device 901 identifies the mid-infrared wavelength corresponding to the generated PT image from the control signal of the laser 201. The control device 901 generates the PT image while sequentially changing the output wavelength of the laser 201, and generates a PT spectrum, for example, by plotting the average PT intensity of the same pixel region against the mid-infrared wavelength. The control device 901 may execute a program to extract feature quantities from the generated phase contrast image and PT image or PT spectrum, or to discover a region of interest on the sample 501 from the feature quantities. The control device 901 may also execute preprocessing programs and analysis programs for the images and spectra necessary for this purpose. The generated phase-contrast image, PT image, PT spectrum, feature quantities, and information regarding the region of interest are stored in the memory device (not shown) of the control device 901 or output to the display 921. The memory device is, for example, a solid-state drive or a hard disk drive. The memory device may also be provided by a server.

[0031] The keyboard 911 and mouse 912 are connected to the control unit 901, and the operator inputs instructions to the control unit 901 by operating the keyboard 911 and mouse 912. The control unit 901 is connected to the LED 101, laser 201, 2-axis scanner 301, stage scanner 512, and photodetector 701, and controls their operation according to the instructions from the operator.

[0032] The display 921 provides visual feedback to the operator's actions and also displays images and text output by the control device 901.

[0033] (Phase difference detection system) Figure 3 is a schematic diagram illustrating the phase detection system of the observation device 1. Phase difference detection in the observation device 1 uses an ejection optical system 10 (phase difference light source unit), a scanning unit 30, a first objective lens 40, a mounting unit 50, a second objective lens 45, a relay unit 60, a detection unit 70 (phase difference detection unit), and a control unit 90.

[0034] As shown by the dotted line in Figure 3, the light emitted by the LED 101 of the emission optical system 10 (phase difference light source) becomes a parallel beam at the collimator 102. The annular beam of light that has passed through the ring slit 103 is focused to a single point toward the pinhole 105 by the relay lens 104. At this time, the first-order and higher diffracted light generated at the edge of the ring slit 103 broadens the focal point. The pinhole 105 blocks the light from the periphery of this focal point while allowing light closer to the center to pass through, thereby increasing the proportion of light that has traveled straight through the ring slit 103 (in other words, zero-order diffracted light). This suppresses the deformation of the annular beam of light, which has been re-formed into an annular shape by the relay lens 106, as it propagates along the subsequent optical path. Therefore, artifacts in the acquired phase difference signal can be suppressed.

[0035] The parallel light beam, whose annular diameter has been expanded or contracted by the relay lens 106, is incident on 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 emitted from the two-axis scanner 301 parallel to the optical axis, or at an angle to the optical axis, has its annular diameter expanded or contracted by the relay lenses 302 and 303, and 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 within the sample surface P501 corresponds to the angle of the light beam emitted from the two-axis scanner 301 with respect to the optical axis. At this time, in addition to the straight-traveling light (in other words, zero-order diffracted light) that travels in a straight line through the sample 501, first-order or higher diffracted light is generated according to the spatial distribution of the refractive index of the sample 501. Therefore, the refractive index of sample 501 changes due to the photothermal effect, which in turn changes the diffracted light.

[0036] The straight-traveling and diffracted light emitted from the sample 501 is collected by the objective lens 451 and emitted as a parallel beam. At this time, the straight-traveling light from the sample 501 becomes an annular shape, and the inside and outside of the annulus become the diffracted light generated in the sample 501. The emission angle of this parallel beam corresponds to the position of the focal point within the sample surface P501, that is, the angle of the light beam emitted from the two-axis scanner 301 with respect to the optical axis. The light beam emitted from the objective lens 451 has its beam diameter expanded or reduced by the relay lenses 601 and 602 of the relay unit 60 and is reflected by the mirror 603. The light beam reflected by the mirror 603 is incident on the relay lens 702 of the first detection unit 70 (phase difference detection unit).

[0037] This light beam is widened or narrowed by relay lenses 702 and 704 to become a parallel light beam again, and then passes through the phase plate 705. At this time, the straight-traveling light from the sample 501 passes through the annular portion and undergoes phase modulation and attenuation, while the diffracted light from the sample 501 passes through the portion other than the annular. The light beam emitted from the phase plate 705 is focused to a single point toward the light-receiving surface P701 of the photodetector 701 by the tube lens 706. At the light-receiving surface P701, the straight-traveling light and the diffracted light from the sample 501 interfere, and the photodetector 701 detects the resulting light intensity. Therefore, the change in diffracted light due to the change in refractive index of the sample 501 caused by the photothermal effect is expressed as a change in light intensity detected by the photodetector 701. The light that reaches the detection surface of the photodetector 701 is sometimes called phase difference detected light.

[0038] The phase plate 705 of the detection unit 70 (phase difference detection unit) of the observation device 1 is positioned conjugate to the pupil plane P451 of the objective lens 451. The angle of the light beam passing through the phase plate 705 with respect to the optical axis changes in accordance with the position of the focal point on the sample surface P501, in other words, the angle of the light beam emitted by 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-traveling light (zero-order diffracted light) at the sample 501 and attenuates the light.

[0039] Figure 4 is a schematic diagram illustrating the relationship between the phase plate 705 of the observation device 1 and the light beam transmitted 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 between the inner cylinder 705_1 and the outer cylinder 705_2 (shaded area in Figure 9). An annular parallel light beam created by the straight-traveling light from the sample 501 is transmitted through this region. Of this annular light beam, the innermost light beam L103_1 and the outermost light beam L103_2 trace concentric cylinders around the principal rays of the parallel light beam (including both the straight-traveling and diffracted light from the sample 501) transmitted through the phase plate 705. The diameters of the cylinders of these luminous beams L103_1 and L103_2 correspond to the inner and outer diameters of the ring slit 103, which is located at a position conjugate to the plane P705 on which the center of the thickness of the phase plate 705 is positioned. Specifically, by multiplying the inner radius I and outer radius E of the ring slit 103 by the imaging magnification m, the radius of luminous beam L103_1 becomes mI and the radius of luminous beam L103_2 becomes mE.

[0040] Figure 4(A) shows the case when the angle of the parallel light beams passing through the phase plate 705 with respect to the optical axis is at its maximum. In Figure 4(A), the principal ray L103_0 makes the maximum angle θ0 with respect to the optical axis 705_0 and intersects on the plane P705 which coincides with the center of the thickness of the phase plate 705. At this time, all of the light beams between L103_1 and L103_2 pass through the region between the cylinders 705_1 and 705_2 of the phase plate 705, so the radii of cylinders 705_1 and 705_2 satisfy the following equations 1 and 2.

[0041]

number

[0042]

number

[0043] In equations 1 and 2, the angle of incidence of the light beam to the phase plate 705 was assumed to be at most 10°, and refraction caused by quarter-wave plates, ND filters, etc., was not considered. More accurately, it is desirable to determine the radius by considering these refractions.

[0044] Figure 4(B) shows the case when the parallel luminous beams passing through the phase plate 705 are parallel to the optical axis. When equations 1 and 2 are satisfied, the entire luminous beam between luminous beams L103_1 and L103_2 passes through the region between the cylinders 705_1 and 705_2 of the phase plate 705.

[0045] Thus, by designing the system so that all straight-traveling light from the sample 501 undergoes phase modulation and 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 and PT image can be suppressed.

[0046] (PT-induced system) Figure 5 is a schematic diagram illustrating the PT induction system using observation device 1. In PT induction using observation device 1, the irradiation optical system 20 (mid-infrared light source), mounting unit 50, and control unit 90 are used.

[0047] As shown by the dashed line in Figure 4, the parallel beam of light emitted by the laser 201 of the mid-infrared light source is reflected by the cylindrical mirror 202, becoming a focused beam of light in the plane parallel to the plane of the paper and a parallel beam of light in the plane perpendicular to the plane of the paper. Next, this beam of light is reflected by the cylindrical mirror 203, becoming a focused beam of light in the plane perpendicular to the plane of the paper as well. This focused beam of light focuses near the mirror 204 in both the plane parallel to and perpendicular to the plane of the paper, and is reflected by the mirror 204. The beam of light reflected by the mirror 204 then enters the lens 205. The beam of light that passes through the lens 205 becomes a parallel beam of light in both the plane parallel to and perpendicular to the plane of the paper. At this time, if the cross-sectional view perpendicular to the optical axis of the beam of light emitted by the laser 201 is circular, then the cross-sectional view of the beam of light that passes through the lens 205 will be elliptical. This is because the cylindrical mirrors 202 and 203 and the lens 205 cause the beam diameter to be magnified or reduced at different magnifications in planes parallel to and perpendicular to the plane of the paper.

[0048] The parallel light beam transmitted through lens 203 is directed toward the sample 501 on the mounting section 50. At this time, in order to prevent interference with the objective lens 451 of the second objective lens 45, the light beam needs to be directed toward the sample 501 at an angle with respect to the optical axis of the phase difference detection system (objective lens 451). Therefore, this light beam tends to create a projected image (shape) on the sample surface P501 that is extended in the direction along the optical axis of lens 205. The projected image of the mid-infrared light beam on the sample surface P501 is adjusted by changing the focal length of the cylindrical mirrors 202, 203 and lens 205, and the angle of the light beam with respect to the optical axis of the phase difference detection system. In the projection region of the mid-infrared light beam on the sample surface P501, molecules absorb mid-infrared light in accordance with their vibrational levels, inducing PT.

[0049] (Area and timing of light irradiation) Figure 6 is a schematic diagram illustrating the region and timing of light irradiated onto sample 501 in observation device 1. Figure 6(A) schematically shows, in order, the time period for acquiring an observation image of the sample surface P501 in the PT-off state (sometimes referred to as a PT-off image) and the time period for acquiring an observation image in the PT-on state (sometimes referred to as a PT-on image). Furthermore, Figure 6(A) schematically shows the time period for heat dissipation, in which heat accumulated on sample surface P501 is dissipated after the PT-on state has ended. As shown in Figure 6(A), in one cycle of photothermal imaging (sometimes referred to as PT imaging), PT-off image acquisition, PT-on image acquisition, and heat dissipation are performed in order. Note that in Figure 6(A), a cell is shown as an example of a sample.

[0050] In the example shown in Figure 6, the observation area can be considered as being divided into m x n elements; that is, the acquired observation image consists of an m x n pixel array. This m x n observation image is acquired by scanning the sample surface P501 for n pixels in each row, then moving to the next row and scanning for n pixels again, and repeating this process. In Figure 6(A), the scanning area on the sample surface P501, indicated by the grid, may be referred to as the phase difference detection area. Also, in Figure 6(A), the mid-infrared light irradiation area is shown in gray. The phase difference detection area is set so that the PT signal detection area is included within the mid-infrared light irradiation area. The method for identifying the mid-infrared light irradiation area and setting the PT signal detection area will be described later.

[0051] Figure 6(B) is an example of a PT imaging timing chart. The vertical axis represents the pixel number of the acquired observation image, and the horizontal axis represents the elapsed time. In an observation image composed of an m x n pixel array, the pixels belonging to the first row are numbered from 1 to n, the pixels belonging to the second row are numbered from n+1 to 2n, and similarly the pixels belonging to the mth row are numbered from m x (n-1)+1 to m x n. The time during which phase-contrast illumination light is irradiated at the position of each element of the sample surface P501 corresponding to each pixel of the observation image is schematically shown by the solid line graph, and the time during which mid-infrared light is irradiated is shown by the gray area.

[0052] As illustrated in Figure 6(B), in one cycle of PT imaging, the sample surface P501 is scanned from the 1st row to the mth row to first acquire the PT-off image. After scanning the mth row is complete, irradiation of the sample 501 with mid-infrared light is started. Then, the system waits for sub-milliseconds to milliseconds until the photothermal effect is induced and saturated (heat accumulation and dissipation balance out). Next, with the mid-infrared light irradiation continuing, the sample surface P501 is scanned from the mth row to the 1st row to acquire the PT-on image. Note that by reversing the scanning direction in the column direction for the PT-off image and the PT-on image, the time required to return the scanning position to the 1st row after scanning the mth row of the PT-off image can be omitted. After acquiring the PT-on image is complete, irradiation with mid-infrared light and phase-contrast illumination light is stopped to dissipate the heat accumulated in the infrared light-irradiated area. The system waits for sub-milliseconds to sub-seconds until it returns to a state where the photothermal effect can be induced again (the state when the PT-off image was initially acquired). If you want to repeat the PT imaging cycle after heat dissipation is complete, start again by acquiring the PT-off image.

[0053] (Generation of infrared-absorbing images) The scanning observation device 1 includes a control unit 90 which includes a demodulation unit 80 that demodulates the signal from the first detection unit 70 to acquire the intensity modulation component of the secondary light from the sample 501 synchronized with the emission of the second light. The scanning observation device 1 also includes an image generation unit 92 which generates an image based on information regarding the irradiation position of the first light and the signal from the first detection unit 70. That is, the image generation unit 92 generates a first image as a phase difference image based on the output of the detection unit 70 when the irradiation operation of the irradiation optical system 20 is off. The image generation unit 92 also generates a second image as a photothermal-induced effect image based on the difference between the output of the detection unit 70 when the irradiation operation of the irradiation optical system 20 is on and the output of the detection unit 70 when the irradiation operation of the irradiation optical system 20 is off. In other words, the image generation unit 92 generates the first image as a phase difference image based on the output from the demodulation unit 80 when the irradiation operation of the irradiation optical system 20 is off. In other words, the image generation unit 92 generates a second image as a photothermal-induced effect image based on the difference between the output from the demodulation unit 80 when the irradiation operation of the irradiation optical system 20 is ON and the output from the demodulation unit 80 when the irradiation operation of the irradiation optical system 20 is OFF.

[0054] (Demodulation unit) Figure 7 illustrates the principle of generating infrared absorption images (PT images) and morphological images (phase contrast images) by the difference processing performed by the demodulation unit 80. In other words, the PT image, or infrared absorption image, is obtained as a difference image obtained by subtracting the phase contrast image under PT-off conditions from the phase contrast image under PT-on conditions. To ensure visibility of such PT images compared to phase contrast images, a display mode using a color palette (color contour) is employed to express contrast using chromaticity from cool to warm colors, so that the PT image has a different display mode from the phase contrast image. On the other hand, the phase contrast image can be obtained by reusing the PT-off image. Since the PT image and the phase contrast image are generated based on signals acquired at the same measurement point and time, each pixel of the PT image and the phase contrast image correspond one-to-one spatially and temporally. The demodulation processing performed by the demodulation unit 80 according to the presence or absence (or intensity change) of mid-infrared irradiation may be performed by difference processing between image data or by difference processing between image signals. In other words, the demodulation unit 80 is configured to perform at least one of the difference processing operations, which is either difference processing between image data or difference processing between image signals, corresponding to when the irradiation operation of the irradiation optical system 20 is ON or OFF.

[0055] (Identification of the mid-infrared light irradiation area and setting of the PT signal detection area) Figure 8 is a schematic diagram illustrating the method for identifying the mid-infrared light irradiation area and setting the PT signal detection area in observation device 1. In observation device 1, before PT imaging of the target sample containing the cells to be observed, it is necessary to identify the mid-infrared light irradiation area and set the phase difference detection area, i.e., the PT detection area, to match it. For this pre-measurement sample 505, for example, water, aqueous solution, or organic solvent can be used. This sample 701 may be referred to as the pre-measurement sample 505. It is also possible to use a region within the target sample that does not contain the cells to be observed as the pre-measurement sample 505. In Figure 7, the phase difference detection areas R505 and R507 are schematically shown as grids, and the mid-infrared light irradiation area R506 is shown in gray.

[0056] As shown in Figure 8(A), PT imaging is performed on the pre-measured sample 701 to identify the mid-infrared light irradiation region. In this case, the phase difference detection region R505 needs to be larger in order to identify the mid-infrared light irradiation region R506. If a significant PT intensity is obtained from the entire acquired PT image, it is considered that the mid-infrared light irradiation region R506 encompasses the phase difference detection region R505. In that case, the scanning range input to the control unit 901 is changed to obtain a wider phase difference detection region R505, or the mid-infrared light source unit 20 is adjusted to obtain a narrower mid-infrared irradiation region R506, and then PT imaging of the pre-measured sample 701 is performed again. If, in the acquired PT image, the region showing a significant PT intensity is surrounded by a region with a PT intensity near zero, the region showing a significant PT intensity can be considered the mid-infrared light irradiation region R506.

[0057] As shown in Figure 8(B), in setting the PT signal detection region, the phase difference detection region R507 is set so as to be included within the identified mid-infrared light irradiation region R506, and PT imaging of the pre-measured sample 701 is performed. At that time, it is desirable to set the proportion of the phase difference detection region R507 to the mid-infrared light irradiation region R506 to be as large as possible in order to maximize the utilization efficiency of mid-infrared light. This phase difference detection region R507 can be rephrased as the PT signal detection region R507. The PT intensity distribution of the acquired PT image of the pre-measured sample 701 can be used for shooting correction in the subsequent PT imaging of the target sample. In this way, by including the phase difference detection region R507 within the mid-infrared light irradiation region R506, irradiation of areas where the PT signal is not detected with phase difference illumination light can be prevented. Therefore, there is an advantage in that the photobleaching of the sample 501, which absorbs visible light, due to visible light can be reduced. Furthermore, it has the advantage of reducing photobleaching due to visible light, fluorescence, and phosphorescence in samples 501 containing dye molecules that emit fluorescence or phosphorescence following the absorption of visible light. Examples of samples 501 containing dye molecules that absorb visible light, or that absorb visible light and emit fluorescence or phosphorescence, include unstained or stained cells or tissues that are populations thereof, as well as organic materials.

[0058] (Observation Flow) Figure 9 illustrates an observation flow using the observation device 1 when sample 501 is a target sample such as cells to be observed, measured, or examined. Figure 9(A) is a flowchart explaining the observation flow, and Figure 9(B) is a diagram explaining the low-magnification field of view R502, high-magnification field of view R503, and region of interest R504 in the flowchart. Figure 9(C) is a diagram showing the schematic configuration of the scanning range acquisition unit.

[0059] As shown in Figure 9(C), the control unit 90 includes an image generation unit 92 that generates a first image captured by a first objective lens 40 and a second objective lens 45 at a predetermined magnification, and displays the first image on the display unit 921. The control unit 90 further includes an input unit 910 that receives input from the operator regarding the region of interest based on the displayed first image, and a scanning range acquisition unit 94 that acquires a scanning range SA0 corresponding to the scanning area of ​​the irradiation position of the first light and commands the scanning unit 30. The control unit 90 further includes an update unit 98 that displays the scanning range SA1 acquired by the scanning range acquisition unit 94 on the display unit and accepts updates to the scanning range SA1. The scanning range acquisition unit 94 acquires the scanning range SA based on the first image displayed on the display unit 921 and the field of view specified by the operator on the screen of the display unit 921.

[0060] First, the sample 501 is held on the stage 511. From this state, the observation flow of the target sample begins. First, the sample 501 is observed using phase contrast observation (a phase contrast image is acquired) in 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 Figure 9(B) is moved. 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 and low-magnification phase contrast observation is continued. 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 in 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 helps to suppress damage to the sample 501.

[0061] Next, a decision is made as to whether to confirm the details of the phase contrast image of region of interest R504 from the low-magnification phase contrast image. (Step S40.) If details are to be confirmed, the field of view is narrowed and real-time phase contrast observation is performed. In that case, the high-magnification phase contrast observation conditions are input to the control device 901 and high-magnification phase contrast observation is started. (Steps S50, S60.) If necessary, the high-magnification field of view R503 shown in Figure 9(B) is moved so that the region of interest R504 is included, and the necessity of PT observation (acquisition of PT image) is considered from the high-magnification phase contrast image. On the other hand, if the details of the phase contrast image of region of interest R504 are not to be confirmed, the necessity of PT observation is considered from the low-magnification phase contrast image. If it is determined from these phase contrast images that PT observation is unnecessary, the process returns to low-magnification phase contrast observation and another region of interest R504 of sample 501 is searched for. On the other hand, if it is determined that PT observation is necessary, the process proceeds to PT observation. (Step S70.) In this way, by smoothly magnifying the region of interest R504 of sample 501 and performing phase contrast observation, the necessity of PT observation can be quickly determined. Therefore, unnecessary PT observations can be reduced, improving measurement efficiency and minimizing damage to sample 501. Irradiation of sample 501 with mid-infrared light can be stopped until the above phase contrast observation is complete. In other words, the phase contrast image can be acquired with only phase contrast illumination light irradiating sample 501.

[0062] Next, the PT observation conditions (in other words, the PT image acquisition conditions) are input to the control device 901, and PT observation is started. (Step S90.) At this stage, it is also acceptable to irradiate the sample 501 with mid-infrared light for the first time. Once simultaneous observation of PT and phase contrast (in other words, observation of PT images and PT-off images) is completed, the series of observation flows is terminated.

[0063] The low-magnification and high-magnification phase-contrast observation conditions include, for example, the light output intensity of LED101, as well as the field of view size, number of integrations, and number of pixels. The PT observation conditions include, for example, the light output intensity, center wavelength, wavelength width, pulse duration of laser201, as well as the field of view size, number of integrations, and number of pixels.

[0064] In the observation flow shown in Figure 9(A), if the region of interest R504 is discovered in step S30, the operator inputs the observation conditions in the subsequent steps S50 and S90. To improve convenience for the operator, a step S35 may be added between steps S30 and S40, in which the operator inputs the location information of the region of interest R504. The location information can be input, for example, by the operator using the mouse 912 to trace the outline of the region of interest R504 or draw a mark on the low-magnification phase-contrast image displayed on the display 921 of the control unit 90. Based on the above location information, the control device 901 may predict appropriate observation conditions and automatically input them in steps S50 and S90, or present them to the operator. By supporting the operator's input of each observation condition in this way, the burden on the operator can be reduced.

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

[0066] In the observation flow shown in Figure 9(A), the control unit 90 may automatically perform the detection of the region of interest R504 and the identification of its location information. This reduces the burden on the operator during observation.

[0067] In the observation device 1 shown in Figure 2, the ring slit 103 is placed in the first ejection optical system 10 (phase difference light source unit) and the phase plate 705 is placed in the first detection unit 70 (phase difference detection unit). However, the phase difference signal can be detected even if the two are swapped in their positions.

[0068] As described above, the observation device 1 according to the first embodiment of the present invention is a scanning observation device that can ensure the reproducibility of adjustments that align the phase difference detection region and the photothermal signal detection region. Furthermore, since the scanning observation device 1 according to the first embodiment of the present invention does not require mechanical operation as a field of view adjustment mechanism arranged on the mirror base, it is possible to improve the usability for the operator by eliminating the need for waiting time to reduce vibrations.

[0069] Note that observation device 1 may be referred to as scanning observation device 1, scanner 1, or scanning microscope 1.

[0070] <Second Embodiment> The configuration of the observation device according to the second embodiment will be described with reference to Figure 10. Figure 10 is a schematic diagram illustrating the configuration of the observation device 2 of this embodiment. The observation device 2 has the same configuration as the observation device 1 of the first embodiment, except for the parts described below. Therefore, the same reference numerals are used for common elements, and redundant explanations are omitted.

[0071] In the observation device 2 shown in Figure 10, the mirror 204 of the mid-infrared light source unit 20 of the observation device 1 is removed, and a single-axis scanner 206 is installed in its place. The control unit 90 of the observation device 2 differs from that of the observation device 1 in that it is electrically connected not only to the laser 201 of the mid-infrared light source unit 20 but also to the single-axis scanner 206. In addition to phase-contrast illumination light, the observation device 2 also scans the sample surface with mid-infrared light.

[0072] (Mid-infrared light source) The uniaxial scanner 206 of the mid-infrared light source unit 20 of the observation device 2 controls the reflection angle when the light beam incident from the cylindrical mirror 203 is reflected toward the lens 205 by changing the angle of the mirror. This controls the height of the parallel beam of mid-infrared light that has passed through the lens 205 with respect to the optical axis of the lens 205. In other words, it controls the position of the projection image of the parallel beam of mid-infrared light irradiated onto the sample surface P501. Therefore, there is a one-to-one correspondence between the angle of the mirror of the uniaxial scanner 206 and the position of the projection image of the mid-infrared light on the sample surface P501, i.e., the mid-infrared light irradiation area. It is desirable that the incident surface of the mirror of the uniaxial scanner 206 (the surface formed by the incident and reflected light) is parallel to the surface of at least one of the cylindrical mirrors 202 and 203 that has curvature. In the example in Figure 10, the incident surface of the mirror of the uniaxial scanner 206 is parallel to the surface of the cylindrical mirror 202 that has curvature (both are planes of paper).

[0073] (Control Unit) The control unit 901 of the control unit 90 of the observation device 2 generates a drive signal for the single-axis scanner 206. The control unit 901 transmits a drive signal to the single-axis scanner 206 that is synchronized with the operation of one of the two mirrors that make up the two-axis scanner 301. For example, in the case of an example similar to Figure 6 in which the observed image consists of an m x n pixel array, the control unit 901 transmits drive signals to the two-axis scanner 301 and the single-axis scanner 206 so that the column-direction scanning of the phase-contrast illumination light and the column-direction scanning of the mid-infrared light are synchronized. The control unit 901 of the observation device 2 also controls the operation of the single-axis scanner 206 according to the instructions input by the operator.

[0074] (Identification of the mid-infrared light irradiation area and setting of the PT signal detection area) Figure 11 is a schematic diagram illustrating the region and timing of light irradiated onto the sample 501 in the observation device 2. Figure 11(A) schematically shows, in order, the state of the sample surface P501 at the start and end times of acquiring the PT-off image, the start time of irradiation with mid-infrared light, and the start and end times of acquiring the PT-on image in one cycle of PT imaging. Similar to Figure 6, in the example of Figure 11, the observation region is considered to be divided into m x n elements. That is, the phase-contrast illumination light scans the sample surface P501 for n pixels in each row, then moves to the next row and scans for n pixels again, repeating this process to acquire an observation image composed of an m x n pixel array.

[0075] As shown in Figure 11(A), in observation device 2, the mid-infrared light irradiation area at each time point becomes a part of the mid-infrared light irradiation area considered for the entire PT imaging cycle. That is, by scanning a mid-infrared light irradiation area that is narrow in the column direction and long in the row direction in the column direction, a mid-infrared light irradiation area encompassing the field of view of the observation image (the area of ​​the corresponding sample surface P501) is obtained. As shown in Figure 11(A), the scanning speed of the mid-infrared light in the column direction and the scanning speed of the phase-contrast illumination light in the column direction are both equal at v. As a result, at any point in the PT on-image acquisition time, the mid-infrared light irradiation area encompasses the irradiation area of ​​the phase-contrast illumination light.

[0076] The duration for which a region of the sample surface P501 corresponding to any row of the observed image is exposed to mid-infrared light is determined by dividing the length w of the mid-infrared light irradiation region, which is narrow in the column direction and long in the row direction, by the scanning speed v in the column direction. Therefore, the observation device 2 controls the duration of mid-infrared light irradiation by adjusting the length w of the mid-infrared light irradiation region in the column direction or the scanning speed v in the column direction. This duration is preferably on the order of sub-milliseconds to milliseconds, where the photothermal effect saturates (heat accumulation and dissipation balance out). The length w of the mid-infrared light irradiation region in the column direction can be adjusted by changing the focal lengths of the cylindrical lenses 202, 203 and lens 205, and the scanning speed v in the column direction can be adjusted by changing the scanning range input to the control device 901.

[0077] In observation device 2, detecting the PT signal at the rear end of the scanning direction in a mid-infrared light illumination area that is narrow in the column direction and long in the row direction results in the longest exposure time to mid-infrared light. In other words, the PT induction time is long, and the utilization efficiency of light is high. Therefore, as shown in Figure 11(A), it is desirable to scan the phase difference illumination light in the row direction at the rear end of the scanning direction in a mid-infrared light illumination area that is narrow in the column direction and long in the row direction.

[0078] Figure 11(B) is an example of a timing chart for PT imaging using observation device 2. Similar to Figure 6(B), the vertical axis represents the pixel number of the acquired observation image, and the horizontal axis represents the elapsed time. The time during which phase-contrast illumination light is irradiated to the position of each element of the sample surface P501 corresponding to each pixel of the observation image is schematically shown by the solid line graph, and the time during which mid-infrared light is irradiated is schematically shown by the gray area. As illustrated in Figure 11(B), in one cycle of PT imaging, the sample surface P501 is scanned from the 1st row to the mth row in order to first acquire a PT-off image. Subsequently, irradiation of the sample 501 with mid-infrared light is started to induce PT. In order for the first mth row to receive mid-infrared light irradiation for the same amount of time as the rows from the m-1th row onward, scanning in the column direction of the mid-infrared irradiation area is started from a position where the area of ​​the sample surface 501 corresponding to the mth row coincides with the front end. When the trailing edge of the mid-infrared illumination area reaches the region of sample surface P501 corresponding to the mth row of the observed image, scanning of the mth row (in the row direction) with phase-contrast illumination light is started. This marks the start of the PT-on image acquisition time. After the scanning of the first row (in the row direction) with phase-contrast illumination light is completed, the mid-infrared light irradiation is stopped, and the PT-on image acquisition time ends. Next, the system waits with light irradiation to sample 501 stopped until the scanning position moves from the first row to the mth row. This completes one cycle of PT imaging. In the next cycle, as shown in Figure 11(B), PT-on and PT-off images are acquired by scanning in the column direction, opposite to the first cycle. In this way, the same heat dissipation time is ensured for each region of sample surface P501 corresponding to each row of the observed image. As shown in Figure 11(B), if the time required for scanning in the column direction is sufficiently long compared to the mid-infrared light irradiation time for each row, a sufficiently long heat dissipation time is ensured compared to the PT induction time. For example, if the mid-infrared light irradiation time for each row is on the order of sub-milliseconds to milliseconds, a heat dissipation time of milliseconds to sub-seconds is ensured. In other words, by the time the mirror of the two-axis scanner 301 is set to the scanning position to start the next cycle of PT imaging, the sample 501 has returned to a state where PT induction is possible again.

[0079] As described above, observation device 2 performs PT induction and heat dissipation by mid-infrared light irradiation in parallel on separate rows within the field of view of the observed image. On the other hand, observation device 1 performed PT induction and heat dissipation sequentially. Therefore, in observation device 1, most rows within the field of view were exposed to mid-infrared light even after PT signal detection was completed, resulting in low light utilization efficiency and concerns about cytotoxicity. In observation device 2, heat dissipation begins immediately after PT signal detection is completed in any row within the field of view of the observed image, thus avoiding unnecessary exposure to mid-infrared light.

[0080] As described above, the observation device 2 according to the second embodiment of the present invention can improve light utilization efficiency and reduce cytotoxicity by synchronously scanning the sample surface with phase-difference illumination light and mid-infrared light. Similar to observation device 1, observation device 2 is a scanning type observation device that can ensure the reproducibility of adjustments that align the phase-difference detection area and the photothermal signal detection area. Furthermore, since observation device 2 does not require mechanical operation as a field-of-view adjustment mechanism arranged on the mirror base, it is possible to improve the usability for the operator by eliminating the waiting time required to wait for vibration reduction.

[0081] <Third Embodiment> The configuration of the observation device according to the third embodiment will be described with reference to Figure 12. Figure 12 is a schematic diagram illustrating the configuration of the observation device 3 of this embodiment. The observation device 3 has the same configuration as the observation device 2 of the second embodiment, except for the parts described below. Therefore, the same reference numerals are used for common elements, and redundant explanations are omitted.

[0082] In the observation device 3 shown in Figure 12, the mirror 603 of the relay unit 60 of observation device 2 is removed, and a two-axis scanner 604 is installed in its place. In addition, the detection unit 70 (phase difference detection unit) is equipped with a pinhole 703 at the position of the intermediate imaging plane P703. The control unit 90 of observation device 3 differs from that of observation device 2 in that it is electrically connected to the two-axis scanner 604 of the relay unit 60. Observation device 3 detects phase difference detection light using an optical system and a pinhole that cancel out the scanning of phase difference illumination light.

[0083] (Relay team) The relay unit 60 of the observation device 3 comprises relay lenses 601 and 602 and a two-axis scanner 603. Light is incident on the two-axis scanner 603 by the relay lenses 601 and 602 at an appropriate beam diameter and maximum angle. The two-axis scanner 603 has two mirrors that oscillate around two mutually orthogonal axes. The angles of these mirrors are controlled to cancel out the angular displacement of the incident light beam relative to the optical axis. That is, one mirror of the two-axis scanner 603 oscillates with respect to one mirror of the two-axis scanner 301, and the other mirror of the two-axis scanner 603 oscillates with respect to the other mirror of the two-axis scanner 301, each at the same frequency 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. This operation of the two-axis scanner is sometimes called reverse scanning (descan). It is desirable that the intermediate position between the two mirrors of the two-axis scanner be conjugate to the pupil plane P451 of the objective lens 451 via relay lenses 601 and 602.

[0084] In particular, when objective lenses 451 and 401 are the same, relay lenses 601 and 602 can be the same as 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, relay lenses 601 and 602 and the two-axis scanner 603 can be arranged symmetrically with respect to the sample surface P501 with respect to relay lenses 303 and 302 and the two-axis scanner 301.

[0085] In the third embodiment having a two-axis scanner 603, the relay unit 60 may be referred to as the reverse scanning unit 60 based on its function. The reverse scanning unit 60 is configured to reverse scan in synchronization with the scanning unit 30 on the optical path between the detection unit 70 and the second objective lens 45. The detection unit 70 (phase difference detection unit) can be described as having a pinhole 105 that blocks a portion of the secondary light, and a pair of relay lenses 104 and 106 arranged on either side of the pinhole 105, on the optical path between the reverse scanning unit 60 and the first photodetector 701.

[0086] (Control Unit) The control unit 901 of the control unit 90 of the observation device 3 generates a drive signal for the two-axis scanner 301. The control unit 901 of the observation device 3 transmits a drive signal to the two-axis scanner 604 that is synchronized with the movement of the two mirrors that make up the two-axis scanner 301. The control unit 901 of the observation device 3 also controls the movement of the two-axis scanner 603 according to the instructions entered by the operator.

[0087] (Phase contrast observation system) In the observation device 3, the straight-traveling light and diffracted light emitted from the sample 501 are collected by the objective lens 451 and emitted as a parallel beam. The emission angle of this parallel beam corresponds to the position of the focal point within the sample surface P501, that is, the angle of the beam emitted from the two-axis scanner 301 with respect to the optical axis. The beam of light emitted from the objective lens 451 is expanded or reduced in diameter by the relay lenses 601 and 602 of the relay unit 60 and incident on the two-axis scanner 603. This beam is reflected by each of the two mirrors of the two-axis scanner 603. Regardless of the incident angle, the beam emitted from the two-axis scanner 603 parallel to or coincides with the optical axis is incident on the relay lens 702 of the detection unit 70 (phase difference detection unit).

[0088] This light beam is focused to a single point towards the pinhole 703 by the relay lens 702. The pinhole 703 blocks the light from the periphery of this focal point, thereby blocking diffracted and scattered light generated outside the focal point on the sample surface P501. This suppresses artifacts in the acquired phase difference signal.

[0089] The light beam that passes through the pinhole 703 has its beam diameter expanded or contracted by the relay lens 704, becoming a parallel light beam again, and then passes through the phase plate 705.

[0090] As described above, the observation device according to the third embodiment of the present invention enables the detection of phase difference detection light transmitted through a pinhole by canceling out the scanning of phase difference illumination light. This suppresses artifacts in the acquired phase difference image and PT image.

[0091] In the observation devices 1 to 3 of the above embodiments, the phase-contrast illumination light may be referred to as the first light, and the mid-infrared light as the second light. The scanning unit 30 may be referred to as the first scanning unit. The phase-contrast illumination light (straight light and diffracted light) emitted from the sample 501 may be referred to as the secondary light. The LED 101 may be referred to as the first light source, and the laser 201 as the second light source. The single-axis scanner 206 may be referred to as the second scanning unit.

[0092] Although the present invention has been described above using 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 or improvements can be made to the above embodiments. It will be clear from the claims that such modified or improved forms may also be included in the technical scope of the present invention. [Explanation of symbols]

[0093] 1, 2, 3 Scanning observation device 10 Exit optical system (phase difference light source) 20 Irradiation optical system (mid-infrared light source) 30 Scanning Unit 40. First objective lens (focusing lens) 45. Second objective lens (light-gathering lens) 50 Mounting section 60 Relay section 70 Detection unit (phase difference detection unit) 90 Control Unit 92 Image generation unit 94 Scanning range acquisition unit

Claims

1. A scanning observation apparatus comprising: an emission optical system that emits a first light containing spatially modulated components; a first scanning unit that scans the first light; a focusing lens that focuses the scanned first light; a scanning range acquisition unit that receives the setting of the scanning area of ​​the first light and commands the first scanning unit; a placement unit on which a sample is placed at the focusing position of the focusing lens; a light-collecting lens located on the opposite side of the placement unit from the focusing lens and for collecting secondary light from the sample; an illumination optical system that irradiates the sample with a second light having a longer wavelength than the first light such that at least a portion of the irradiation area of ​​the sample by the focusing lens overlaps; a detection unit that detects the secondary light through the light-collecting lens; and a demodulation unit that demodulates the signal from the detection unit to acquire the intensity modulated component of the secondary light synchronized with the emission of the second light.

2. The scanning observation apparatus according to claim 1, wherein the intensity modulation of the secondary light is caused by the absorption of the second light by the sample.

3. The scanning observation apparatus according to claim 1 or 2, wherein the illumination optical system comprises a second light source that emits light with a wavelength of 2 μm to 30 μm.

4. The scanning observation apparatus according to claim 1 or 2, wherein the irradiation area of ​​the second light in the sample includes the irradiation area of ​​the first light.

5. A scanning observation apparatus according to any one of claims 1, 2, or 4, further comprising a second scanning unit for scanning the second light.

6. The scanning observation apparatus according to claim 5, wherein the first scanning unit and the second scanning unit are synchronized.

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

8. The scanning observation apparatus according to claim 1 or 2, wherein the emission optical system comprises a modulation optical element that modulates the intensity component of the first light in an annular manner, and the detection unit comprises a demodulation optical element having an annular portion that demodulates the intensity component and phase difference component of the secondary light.

9. The scanning observation apparatus according to claim 1 or 2, wherein the emission optical system comprises a pinhole that blocks a portion of the focused primary light.

10. The scanning observation apparatus according to claim 1 or 2, wherein the ejection optical system comprises a modulation optical element having an annular portion for modulating the intensity component of the first light, and the detection unit comprises a demodulation optical element for separating and demodulating the intensity component and the phase difference component of the first light, respectively.

11. The scanning observation apparatus according to claim 1 or 2, further comprising a reverse scanning unit that scans in reverse in synchronization with the first scanning unit, on the optical path between the detection unit and the light-gathering lens.

12. The scanning observation apparatus according to claim 11, wherein the reverse scanning unit focuses the secondary light toward the detection unit, and the detection unit is provided with a pinhole that blocks a portion of the focused secondary light.

13. The scanning range acquisition unit acquires the scanning range based on a command from the operator, according to the scanning type observation apparatus according to claim 1 or 2.

14. The scanning observation apparatus according to claim 1 or 2, further comprising a condensing lens with a predetermined magnification and an image generation unit that generates a first image captured by the light-gathering lens and displays the first image on a display unit.

15. The scanning observation apparatus according to claim 14, wherein the scanning range acquisition unit acquires the scanning range based on the first image displayed on the display unit and based on the field of view specified by the operator on the screen of the display unit.

16. The scanning observation apparatus according to claim 15, further comprising an update unit that displays the scanning range acquired by the scanning range acquisition unit on the display unit and accepts updates to the scanning range.

17. The demodulation unit is configured to perform at least one of the following difference processing operations: difference processing between image data and difference processing between image signals, corresponding to when the irradiation operation of the irradiation optical system is ON and when it is OFF. The scanning observation apparatus according to claim 14, wherein the image generation unit generates a first image as a phase difference image based on the output of the detection unit when the irradiation operation of the irradiation optical system is off, and generates a second image as a photothermal induced effect image based on the difference between the output of the detection unit when the irradiation operation of the irradiation optical system is on and the output of the detection unit when it is off.

18. The scanning observation apparatus according to claim 1 or 2, wherein the demodulation unit is configured to perform difference processing of at least one of the difference processing between image data and difference processing between image signals, corresponding to when the irradiation operation of the irradiation optical system is ON and when it is OFF.

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