Scanning microscope unit, scanning microscope, and method for calibrating scanning microscope unit

Abstract

This scanning microscope unit comprises: a light source which outputs irradiation light; a light detector which detects observation light that is produced from an observation target in accordance with irradiation thereof with the irradiation light; a MEMS mirror which causes the irradiation light to scan the observation target and which directs the observation light to the light detector; a scan lens which guides the irradiation light that has been scanned by the MEMS mirror to a microscope optical system and which guides the observation light that has been formed into an image by the microscope optical system to the MEMS mirror; and a frame member which formed in a frame-like shape so as to define an aperture, and which is disposed to the microscope optical system side of the scan lens such that the irradiation light and the observation light pass through the aperture. The frame member has a calibration part that is provided at the sides that define the aperture, and that generates correction light including sensitivity wavelengths of the light detector, in accordance with entry of the irradiation light.

Description

[Technical Field]

[0001] One aspect of the present disclosure relates to a scanning microscope unit, a scanning microscope, and a method for calibrating a scanning microscope unit. [Background technology]

[0002] Patent Document 1 describes a scanning microscope unit that constitutes a scanning microscope when attached to a connection port of a microscope having a microscope optical system. The scanning microscope unit described in Patent Document 1 includes a MEMS (Micro Electro Mechanical System) mirror that scans the illumination light output from a light source over an observation object, and a photodetector that detects observation light generated from the observation object in response to illumination with the illumination light. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2020 / 196782 Summary of the Invention [Problem to be solved by the invention]

[0004] In the scanning microscope unit described above, in order to image a desired field of view, for example, during manufacturing, the drive signal applied to the MEMS mirror is adjusted so that the oscillation angle of the MEMS mirror is appropriate relative to the sampling timing of the photodetector. However, the amplitude or phase of the oscillation of the MEMS mirror may change over time, which may cause a shift in the field of view.

[0005] Therefore, an object of one aspect of the present disclosure is to provide a scanning microscope unit, a scanning microscope, and a method for calibrating a scanning microscope unit that can suppress the occurrence of field of view deviation. [Means for solving the problem]

[0006] A scanning microscope unit according to one aspect of the present disclosure is a scanning microscope unit that constitutes a scanning microscope by being attached to a connection port of a microscope having a microscope optical system, and includes: a light source that outputs illumination light; a photodetector that detects observation light generated from an object of observation in response to illumination with the illumination light; a MEMS mirror that scans the object of observation with the illumination light output from the light source and directs the observation light generated from the object of observation in response to illumination with the illumination light toward the photodetector; a scan lens that directs the illumination light scanned by the MEMS mirror to the microscope optical system and directs the observation light imaged by the microscope optical system to the MEMS mirror; and a frame member that is formed in a frame shape to define an opening and is positioned on the microscope optical system side of the scan lens so that the illumination light and observation light pass through the opening, the frame member being provided on a side that defines the opening and having a calibration unit that generates calibration light including a sensitivity wavelength of the photodetector in response to incidence of the illumination light.

[0007] This scanning microscope unit includes a frame member formed in a frame shape to define an aperture and positioned on the microscope optical system side of the scan lens so that illumination light and observation light pass through the aperture. The frame member also includes a calibration unit disposed on a side defining the aperture, generating calibration light including a wavelength to which the photodetector is sensitive in response to the incidence of illumination light. This allows, for example, the illumination light to be scanned within a scan area on a plane along the frame member using a MEMS mirror, the calibration light generated by the calibration unit in response to the incidence of illumination light using a photodetector, and at least one of the amplitude and phase of the oscillation of the MEMS mirror to be adjusted based on the detection result from the photodetector. By adjusting (calibrating) at least one of the amplitude and phase in this manner, the occurrence of the field of view deviation described above can be suppressed. Therefore, this scanning microscope unit can suppress the occurrence of field of view deviation.

[0008] The frame member may have a plate member that generates calibration light in response to the incidence of irradiation light and a cover member that is disposed on the plate member and covers the plate member, and the calibration unit may be formed by a part of the plate member that is exposed through an exposure opening formed in the cover member. In this case, it is possible to accurately detect the edge of the calibration unit, thereby improving the accuracy of the calibration. Furthermore, it is possible to accurately form the calibration unit, thereby improving the accuracy of the calibration.

[0009] The calibration unit may include a fluorescent member, in which case calibration can be performed in a scanning microscope unit for fluorescence observation.

[0010] The photodetector may detect, as observation light, fluorescence emitted from the object to be observed in response to irradiation with the illumination light, in which case fluorescence observation can be performed.

[0011] The frame member may have a first side and a second side facing the first side with the opening therebetween, and the calibration unit may include a first calibration unit provided on the first side and a second calibration unit provided on the second side. In this case, it is possible to reliably adjust both the amplitude and phase of the MEMS mirror.

[0012] The calibration portion may extend along the direction in which the side portion extends, thereby widening the area in which calibration can be performed.

[0013] The frame member may have a first side and a third side extending in a direction intersecting the extension direction of the first side, and the calibration unit may include a first calibration unit provided on the first side and a third calibration unit provided on the third side. In this case, it is possible to perform calibration not only for scanning along one direction but also for scanning along a direction intersecting that direction.

[0014] The MEMS mirror scans the irradiated light within a scan area in a plane along the frame member, and the width of the opening in the frame member may be narrower than the width of the scan area, in which case calibration can be performed by positioning a calibration unit within the scan area.

[0015] The MEMS mirror is configured to be oscillating about a first axis and a second axis, and scans the irradiated light along a first direction within a scan area in a plane along the frame member by resonating about the first axis, and changes the scanning position in a second direction intersecting with the first direction by rotating about the second axis, and the calibration unit may be provided on a side extending along the second direction. In this case, although the amplitude and phase of the oscillation about the first axis that resonates easily change, it is possible to calibrate the scan by the oscillation about the first axis.

[0016] The scanning microscope unit of the present invention may further include a housing to which the scan lens is fixed, an attachment part for attaching the housing to the connection port, and a movable part for supporting the housing so that the angle of the housing relative to the attachment part can be changed. In this case, by changing the angle of the housing relative to the attachment part, the optical axis of the scan lens can be aligned with the optical axis of the microscope optical system. As a result, imaging can be achieved while maintaining signal strength and resolution.

[0017] The frame member may be disposed on the image plane of the microscope optical system, in which case the MEMS mirror is used to scan the illumination light within a scan area on the image plane, thereby adjusting at least one of the amplitude and phase of the oscillation of the MEMS mirror.

[0018] A scanning microscope according to the present invention includes the scanning microscope unit described above and a microscope having a microscope optical system and a connection port. With this scanning microscope, it is possible to suppress the occurrence of field of view shift for the reasons described above.

[0019] A method for calibrating a scanning microscope unit according to one aspect of the present disclosure is a method for calibrating the above-mentioned scanning microscope unit, comprising: a first step of scanning illumination light within a scan area in a plane along a frame member using a MEMS mirror, wherein at least a portion of a calibration unit is positioned within the scan area; a second step of detecting calibration light generated from the calibration unit in response to the incidence of illumination light using a photodetector; and a third step of adjusting at least one of the amplitude and phase of the oscillation of the MEMS mirror based on the detection result of the photodetector.

[0020] In this method for calibrating a scanning microscope unit, a MEMS mirror is used to scan an illumination light within a scan area on a plane along a frame member, a photodetector is used to detect calibration light generated from a calibration unit in response to the incidence of the illumination light, and at least one of the amplitude and phase of the oscillation of the MEMS mirror is adjusted based on the detection result of the photodetector. By adjusting (calibrating) at least one of the amplitude and phase in this manner, it is possible to suppress the occurrence of field of view shift as described above. Therefore, this method for calibrating a scanning microscope unit makes it possible to suppress the occurrence of field of view shift. [Effects of the Invention]

[0021] According to one aspect of the present disclosure, it is possible to provide a scanning microscope unit, a scanning microscope, and a method for calibrating a scanning microscope unit that can suppress the occurrence of field of view deviation. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a schematic configuration diagram of a confocal microscope according to an embodiment. [Figure 2] 10A and 10B are diagrams illustrating a refraction state of observation light in a dichroic mirror. [Figure 3] FIG. 2 is a cross-sectional view showing a mounting structure of a confocal microscope unit to a microscope. [Figure 4] FIG. 4 is a view of the periphery of the frame member as seen from the scan lens side. [Figure 5] FIG. 5 is a cross-sectional view taken along line VV in FIG. [Figure 6] FIG. 10 is a view of the frame member as seen from the light guide direction. [Figure 7] 10 is a diagram showing the relationship between a drive signal applied to a MEMS mirror, a swing angle of the MEMS mirror, and sampling timing of a photodetector. FIG. [Figure 8] Graph (a) shows the change in the deflection angle of the MEMS mirror over time, and graph (b) shows the change in the phase of the MEMS mirror over time. [Figure 9] 10A and 10B are diagrams for explaining a method for calibrating the amplitude and phase of a MEMS mirror. [Figure 10] FIG. 10(a) is a diagram showing a first modified example, and FIG. 10(b) is a diagram showing a second modified example. [Figure 11] FIG. 10(a) is a diagram showing a third modified example, and FIG. 10(b) is a diagram showing a fourth modified example. DETAILED DESCRIPTION OF THE INVENTION

[0023] Hereinafter, an embodiment of the present disclosure will be described in detail with reference to the drawings. In the following description, the same or equivalent elements will be designated by the same reference numerals, and redundant description will be omitted.

[0024] FIG. 1 shows a confocal microscope A, a type of scanning microscope. The confocal microscope A is used to acquire images that enable the construction of an optical tomographic image of an observation object M. The confocal microscope A is configured by connecting a confocal microscope unit 1, which is a scanning microscope unit, to a connection port PT of a microscope 50 for connecting an external unit. The microscope 50 has a microscope optical system R including an imaging lens 51 and an objective lens 52. The confocal microscope unit 1 irradiates an observation object M placed on a stage or the like of the microscope 50 with illumination light via the microscope optical system R, and receives (detects) observation light generated from the observation object M in response to the illumination light via the microscope optical system R, thereby generating and outputting an optical tomographic image. In this example, the observation object M is a sample that generates fluorescence as observation light when irradiated with illumination light, and the illumination light is excitation light for exciting the sample.

[0025] The confocal microscope unit 1 comprises a main housing 2, a lens barrel (housing) 3 that forms part of the main housing 2 and is detachably connected to a connection port PT of the microscope 50, a MEMS (Micro Electro Mechanical System) mirror 4 fixed within the main housing 2, a fixed mirror 5, first to fourth subunits 6a to 6d, and a scan lens 7 fixed within the lens barrel 3.

[0026] The scan lens 7 is disposed within the lens barrel 3. The scan lens 7 relays the reflecting surface of the MEMS mirror 4 to the pupil position of the objective lens 52 and focuses the illumination light on the primary image plane of the microscope optical system R of the microscope 50. The scan lens 7 guides the illumination light scanned by the MEMS mirror 4 to the microscope optical system R, thereby irradiating the observation object M, and guides the observation light generated from the observation object M in response to this to the MEMS mirror 4. In detail, the scan lens 7 is configured to image the pupil of the objective lens 52 on the MEMS mirror 4, and guides the observation light imaged by the objective lens 52 and imaging lens 51 of the microscope 50 to the MEMS mirror 4.

[0027] The MEMS mirror 4 is disposed within the main housing 2. The MEMS mirror 4 is, for example, an optical scanning element (scan mirror) having a reflector configured to be swingable about a first axis and a second axis that are perpendicular to each other. The MEMS mirror 4 is formed by processing a semiconductor substrate using MEMS technology (patterning, etching, etc.). By continuously changing the angle of the reflector, the MEMS mirror 4 scans the illumination light output from the first to fourth subunits 6a to 6d over the observation object M, and also guides the observation light generated from the observation object M in response to the illumination light toward the first to fourth subunits 6a to 6d.

[0028] The fixed mirror 5 is fixed inside the main housing 2. The fixed mirror 5 reflects the illumination light output from the first to fourth subunits 6a to 6d toward the MEMS mirror 4, and also reflects the observation light reflected by the MEMS mirror 4 toward the first to fourth subunits 6a to 6d coaxially with the illumination light. The fixed mirror 5 may include two fixed mirrors 5a and 5b, as shown in FIG. 3.

[0029] The first subunit 6a includes a base plate 8a, a dichroic mirror (first beam splitter) 9a arranged on the base plate 8a, a light source 10a, a dichroic mirror 11a, a pinhole plate (first aperture member) 12a, and a photodetector (first photodetector) 13a. The dichroic mirror 9a is fixed to the fixed mirror 5 on the side of the observation light reflection direction. The dichroic mirror 9a reflects the first illumination light with wavelength λ1 emitted by the first subunit 6a and the first observation light with a wavelength range Δλ1 generated from the observation object M in response to the first illumination light, and transmits light with wavelengths longer than the first illumination light and the first observation light. The dichroic mirror 11a is arranged on the side of the dichroic mirror 9a on the side of the first observation light reflection direction, and transmits the first observation light and reflects the first illumination light.

[0030] The light source 10a outputs a first illumination light. For example, the light source 10a is a laser diode, and the first illumination light is laser light. The light source 10a is positioned so that the first illumination light is reflected by the dichroic mirror 11a toward the dichroic mirror 9a coaxially with the first observation light. The pinhole plate 12a is positioned so that its pinhole position coincides with a conjugate position of the spot of the first illumination light on the observation object M, and limits the luminous flux of the first observation light. The pinhole plate 12a, together with the light source 10a and other components, constitutes a confocal optical system. The pinhole diameter of the pinhole plate 12a is configured to be externally adjustable, thereby changing the resolution and signal intensity of the image detected by the photodetector 13a. The photodetector 13a has a detection surface positioned opposite the pinhole plate 12a and receives and detects the first observation light that has passed through the pinhole plate 12a. The photodetector 13a is a photomultiplier tube, a photodiode, an avalanche photodiode, a multi-pixel photon counter (MPPC), a hybrid photodetector (HPD), an area image sensor, or the like.

[0031] The second to fourth subunits 6b to 6d have the same configuration as the first subunit 6a. Specifically, the second subunit 6b includes a base plate 8b, a dichroic mirror (second beam splitter) 9b, a light source 10b, a dichroic mirror 11b, a pinhole plate (second aperture member) 12b, and a photodetector (second photodetector) 13b. The dichroic mirror 9b reflects the second illumination light having a wavelength λ2 (>λ1) emitted by the second subunit 6b and the second observation light having a wavelength range Δλ2 generated from the observation object M in response to the second illumination light, while transmitting light having a wavelength longer than the second illumination light and the second observation light. The dichroic mirror 11b transmits the second observation light having a wavelength range Δλ2 and reflects the second illumination light having a wavelength λ2 shorter than the wavelength range Δλ2.

[0032] The light source 10b outputs a second illumination light. The pinhole plate 12b is disposed so that its pinhole position coincides with a conjugate position of the spot of the second illumination light on the observation object M, and limits the luminous flux of the second observation light. The photodetector 13b has a detection surface disposed opposite the pinhole plate 12b, and receives and detects the second observation light that has passed through the pinhole plate 12b.

[0033] The third subunit 6c includes a base plate 8c, a dichroic mirror (third beam splitter) 9c, a light source 10c, a dichroic mirror 11c, a pinhole plate (third aperture member) 12c, and a photodetector (third photodetector) 13c. The dichroic mirror 9c reflects the third illumination light having a wavelength λ3 (>λ2) emitted by the third subunit 6c and the third observation light having a wavelength range Δλ3 generated from the observation object M in response to the third illumination light, while transmitting light having a wavelength longer than the third illumination light and the third observation light. The dichroic mirror 11c transmits the third observation light having a wavelength range Δλ3 and reflects the third illumination light having a wavelength λ3 shorter than the wavelength range Δλ3.

[0034] The light source 10c outputs a third illumination light. The pinhole plate 12c is positioned so that its pinhole coincides with a conjugate position of the spot of the third illumination light on the observation object M, and limits the luminous flux of the third observation light. The photodetector 13c has a detection surface positioned opposite the pinhole plate 12c, and receives and detects the third observation light that has passed through the pinhole plate 12c.

[0035] The fourth subunit 6d includes a base plate 8d, a total reflection mirror 9d, a light source 10d, a dichroic mirror 11d, a pinhole plate (fourth aperture member) 12d, and a photodetector (fourth photodetector) 13d. The total reflection mirror 9d reflects the fourth illumination light having a wavelength λ4 (>λ3) emitted by the fourth subunit 6d and the corresponding fourth observation light having a wavelength range Δλ4 emitted from the observation object M. The dichroic mirror 11d transmits the fourth observation light having a wavelength range Δλ4 and reflects the fourth illumination light having a wavelength λ4 shorter than the wavelength range Δλ4.

[0036] The light source 10d outputs a fourth illumination light. The pinhole plate 12d is positioned so that its pinhole coincides with a conjugate position of the spot of the fourth illumination light on the observation object M, and limits the luminous flux of the fourth observation light. The photodetector 13d has a detection surface positioned opposite the pinhole plate 12d, and receives and detects the fourth observation light that has passed through the pinhole plate 12d.

[0037] The first to fourth subunits 6a to 6d are fixed in the main housing 2 so as to be lined up in this order in a direction away from the fixed mirror 5 along the light-guiding direction of the first to fourth observation light by the MEMS mirror 4 and the fixed mirror 5, and so that the dichroic mirrors 9a to 9c and the total reflection mirror 9d are positioned on the optical paths of the first to fourth observation light. Specifically, the second to fourth subunits 6b to 6d are arranged so as to be shifted by a shift distance d relative to the first to third subunits 6a to 6c, respectively, in a direction perpendicular to the light-guiding direction of the second to fourth observation light, using the central positions of the dichroic mirrors 9a to 9c and the total reflection mirror 9d as references.

[0038] The shift distance d is set to be approximately equal to the shift amount δ in the direction perpendicular to the optical path of the observation light transmitted through the dichroic mirrors 9a to 9c, which occurs due to the refraction of the observation light in each of the dichroic mirrors 9a to 9c. In this embodiment, the thicknesses of the mirror members constituting the dichroic mirrors 9a to 9c are set to be the same, so the shift amounts occurring in the dichroic mirrors 9a to 9c are approximately the same. Therefore, the shift distance d between any two adjacent subunits among the first to fourth subunits 6a to 6d is also set to be the same.

[0039] The shift distance d is set according to the thickness and refractive index of the mirror members that make up the dichroic mirrors 9a to 9c. Specifically, if the thickness of the mirror members is t, the refractive index of the mirror members is n, the angle of incidence of the observation light incident on the mirror members is θ, and the angle of refraction into the mirror members is φ, the shift amount δ of the observation light caused by the mirror members has the relationship shown in Figure 2 and is calculated by the following formula (1). The shift distance d is set according to this shift amount δ. δ=t sin(θ-φ) / cosφ …(1)

[0040] The mounting structure of the confocal microscope unit 1 on the microscope 50 will be described with reference to Figure 3. As shown in Figure 3, the scan lens 7 is fixed inside the lens barrel 3, and a tilt adjustment mechanism 23 including an attachment part 21 and a movable part 22 that are integrated with each other is provided inside the tip of the lens barrel 3. Although shown in a simplified form in Figure 3, the scan lens 7 is actually made up of multiple lenses.

[0041] Attachment unit 21 is formed in a ring shape and protrudes from the tip of lens barrel 3, and has a structure on the tip side that allows it to be attached to a connection port PT for connecting a camera of microscope 50 (for example, a structure compatible with a C-mount). Movable unit 22 is continuous with the base end side of attachment unit 21. Movable unit 22 is formed in a roughly ring shape, and the outer surface of movable unit 22 forms a spherical sliding surface. A spherical sliding surface 24 that corresponds to the outer surface shape of movable unit 22 is formed on the inner surface of the tip of lens barrel 3. Here, the outer surface of movable unit 22 and the inner surface of lens barrel 3 are shaped so that when movable unit 22 is fitted into lens barrel 3 and attachment unit 21 is connected to the connection port PT of microscope 50, the center C of a sphere containing these shapes is located on the image plane FS of the microscope optical system R of microscope 50.

[0042] According to the mounting structure in which the tilt adjustment mechanism 23 is fitted into the tip side of the lens barrel 3, when the confocal microscope unit 1 is attached to the microscope 50, the angle of the lens barrel 3 relative to the attachment part 21 can be changed by sliding the movable part 22 along the sliding surface 24 of the lens barrel 3. At this time, because the outer surface of the movable part 22 and the inner surface of the lens barrel 3 are formed spherically, the lens barrel 3 can be rotated relative to the attachment part 21, and the angle of the central axis of the lens barrel 3 relative to the central axis of the attachment part 21 can be adjusted two-dimensionally. In other words, the tilt adjustment mechanism 23 is configured to change the angle of the lens barrel 3 relative to the attachment part 21 so that the optical axis of the microscope optical system R of the microscope 50 and the optical axis of the scan lens 7 are parallel to each other.

[0043] A support member 30 that supports a frame member 40 is provided inside the lens barrel 3. The support member 30, together with the lens barrel 3, constitutes a housing that houses the scan lens 7. The support member 30 has a cylindrical portion 31 and an annular flange portion 32 that extends outward from the base end of the cylindrical portion 31. The support member 30 is fixed to the inner surface of the lens barrel 3 at the flange portion 32 so that it is located on the image plane FS side of the microscope optical system R with respect to the scan lens 7. The tip of the cylindrical portion 31 is located within the movable portion 22. The frame member 40 is fixed to the tip of the cylindrical portion 31 and is located within the movable portion 22.

[0044] 4 and 5, the frame member 40 has a plate member 41 and a covering member 42. The plate member 41 is, for example, a fluorescent plate (fluorescent member) formed in a plate shape from a fluorescent material, and generates fluorescence in response to incidence of irradiated light. The fluorescence generated from the plate member 41 is used as calibration light in calibrating the confocal microscope unit 1, as will be described later.

[0045] The covering member 42 is a mask member that is placed on the plate member 41 and covers the surface of the plate member 41 that faces the support member 30. The covering member 42 is formed in a plate (layer) shape from, for example, aluminum, iron, or stainless steel. The surface of the covering member 42 is subjected to a black treatment (anti-reflective treatment) to suppress light reflection.

[0046] The frame member 40 has an opening 43. That is, the frame member 40 (the plate member 41 and the covering member 42) is formed in a frame shape so as to define the opening 43. The frame member 40 is disposed on the microscope optical system R side (the opposite side to the MEMS mirror 4) of the scan lens 7 on the optical axis of the scan lens 7 so that the illumination light and the observation light pass through the opening 43. More specifically, the frame member 40 is disposed at the image plane FS of the microscope optical system R, and the surface of the plate member 41 facing the scan lens 7 is located on the image plane FS. The opening 43 is formed, for example, in a rectangular shape whose length along the first direction D1 is longer than its length along the second direction D2.

[0047] The frame member 40 has a first side portion 40a, a second side portion 40b, a third side portion 40c, and a fourth side portion 40d that define an opening 43. The first side portion 40a and the second side portion 40b extend along the second direction D2 and face each other with the opening 43 in between. The third side portion 40c and the fourth side portion 40d extend along the first direction D1 that is perpendicular to the second direction D2 and face each other with the opening 43 in between.

[0048] Two rectangular openings (exposure openings) 42a are formed in the covering member 42. The two openings 42a are formed in the covering member 42 in the portions that define the first side 40a and the second side 40b, respectively. The formation of the openings 42a exposes a portion of the plate member 41 toward the scan lens 7, allowing irradiation light to be incident on a portion of the plate member 41. This forms a calibration unit 44 that generates calibration light in response to the incidence of irradiation light. That is, the calibration unit 44 is formed by a portion of the plate member 41 that is exposed through the openings 42a formed in the covering member 42. In this example, the calibration unit 44 includes a first calibration unit 44a provided on the first side 40a and a second calibration unit 44b provided on the second side 40b. The first calibration unit 44a and the second calibration unit 44b are positioned on the same straight line parallel to the first direction D1. Each of the first calibration unit 44a and the second calibration unit 44b is formed in a rectangular shape (a square shape in this example).

[0049] With reference to FIG. 6, scanning of the illumination light by the MEMS mirror 4 will be described. In the confocal microscope unit 1, the MEMS mirror 4 rotates, thereby scanning the illumination light within a scan area in a plane along the frame member 40 (in this example, the plane along the surface of the plate member 41 on the scan lens 7 side, which is located on the image plane FS). In particular, the MEMS mirror 4 rotates about a first axis, thereby scanning the illumination light along a first direction D1 within the scan area A1 on the image plane FS. Furthermore, the MEMS mirror 4 rotates about a second axis, thereby changing the scanning position in the second direction D2. By scanning the illumination light along the first direction D1 while sequentially changing the scanning position in the second direction D2, the illumination light can be scanned over the entire scan area A1. In this example, the MEMS mirror 4 resonates around the first axis (i.e., oscillates at high speed at the resonant frequency level) and performs linear (non-resonant) motion around the second axis. The width of the opening 43 of the frame member 40 in the first direction D1 is narrower than the width of the scan area A1 in the first direction D1, and a part of the inside of the calibration unit 44 is located within the scan area A1. In other words, the MEMS mirror 4 is driven so that the calibration light is scanned within the scan area A1, which has a width wider than the opening 43, and so that at least a part of the calibration unit 44 is located within the scan area A1.

[0050] When observing the observation object M, observation light is detected from an effective area A2 set inside the scan area A1. Therefore, the oscillation angle of the MEMS mirror 4 is adjusted relative to the sampling timing of the photodetectors 13a to 13d. As shown in FIG. 7, the MEMS mirror 4 operates at a deflection angle according to the intensity of the drive signal S1, with a certain phase difference from the drive signal S1. In this example, the drive signal S1 is a pulse signal. The sampling timing of the photodetectors 13a to 13d is synchronized with the horizontal synchronization signal S2. In this example, the photodetectors 13a to 13d detect the observation light over the range indicated by the arrows in FIG. 7. Note that FIG. 7 shows the oscillation angle of the MEMS mirror 4 around the first axis.

[0051] On the other hand, the oscillation angle (amplitude) and phase of the oscillation of the MEMS mirror 4 may change over time due to aging and other factors. Figures 8(a) and 8(b) show measurement results of the oscillation angle and phase when the MEMS mirror 4 is continuously driven with a constant drive signal S1 for one month (30 days). As shown in Figures 8(a) and 8(b), the oscillation angle decreases and the phase changes over time. If the oscillation angle and phase change, there is a concern that the field of view may shift from the effective area A2.

[0052] Therefore, in the calibration method for the confocal microscope unit 1 according to the embodiment, the amplitude and phase of the MEMS mirror 4 are calibrated based on the calibration light from the calibration unit 44. This calibration is performed, for example, every time the confocal microscope unit 1 is activated. During calibration, first, the MEMS mirror 4 is used to scan the illumination light within the scan area A1 on the imaging plane FS, which is a plane along the frame member 40 (first step). In the first step, the MEMS mirror 4 is driven so that at least a portion of the calibration unit 44 is positioned within the scan area A1. Following or simultaneously with the first step, the photodetectors 13a to 13d are used to detect the calibration light generated from the calibration unit 44 in response to the incidence of the illumination light (second step). In the second step, it is sufficient that the calibration light is detected by at least one of the photodetectors 13a to 13d. For example, the calibration light may be detected only by the photodetector 13a. Next, at least one of the amplitude and phase of the oscillation of the MEMS mirror 4 is adjusted based on the detection results of the photodetectors 13a to 13d (third step).

[0053] An example of a method for adjusting the amplitude and phase of the oscillation of the MEMS mirror 4 based on the intensity signals of the calibration light, which are the detection results of the photodetectors 13a to 13d, will be described below with reference to FIG. 9. The horizontal synchronization signal S2 and the effective area A2 are shown in the upper part of FIG. 9. Examples of the intensity signals of the calibration light in states C1 to C5 are also shown. The pulse P1 is formed by the calibration light from the first calibration unit 44a, and the pulse P2 is formed by the calibration light from the second calibration unit 44b. State C1 is an appropriate state (target state), and is, for example, the state immediately after the drive signal S1 is adjusted during manufacturing.

[0054] As an example, assume that an intensity signal in state C2 is detected. In state C2, both the deflection angle and phase of the MEMS mirror 4 are deviated from those in state C1. The deflection angle of the MEMS mirror 4 corresponds to the distance between pulses P1 and P2, and the phase of the MEMS mirror 4 corresponds to the rising positions of pulses P1 and P2. First, the phase of the drive signal S1 is changed to align the rising position of pulse P1 with the position in state C1, as shown in state C3. Next, the amplitude of the drive signal S1 is changed to align the distance between pulses P1 and P2 with the distance in state C1, as shown in state C4. Next, the phase of the drive signal S1 is changed again to align the rising position of pulse P1 with the position in state C1, as shown in state C5. Through the above steps, the deflection angle and phase of the MEMS mirror 4 can be adjusted (calibrated) to the same state as state C1, which is the target state. In this calibration, adjustment is performed so that the width W between the rising positions of the pulses P1 and P2 and the reference position based on the horizontal synchronization signal S2 matches a target value (a saved value stored as a target value during manufacturing). [Action and effect]

[0055] The confocal microscope unit 1 includes a frame member 40 formed in a frame shape to define an opening 43 and disposed on the microscope optical system R side of the scan lens 7 so that the illumination light and observation light pass through the opening 43. The frame member 40 also includes a first calibration unit 44a disposed on a first side 40a defining the opening 43, which generates calibration light in response to the incidence of the illumination light. This allows, for example, the MEMS mirror 4 to scan the illumination light within a scan area A1 on the imaging surface FS (a plane along the frame member 40), the photodetectors 13a-13d to detect the calibration light generated from the calibration unit 44 in response to the incidence of the illumination light, and at least one of the amplitude and phase of the oscillation of the MEMS mirror 4 to be adjusted based on the detection results of the photodetectors 13a-13d. By adjusting (calibrating) at least one of the amplitude and phase in this manner, the occurrence of the field of view deviation described above can be suppressed. Therefore, the confocal microscope unit 1 can suppress the occurrence of field of view deviation.

[0056] The calibration section 44 is formed by a part of the plate member 41 exposed from an opening (exposure opening) 42a formed in the covering member 42. This makes it possible to detect the edge of the calibration section 44 well, thereby improving the accuracy of calibration. Furthermore, since the opening 42a can be formed in the covering member 42 with high accuracy, it is possible to form the calibration section 44 with high accuracy. This also improves the accuracy of calibration.

[0057] The calibration section 44 includes a plate member 41, which is a fluorescent member, so that calibration can be performed in the confocal microscope unit 1 for fluorescence observation.

[0058] The photodetectors 13a to 13d detect, as observation light, the fluorescence generated from the observation object M in response to the irradiation of the illumination light, thereby making it possible to perform fluorescence observation.

[0059] The calibration unit 44 includes a first calibration unit 44a provided on the first side 40a and a second calibration unit 44b provided on the second side 40b. This makes it possible to reliably adjust both the amplitude and phase of the MEMS mirror 4 even if there is a deviation in the arrangement of each unit due to, for example, a manufacturing error.

[0060] The width of the opening 43 of the frame member 40 is narrower than the width of the scan area A1, which makes it possible to position the calibration unit 44 within the scan area A1 and perform calibration.

[0061] The MEMS mirror 4 is configured to be able to swing around a first axis and a second axis, and by resonating around the first axis, scans the illumination light along a first direction D1 within a scan area A1 on the imaging surface FS (a plane along the frame member 40), and by rotating around the second axis, changes the scanning position in a second direction D2 intersecting with the first direction D1. A calibration unit 44 is provided on the first side 40a extending along the second direction D2. Although the amplitude and phase of the oscillation around the first axis (resonance axis) that resonates are prone to change, the confocal microscope unit 1 makes it possible to calibrate the scan by oscillation around the first axis.

[0062] The confocal microscope unit 1 includes an attachment part 21 for attaching the lens barrel 3 to the connection port PT, and a movable part 22 that supports the lens barrel 3 so that the angle of the lens barrel 3 relative to the attachment part 21 can be changed. This makes it possible to align the optical axis of the scan lens 7 with the optical axis of the microscope optical system R by changing the angle of the lens barrel 3 relative to the attachment part 21. As a result, imaging can be achieved while maintaining signal intensity and resolution.

[0063] The frame member 40 is disposed on the imaging plane FS of the microscope optical system R. This makes it possible to adjust at least one of the amplitude and phase of the oscillation of the MEMS mirror 4 by scanning the irradiated light within the scan area A1 on the imaging plane FS using the MEMS mirror 4. [Variations]

[0064] 10(a), the first calibration unit 44a and the second calibration unit 44b do not have to be positioned on the same straight line parallel to the first direction D1. That is, the positions of the first calibration unit 44a and the second calibration unit 44b in the second direction D2 may be different from each other. Even with this first modification, calibration can be performed in the same way as in the above embodiment, and the occurrence of field of view shift can be suppressed.

[0065] As in the second modified example shown in FIG. 10(b), the first calibration unit 44a may extend along the extension direction (second direction D2) of the first side 40a on which the first calibration unit 44a is provided. Similarly, the second calibration unit 44b may extend along the extension direction (second direction D2) of the second side 40b on which the second calibration unit 44b is provided. In this example, the first calibration unit 44a and the second calibration unit 44b extend straight along the second direction D2. In this example, each of the first calibration unit 44a and the second calibration unit 44b is formed in a rectangular shape with a long side along the second direction. This second modified example also allows calibration to be performed in the same way as in the above embodiment, and can suppress the occurrence of field of view deviation. Furthermore, the area that can be calibrated can be widened.

[0066] In a third modified example shown in FIG. 11(a), the calibration unit 44 further includes a third calibration unit 44c provided on the third side 40c and a fourth calibration unit 44d provided on the fourth side 40d. The third calibration unit 44c and the fourth calibration unit 44d are located on the same straight line parallel to the second direction D2. This third modified example also enables calibration to be performed in the same manner as in the above embodiment, and can suppress the occurrence of field of view deviation. Furthermore, it is possible to perform calibration not only for scanning along one direction (first direction D1) but also for scanning along a direction intersecting that direction (second direction D2).

[0067] 11(b), the calibration unit 44 may include only the first calibration unit 44a. This first modification also makes it possible to adjust at least one of the amplitude and phase of the oscillation of the MEMS mirror 4, thereby suppressing the occurrence of field of view deviation.

[0068] The present disclosure is not limited to the above-described embodiments and modifications. For example, the materials and shapes of the components are not limited to those described above, and various materials and shapes can be used. In the above-described embodiments, the frame member 40 is disposed at the image plane FS of the microscope optical system R. However, the frame member 40 need only be disposed along a predetermined plane perpendicular to the optical axis of the scan lens 7, and does not necessarily have to be disposed at the image plane FS.

[0069] In the above embodiment, the calibration unit 44 generates fluorescence in response to the incidence of irradiation light, but the calibration unit 44 may generate calibration light including the sensitivity wavelength of at least one of the photodetectors 13 a to 13 d so that the calibration light can be detected by at least one of the photodetectors 13 a to 13 d. From another perspective, the calibration unit 44 may generate calibration light including the wavelength of the observation light.

[0070] The observation object M may be one that generates light other than fluorescence as observation light in response to the irradiated light. In this case, the calibration unit may also be one that generates light other than fluorescence in response to the irradiated light. The observation light may be reflected light generated from the observation object M in response to irradiation with the irradiated light. The scanning microscope unit of the present invention is not limited to the confocal microscope A, and may be applied to a general fluorescence microscope, a reflective microscope, or the like, as long as it is a scanning microscope using a MEMS mirror. The tilt adjustment mechanism 23 may be omitted.

[0071] In the above embodiment, a pinhole plate is used as the diaphragm member to configure the confocal optical system, but the diaphragm member may be any optical element that limits the light beam, such as an iris diaphragm or a fiber core. When a fiber output type light source is used, the position of the fiber core end face can be set as the diaphragm position (the position where the light beam is limited). Laser light sources such as solid-state lasers and diode lasers can also be used. In this case, the position of the beam waist of these laser light sources can be set as the diaphragm position, and the light source itself will serve as the diaphragm member.

[0072] The configurations of frame member 40 and calibration unit 44 are not limited to the above examples. For example, calibration unit 44 may be configured with a fluorescent member attached to the surface of a frame member made of a metal material. Frame member 40 may be formed integrally with a housing that houses scan lens 7. That is, frame member 40 and the housing that houses scan lens 7 may be configured with a single member. For example, calibration unit 44 may be configured by attaching a fluorescent member to a frame-shaped portion (frame member) that is provided in the housing and defines an opening. [Explanation of symbols]

[0073] 1...confocal microscope unit (scanning microscope unit), 3...lens barrel (housing), 4...MEMS mirror, 7...scan lens, 10a to 10d...light source, 13a to 13d...photodetector, 21...attachment portion, 22...movable portion, 40...frame member, 40a...first side portion, 40b...second side portion, 40c...third side portion, 41...plate member, 42...covering member, 42a...opening (exposure opening), 43...opening, 44...calibration portion, 44a...first calibration portion, 44b...second calibration portion, 44c...third calibration portion, 50...microscope, A...confocal microscope (scanning microscope), A1...scan area, D1...first direction, D2...second direction, FS...imaging plane, M...observation object, PT...connection port, R...microscope optical system.

Claims

1. A scanning microscope unit that constitutes a scanning microscope by being attached to a connection port of a microscope having a microscope optical system, a light source that outputs irradiation light; a photodetector for detecting observation light generated from an observation object in response to irradiation with the irradiation light; a MEMS mirror that scans the illumination light output from the light source on the observation object and guides the observation light generated from the observation object in response to irradiation with the illumination light toward the photodetector; a scan lens that guides the illumination light scanned by the MEMS mirror to the microscope optical system and guides the observation light imaged by the microscope optical system to the MEMS mirror; a frame member formed in a frame shape to define an opening, and arranged on the microscope optical system side with respect to the scan lens so that the illumination light and the observation light pass through the opening; the frame member has a calibration portion provided on a side portion that defines the opening, the calibration portion generating calibration light including a sensitivity wavelength of the photodetector in response to the incidence of the irradiation light; a scanning microscope unit that adjusts at least one of the amplitude and phase of the oscillation of the MEMS mirror based on the time waveform of the intensity of the calibration light detected by the photodetector;

2. the frame member includes a plate member that generates the calibration light in response to the incidence of the irradiation light, and a covering member that is disposed on the plate member and covers the plate member, 2. The scanning microscope unit according to claim 1, wherein the calibration section is configured by a part of the plate member exposed through an exposure opening formed in the cover member.

3. The scanning microscope unit according to claim 1 , wherein the calibration unit includes a fluorescent member.

4. 4. The scanning microscope unit according to claim 1, wherein the photodetector detects, as the observation light, fluorescence generated from the object to be observed in response to irradiation with the illumination light.

5. the frame member has a first side portion and a second side portion facing the first side portion across the opening, 5. A scanning microscope unit according to claim 1, wherein the calibration unit includes a first calibration unit provided on the first side portion and a second calibration unit provided on the second side portion.

6. 6. The scanning microscope unit according to claim 1, wherein the calibration section extends along the extension direction of the side section.

7. the frame member has a first side portion and a third side portion extending in a direction intersecting with the extending direction of the first side portion, 7. A scanning microscope unit according to claim 1, wherein the calibration unit includes a first calibration unit provided on the first side portion and a third calibration unit provided on the third side portion.

8. the MEMS mirror scans the irradiation light within a scan area in a plane along the frame member; 8. The scanning microscope unit according to claim 1, wherein the width of the opening of the frame member is narrower than the width of the scan area.

9. the MEMS mirror is configured to be swingable around a first axis and a second axis, and scans the irradiation light along a first direction within a scan area in a plane along the frame member by resonating around the first axis, and changes a scanning position in a second direction intersecting with the first direction by rotating around the second axis; 9. The scanning microscope unit according to claim 1, wherein the calibration section is provided on the side extending along the second direction.

10. a housing to which the scan lens is fixed; an attachment portion for attaching the housing to the connection port; 10. The scanning microscope unit according to claim 1, further comprising: a movable part that supports the housing so that an angle of the housing relative to the attachment part can be changed.

11. 11. The scanning microscope unit according to claim 1, wherein the frame member is disposed on an image plane of the microscope optical system.

12. A scanning microscope unit according to any one of claims 1 to 11; a microscope having the microscope optical system and the connection port.

13. A method for calibrating a scanning microscope unit according to any one of claims 1 to 11, comprising: a first step of scanning the illumination light within a scan area in a plane along the frame member using the MEMS mirror, wherein at least a portion of the calibration unit is located within the scan area; a second step of detecting the calibration light generated from the calibration unit in response to the incidence of the irradiation light using the photodetector; and a third step of adjusting at least one of the amplitude and phase of the oscillation of the MEMS mirror based on the time waveform of the intensity of the calibration light detected by the photodetector.

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