Scanning microscope unit, scanning microscope, and method for correcting a scanning microscope unit

Abstract

A scanning microscope unit includes: a light source that outputs irradiation light; a light detector that detects observation light generated from an observation object in response to irradiation of the irradiation light; a MEMS mirror that scans the irradiation light on the observation object and guides the observation light toward the light detector; a scanning lens that guides the irradiation light scanned by the MEMS mirror to a microscope optical system and guides the observation light imaged by the microscope optical system to the MEMS mirror; and a frame member that is formed in a frame shape in a manner of defining an opening, and is disposed on a microscope optical system side with respect to the scanning lens in a manner of passing the irradiation light and the observation light through the opening. The frame member has a correction portion that is provided to a side portion that defines the opening, and generates correction light containing a wavelength of sensitivity of the light detector in response to incidence of the irradiation light.

Description

Technical Field

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

[0002] Patent Document 1 describes a scanning microscope unit that is installed at the connection port of a microscope having a microscope optical system, thereby constituting a scanning microscope. The scanning microscope unit described in Patent Document 1 includes: a MEMS (MicroElectroMechanical System) mirror that scans the object being observed with illumination light output from a light source; and a photosensor that detects the observation light generated from the object being observed in response to the illumination light.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: International Publication No. 2020 / 196782 Summary of the Invention

[0006] The technical problem that the invention aims to solve

[0007] In scanning microscope units like those described above, in order to image the desired field of view, for example, the drive signal applied to the MEMS mirror is adjusted during manufacturing so that the swing angle of the MEMS mirror relative to the sampling timing of the photodetector is appropriate. However, there are cases where the amplitude or phase of the MEMS mirror's swing changes over time, in which case there is a concern about field of view shift.

[0008] Therefore, one aspect of the object of the present invention is to provide a scanning microscope unit, a scanning microscope, and a correction method for the scanning microscope unit capable of suppressing the generation of field of view shift.

[0009] Technical means to solve the problem

[0010] One aspect of the present invention is a scanning microscope unit that is configured as a scanning microscope by being installed at the connection port of a microscope having a microscope optical system. The scanning microscope unit comprises: a light source that outputs illumination light; a photodetector that detects observation light generated from an object in response to illumination; a MEMS mirror that scans the object with the illumination light output from the light source and guides the observation light generated from the object in response to illumination toward the photodetector; a scanning 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; and a frame member formed in a frame shape with a defined opening, such that the illumination light and observation light are disposed relative to the scanning lens on the microscope optical system side through the opening. The frame member has a correction section disposed at the edge of the defined opening, which generates correction light including the sensitivity wavelength of the photodetector in response to the incident illumination light.

[0011] This scanning microscope unit includes a frame member formed in a frame shape with a defined opening, allowing illumination light and observation light to pass through the opening and be positioned relative to the scanning lens on the microscope optical system side. Furthermore, the frame member has a correction section located at the edge of the defined opening, which generates correction light containing the sensitivity wavelength of a photodetector in response to the incident illumination light. Thus, for example, it is possible to scan the illumination light within a scanning area along the plane of the frame member using a MEMS mirror, detect the correction light generated from the correction section in response to the incident illumination light using a photodetector, and adjust at least one of the amplitude and phase of the MEMS mirror's oscillation based on the photodetector's detection result. By adjusting (correcting) at least one of the amplitude and phase in this way, the generation of field-of-view shift as described above can be suppressed. Therefore, according to this scanning microscope unit, the generation of field-of-view shift can be suppressed.

[0012] Alternatively, the frame member may include: a plate member that generates correction light in response to incident illumination light; and a cover member disposed on the plate member and covering the plate member, wherein the correction portion is composed of a portion of the plate member exposed from an exposed opening formed in the cover member. In this case, the edge of the correction portion can be detected well, and the accuracy of correction can be improved. Furthermore, the accuracy of correction can also be improved by forming the correction portion with good precision.

[0013] The calibration unit may also include a fluorescence component. In this case, calibration can be performed within a scanning microscope unit used for fluorescence observation.

[0014] A photodetector can also detect fluorescence emitted from the object in response to illumination as observation light. In this case, fluorescence observation can be performed.

[0015] Alternatively, the frame component may have a first side and a second side opposite the first side through an opening, and the correction section may include a first correction section disposed on the first side and a second correction section disposed on the second side. In this case, both the amplitude and phase of the MEMS mirror can be reliably adjusted.

[0016] The correction section can also extend along the direction of the edge. In this case, the area that can be corrected can be expanded.

[0017] Alternatively, the frame member may have a first side and a third side extending in a direction intersecting the extending direction of the first side, and the correction unit may include a first correction unit disposed on the first side and a third correction unit disposed on the third side. In this case, correction can be performed not only for scans along one direction, but also for scans along directions intersecting that direction.

[0018] Alternatively, the MEMS mirror scans the illumination light within a scanning area along the plane of the frame component, where the width of the opening in the frame component is narrower than the width of the scanning area. In this case, the correction unit can be positioned within the scanning area to perform correction.

[0019] Alternatively, the MEMS mirror can be configured to oscillate around a first axis and a second axis. By resonating around the first axis, it scans illumination along a first direction within a scanning area along the plane of the frame member. Furthermore, by rotating around the second axis, the scanning position in a second direction intersecting the first direction changes. A correction section is disposed on the edge extending along the second direction. In this case, the amplitude and phase of the oscillation around the first axis during the resonant operation are easily varied, but correction can be performed on the scan based on the oscillation around the first axis.

[0020] The scanning microscope unit of the present invention may further include: a housing on which a scanning lens is fixed; an attachment for mounting the housing to a connection port; and a movable part for supporting the housing in a manner that allows changing the angle of the housing relative to the attachment. In this case, by changing the angle of the housing relative to the attachment, the optical axis of the scanning lens can be aligned with the direction of the optical axis of the microscope optical system. As a result, imaging that maintains both signal strength and resolution can be achieved.

[0021] The frame component can also be configured on the imaging surface of the microscope optical system. In this case, it is possible to use a MEMS mirror to scan the illumination light within the scanning area of ​​the imaging surface and adjust at least one of the amplitude and phase of the MEMS mirror's oscillation.

[0022] The scanning microscope of the present invention includes the aforementioned scanning microscope unit and a microscope having a microscope optical system and a connection port. According to this scanning microscope, for the reasons described above, the generation of field of view shift can be suppressed.

[0023] A method for calibrating a scanning microscope unit according to one aspect of the present invention includes the following steps: a first step of scanning illumination light in a scanning area along the plane of a frame member using a MEMS mirror, wherein at least a portion of the calibration unit is located in the scanning area; a second step of detecting calibration light generated from the calibration unit in response to the incident illumination light using a photodetector; and a third step of adjusting at least one of the amplitude and phase of the swing of the MEMS mirror based on the detection result of the photodetector.

[0024] In the calibration method of this scanning microscope unit, a MEMS mirror scans the illumination light within a scanning area along the plane of the frame member, a photodetector detects the calibration light generated from the calibration unit in response to the incident illumination light, and at least one of the amplitude and phase of the MEMS mirror's oscillation is adjusted based on the photodetector's detection result. By adjusting (correcting) at least one of the amplitude and phase in this way, the generation of field of view shift as described above can be suppressed. Therefore, according to the calibration method of this scanning microscope unit, the generation of field of view shift can be suppressed.

[0025] The effects of the invention

[0026] According to one aspect of the present invention, a scanning microscope unit, a scanning microscope, and a correction method for the scanning microscope unit are provided that can suppress the generation of field of view shift. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the confocal microscope according to the implementation method.

[0028] Figure 2 It is a diagram showing the refraction state of the observed light in a dichroic mirror.

[0029] Figure 3 This is a cross-sectional view showing the mounting structure of the confocal microscope unit relative to the microscope.

[0030] Figure 4 This is a view of the periphery of the frame component from the scanning lens side.

[0031] Figure 5 It is along Figure 4 A cross-sectional view of the VV line.

[0032] Figure 6 This is a diagram of the frame component viewed from the direction of the light guide.

[0033] Figure 7 This is a graph showing the relationship between the driving signal applied to the MEMS mirror, the swing angle of the MEMS mirror, and the sampling timing of the photodetector.

[0034] Figure 8 (a) is a graph showing the change in the swing angle of the MEMS mirror over time, and (b) is a graph showing the change in the phase of the MEMS mirror over time.

[0035] Figure 9 This is a diagram used to illustrate the method for correcting the amplitude and phase of a MEMS mirror.

[0036] Figure 10 (a) is a diagram representing the first variation, and (b) is a diagram representing the second variation.

[0037] Figure 11 (a) is a diagram representing the third variation, and (b) is a diagram representing the fourth variation. Detailed Implementation

[0038] Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying drawings. In the following description, the same or equivalent elements are referred to by the same reference numerals, and repeated descriptions are omitted.

[0039] exist Figure 1 The image shows a confocal microscope A, a type of scanning microscope. The confocal microscope A is used to acquire an optical tomographic image of an object M. The confocal microscope A is configured by connecting a scanning microscope unit, namely the confocal microscope unit 1, to an external unit PT of the microscope 50 via a connection port. The microscope 50 has a microscope optical system R including an imaging lens 51 and an objective lens 52. The confocal microscope unit 1 illuminates the object M, which is positioned on the stage of the microscope 50, with illumination light via the microscope optical system R, and receives (detects) the observation light generated from the object M in response to the illumination light, generating and outputting an optical tomographic image. In this example, the object M is a sample that fluoresces as observation light when illuminated by illumination light, and the illumination light is excitation light used to excite the sample.

[0040] The confocal microscope unit 1 includes: a main housing 2; a microscope tube (housing) 3, which forms part of the main housing 2 and is detachably connected to the connection port PT of the microscope 50; a MEMS (Micro Electro Mechanical System) mirror 4, which is fixed inside the main housing 2; a fixed mirror 5; first to fourth sub-units 6a to 6d; and a scanning lens 7, which is fixed inside the microscope tube 3.

[0041] A scanning lens 7 is disposed within the microscope tube 3. The scanning 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 onto the primary imaging surface of the microscope optics system R of the microscope 50. The scanning lens 7 illuminates the object M by guiding the illumination light scanned by the MEMS mirror 4 to the microscope optics system R, and guides the observation light generated from the object M in response to this to the MEMS mirror 4. Specifically, the scanning lens 7 is configured to image the pupil of the objective lens 52 onto the MEMS mirror 4, and guide the observation light imaged by the objective lens 52 and the imaging lens 51 of the microscope 50 to the MEMS mirror 4.

[0042] MEMS mirror 4 is disposed within the main housing 2. MEMS mirror 4 is, for example, a light scanning element (scanning mirror) having a reflector configured to oscillate around mutually orthogonal first and second axes. MEMS mirror 4 is formed by processing a semiconductor substrate using MEMS technology (patterning, etching, etc.). MEMS mirror 4 scans the object M with illumination light output from the first to fourth sub-units 6a to 6d by continuously changing the angle of the reflector, and guides the observation light generated from the object M in response to the illumination light toward the first to fourth sub-units 6a to 6d.

[0043] The fixed mirror 5 is fixed inside the main housing 2. The fixed mirror 5 causes the illumination light output from the first to fourth sub-units 6a to 6d to be reflected towards the MEMS mirror 4, and also reflects the observation light reflected by the MEMS mirror 4 coaxially with the illumination light towards the first to fourth sub-units 6a to 6d. Figure 3 As shown, the fixed mirror 5 may also include two fixed mirrors 5a and 5b.

[0044] The first subunit 6a includes a base plate 8a, a dichroic mirror (first beam splitter) 9a disposed on the base plate 8a, a light source 10a, a dichroic mirror 11a, a pinhole plate (first aperture component) 12a, and a photodetector (first photodetector) 13a. The dichroic mirror 9a is fixed on the side of the fixed mirror 5 in the direction of reflection of the observation light. The dichroic mirror 9a reflects the first illumination light with wavelength λ1 irradiated by the first subunit 6a and the first observation light with wavelength range Δλ1 generated from the observed object M in response to this, allowing light with wavelengths longer than the first illumination light and the first observation light to pass through. The dichroic mirror 11a is disposed on the side of the dichroic mirror 9a in the direction of reflection of the first observation light, allowing the first observation light to pass through and reflecting the first illumination light.

[0045] Light source 10a outputs first illumination light. For example, light source 10a is a laser diode, and the first illumination light is a laser. Light source 10a is configured such that the first illumination light is reflected coaxially by dichroic mirror 11a and the first observation light to dichroic mirror 9a. Pinhole plate 12a is configured such that its pinhole position coincides with the conjugate position of the spot of the first illumination light on the object M being observed, thus limiting the beam of the first observation light. Pinhole plate 12a, together with light source 10a, constitutes a confocal optical system. Pinhole plate 12a is configured such that the diameter of the pinhole can be adjusted externally, thereby changing the resolution and signal strength of the image detected by photodetector 13a. Photodetector 13a has a detection surface configured opposite to pinhole plate 12a, and receives and detects the first observation light that has passed through pinhole plate 12a. The photodetector 13a is a photomultiplier tube, photodiode, avalanche photodiode, MPPC (Multi-Pixel Photon Counter), HPD (Hybrid Photo Detector), or area image sensor, etc.

[0046] The second to fourth subunits 6b to 6d have the same structure as the first subunit 6a. That is, the second subunit 6b has a base plate 8b, a dichroic mirror (second beam splitter) 9b, a light source 10b, a dichroic mirror 11b, a pinhole plate (second aperture component) 12b, and a photodetector (second photodetector) 13b. The dichroic mirror 9b reflects the second illumination light with a wavelength λ2 (>λ1) irradiated by the second subunit 6b and the second observation light with a wavelength range Δλ2 generated from the observed object M in response to this, allowing light with a wavelength longer than the second illumination light and the second observation light to pass through. The dichroic mirror 11b allows the second observation light with a wavelength range Δλ2 to pass through and reflects the second illumination light with a wavelength λ2 shorter than the wavelength range Δλ2.

[0047] Light source 10b outputs a second illumination light. Pinhole plate 12b is configured such that the position of its pinhole coincides with the conjugate position of the spot of the second illumination light on the object being observed M, thus limiting the beam of the second observation light. Photodetector 13b has a detection surface configured opposite to pinhole plate 12b, receiving and detecting the second observation light that has passed through pinhole plate 12b.

[0048] 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 component) 12c, and a photodetector (third photodetector) 13c. The dichroic mirror 9c reflects the third illumination light with wavelength λ3 (>λ2) emitted by the third subunit 6c and the third observation light with wavelength range Δλ3 generated from the observed object M in response, allowing light with wavelengths longer than both the third illumination light and the third observation light to pass through. The dichroic mirror 11c allows the third observation light with wavelength range Δλ3 to pass through and reflects the third illumination light with wavelength λ3 shorter than Δλ3.

[0049] The light source 10c outputs a third illumination light. The pinhole plate 12c is configured such that the position of its pinhole coincides with the conjugate position of the spot of the third illumination light of the observed object M, thus limiting the beam of the third observation light. The photodetector 13c has a detection surface configured opposite to the pinhole plate 12c, and receives and detects the third observation light that has passed through the pinhole plate 12c.

[0050] The fourth subunit 6d comprises a base plate 8d, a total reflection mirror 9d, a light source 10d, a dichroic mirror 11d, a pinhole plate (fourth aperture component) 12d, and a photodetector (fourth photodetector) 13d. The total reflection mirror 9d reflects the fourth illumination light of wavelength λ4 (>λ3) emitted by the fourth subunit 6d and, in response, the fourth observation light of wavelength range Δλ4 generated from the observed object M. The dichroic mirror 11d allows the fourth observation light of wavelength range Δλ4 to pass through and reflects the fourth illumination light of wavelength λ4, which is shorter than wavelength range Δλ4.

[0051] The light source 10d outputs a fourth illumination light. The pinhole plate 12d is configured such that the position of its pinhole coincides with the conjugate position of the spot of the fourth illumination light of the observed object M, thus limiting the beam of the fourth observation light. The photodetector 13d has a detection surface configured opposite to the pinhole plate 12d, and receives and detects the fourth observation light that has passed through the pinhole plate 12d.

[0052] The first to fourth subunits 6a to 6d are arranged sequentially along the light guiding direction of the first to fourth observation lights based on the MEMS mirror 4 and the fixed mirror 5, in a direction away from the fixed mirror 5, and are fixed within the main housing 2 with the dichroic mirrors 9a to 9c and the total reflection mirror 9d located in the light path of the first to fourth observation lights. Specifically, the second to fourth subunits 6b to 6d are respectively configured relative to the first to third subunits 6a to 6c, with the center positions of the dichroic mirrors 9a to 9c and the total reflection mirror 9d as a reference, offset by an offset distance d in a direction perpendicular to the light guiding direction of the second to fourth observation lights.

[0053] The offset distance d is set such that, in the optical path of the observation light transmitted through the dichroic mirrors 9a to 9c, it is approximately equal to the offset δ generated by the refraction of the observation light in each of the dichroic mirrors 9a to 9c in a direction perpendicular to the optical path. In this embodiment, the thickness of the mirror components constituting the dichroic mirrors 9a to 9c is set to be the same, so the offset generated in the dichroic mirrors 9a to 9c is approximately the same. Therefore, the offset distance d between two adjacent subunits in the first to fourth subunits 6a to 6d is also set to be the same.

[0054] The offset distance d is set based on the thickness and refractive index of the mirror components constituting dichroic mirrors 9a to 9c. Specifically, assuming the thickness of the mirror component is t, the refractive index of the mirror component is n, the incident angle of the observation light incident on the mirror component is θ, and the refraction angle into the mirror component is φ, the offset δ of the observation light based on the mirror component has… Figure 2 The relationship shown is calculated using the following equation (1). The offset distance d is set based on this offset δ.

[0055] δ=t·sin(θ-φ) / cosφ…(1)

[0056] Reference Figure 3 The mounting structure of the confocal microscope unit 1 relative to the microscope 50 will be described. For example... Figure 3 As shown, the scanning lens 7 is fixed inside the lens barrel 3, and a rocking adjustment mechanism 23 with an integrated accessory part 21 and a movable part 22 is provided on the inner side of the front end of the lens barrel 3. Although in Figure 3 The simplified representation is shown, but the scanning lens 7 is actually composed of multiple lenses.

[0057] The accessory section 21 is formed in a ring shape and protrudes from the front end of the microscope tube 3. It has a connection port PT (e.g., a C-mount configuration) on the front end side for mounting to the camera of the microscope 50. The movable section 22 is continuous with the base end side of the accessory section 21. The movable section 22 is generally ring-shaped, and its outer surface is a spherical sliding surface. A spherical sliding surface 24 corresponding to the shape of the outer surface of the movable section 22 is formed on the inner surface of the front end of the microscope tube 3. Here, the outer surface of the movable section 22 and the inner surface of the microscope tube 3 have the following shapes: when the movable section 22 is embedded in the microscope tube 3 and the accessory section 21 is connected to the connection port PT of the microscope 50, the center C of the spherical surface containing these shapes is located on the imaging plane FS of the microscope optical system R of the microscope 50.

[0058] According to the mounting structure where the rocking adjustment mechanism 23 is embedded in the front end of the microscope tube 3, when the confocal microscope unit 1 is mounted on the microscope 50, the angle of the microscope tube 3 relative to the accessory part 21 can be changed by sliding the movable part 22 relative to the sliding surface 24 of the microscope tube 3. At this time, since the outer surface of the movable part 22 and the inner surface of the microscope tube 3 are spherical, the microscope tube 3 can be rotated relative to the accessory part 21, and the angle of the central axis of the microscope tube 3 relative to the central axis of the accessory part 21 can be adjusted in two dimensions. That is, the rocking adjustment mechanism 23 is configured to change the angle of the microscope tube 3 relative to the accessory part 21, so that the optical axis of the microscope optical system R of the microscope 50 is parallel to the optical axis of the scanning lens 7.

[0059] A support member 30 for a support frame member 40 is provided inside the microscope tube 3. The support member 30, together with the microscope tube 3, forms a housing for housing the scanning lens 7. The support member 30 has a cylindrical portion 31 and an annular flange portion 32 extending outward from the base end of the cylindrical portion 31. The support member 30 is fixed to the inner surface of the microscope tube 3 at the flange portion 32, such that it is located on the imaging plane FS side of the microscope optical system R relative to the scanning lens 7. The front end of the cylindrical portion 31 is located within the movable portion 22. The frame member 40 is fixed to the front end of the cylindrical portion 31 and is located within the movable portion 22.

[0060] like Figure 4 and Figure 5 As shown, the frame component 40 has a plate component 41 and a cover component 42. The plate component 41 is, for example, a fluorescent plate (fluorescent component) formed in the shape of a plate from a fluorescent material, which generates fluorescence in response to the incident illumination light. As described later, the fluorescence generated from the plate component 41 is used as correction light in the correction of the confocal microscope unit 1.

[0061] The cover member 42 is a mask member disposed on the plate member 41 and covering the surface of the plate member 41 on the side of the support member 30. The cover member 42 is formed into a plate (layer) shape, for example, from aluminum, iron, or stainless steel. A black treatment (non-reflective treatment) is applied to the surface of the cover member 42 to suppress light reflection.

[0062] The frame member 40 has an opening 43. That is, the frame member 40 (plate member 41 and cover member 42) is formed in a frame shape to define the opening 43. The frame member 40 is disposed on the side of the microscope optical system R (opposite to the MEMS mirror 4) relative to the scanning lens 7 on the optical axis of the scanning lens 7, such that the illumination light and observation light pass through the opening 43. More specifically, the frame member 40 is disposed on the imaging plane FS of the microscope optical system R, and the surface of the plate member 41 on the scanning lens 7 side is located on the imaging plane FS. The opening 43 is formed, for example, as a rectangular shape in which the length along the first direction D1 is longer than the length along the second direction D2.

[0063] The frame member 40 has a first side 40a, a second side 40b, a third side 40c, and a fourth side 40d that define an opening 43. The first side 40a and the second side 40b extend along a second direction D2 and are opposite to each other across the opening 43. The third side 40c and the fourth side 40d extend along a first direction D1 that is perpendicular to the second direction D2 and are opposite to each other across the opening 43.

[0064] Two rectangular openings (exposed openings) 42a are formed in the cover member 42. The two openings 42a are respectively formed in the portions of the cover member 42 that constitute the first side 40a and the second side 40b. By forming the openings 42a, a portion of the plate member 41 is exposed on the scanning lens 7 side, allowing illumination light to enter this portion of the plate member 41. Thus, a correction section 44 is formed that generates correction light in response to the incident illumination light. That is, the correction section 44 is composed of the portion of the plate member 41 exposed from the openings 42a formed in the cover member 42. In this example, the correction section 44 includes a first correction section 44a disposed in the first side 40a and a second correction section 44b disposed in the second side 40b. The first correction section 44a and the second correction section 44b are located on the same straight line parallel to the first direction D1. The first correction section 44a and the second correction section 44b are each formed in a rectangular shape (square in this example).

[0065] Reference Figure 6 The scanning of illumination light based on MEMS mirror 4 will be explained. In the confocal microscope unit 1, illumination light is scanned within a scanning area along the plane of the frame member 40 (in this example, along the surface of the scanning lens 7 in the plate member 41, located on the imaging plane FS) by rotating MEMS mirror 4. Specifically, illumination light is scanned along the first direction D1 within the scanning area A1 of the imaging plane FS by rotating MEMS mirror 4 around the first axis. Furthermore, the scanning position in the second direction D2 is changed by rotating MEMS mirror 4 around the second axis. By sequentially changing the scanning position in the second direction D2 and scanning illumination light along the first direction D1, illumination light can be scanned throughout the scanning area A1. In this example, MEMS mirror 4 resonates around the first axis (i.e., oscillates at a high speed at a resonant frequency) and moves linearly (non-resonantly) 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 scanning area A1 in the first direction D1, and a portion of the inner side of the correction section 44 is located within the scanning area A1. In other words, the MEMS mirror 4 is driven to scan the correction light within a scanning region A1 that is wider than the opening 43, and at least a portion of the correction section 44 is located within the scanning region A1.

[0066] During observation of the object M, observation light from the effective region A2, set inside the scanning region A1, is detected. Therefore, the swing angle of the MEMS mirror 4 relative to the sampling timing of the photodetectors 13a-13d is adjusted. For example... Figure 7 As shown, the MEMS mirror 4 operates with a swing angle corresponding to the intensity of the drive signal S1, and has a certain phase difference with the drive signal S1. In this example, the drive signal S1 is a pulse signal. The sampling timing of the photodetectors 13a-13d is synchronized with the horizontal synchronization signal S2. In this example, throughout... Figure 7 Within the range indicated by the arrows, photodetectors 13a to 13d detect the observed light. Additionally, in... Figure 7 The image shows the swing angle of the MEMS mirror 4 around the first axis.

[0067] On the other hand, the swing angle (amplitude) and phase of the MEMS mirror 4 change over time due to aging and other factors. Figure 8 (a) and Figure 8 Figure (b) shows the measurement results of the swing angle and phase when the MEMS mirror 4 was continuously driven with a constant drive signal S1 over a period of 1 month (30 days). Figure 8 (a) and Figure 8 As shown in (b), the swing angle decreases and the phase changes over time. If both the swing angle and phase change, there is a concern that the field of view may shift from the effective region A2.

[0068] Therefore, in the calibration method of the confocal microscope unit 1 in this embodiment, the amplitude and phase of the MEMS mirror 4 are calibrated based on the calibration light from the calibration unit 44. For example, this calibration is performed whenever the confocal microscope unit 1 is started. During calibration, firstly, the MEMS mirror 4 is used to scan the illumination light within the scanning area A1 along the plane of the frame member 40, i.e., the imaging plane FS (first step). In the first step, the MEMS mirror 4 is driven such that at least a portion of the calibration unit 44 is located within the scanning area A1. Then, or simultaneously with the first step, photodetectors 13a to 13d are used to detect the calibration light generated from the calibration unit 44 in response to the incident illumination light (second step). In the second step, the calibration light can be detected using only one of the photodetectors 13a to 13d; for example, the calibration light can be detected using only photodetector 13a. Next, at least one of the amplitude and phase of the swing of the MEMS mirror 4 is adjusted based on the detection results of the photodetectors 13a to 13d (third step).

[0069] The following is for reference Figure 9 An example of a method for adjusting the amplitude and phase of the oscillation of the MEMS mirror 4 based on the detection results of photodetectors 13a-13d, i.e., the intensity signal of the correction light, is explained. Figure 9 The upper part shows the horizontal synchronization signal S2 and the effective area A2. Furthermore, examples of the intensity signals of the correction light under states C1 to C5 are shown. Pulse P1 is formed by correction light from the first correction unit 44a, and pulse P2 is formed by correction light from the second correction unit 44b. State C1 is the appropriate state (target state), for example, the state immediately after the drive signal S1 was adjusted during manufacturing.

[0070] As an example, suppose the intensity signal of state C2 is detected. In state C2, both the swing angle and phase of MEMS mirror 4 deviate from state C1. The swing angle of MEMS mirror 4 corresponds to the distance between pulses P1 and P2, and the phase of MEMS mirror 4 corresponds to the rising position of pulses P1 and P2. First, the phase of drive signal S1 is changed, as shown in state C3, so that the rising position of pulse P1 is aligned with the position in state C1. Next, the amplitude of drive signal S1 is changed, as shown in state C4, so that the distance between pulses P1 and P2 matches the distance in state C1. Then, the phase of drive signal S1 is changed again, as shown in state C5, so that the rising position of pulse P1 is aligned with the position in state C1. Through the above steps, the swing angle and phase of MEMS mirror 4 can be adjusted (corrected) to be the same as the target state, i.e., state C1. In this correction, the adjustment is performed so that the width W between the rising position of pulses P1 and P2 and the reference position based on the horizontal synchronization signal S2 is consistent with the target value (a saved value stored as the target value during manufacturing).

[0071] [Functions and Effects]

[0072] The confocal microscope unit 1 includes a frame member 40 formed in the shape of a defined opening 43, which is disposed on the R side of the microscope optical system relative to the scanning lens 7 so that both illumination light and observation light pass through the opening 43. Furthermore, the frame member 40 has a first correction section 44a, which is disposed at the first edge 40a of the defined opening 43 and generates correction light in response to the incident illumination light. Thus, for example, the MEMS mirror 4 can scan the illumination light within the scanning area A1 of the imaging plane FS (along the plane of the frame member 40), and the correction light generated from the correction section 44 in response to the incident illumination light can be detected using photodetectors 13a-13d. Based on the detection results of the photodetectors 13a-13d, at least one of the amplitude and phase of the oscillation of the MEMS mirror 4 can be adjusted. By adjusting (correcting) at least one of the amplitude and phase in this way, the generation of field of view shift as described above can be suppressed. Therefore, according to the confocal microscope unit 1, the generation of field of view shift can be suppressed.

[0073] The correction section 44 is composed of a portion of a plate member 41 exposed from the opening (exposed opening) 42a formed in the cover member 42. This allows for accurate detection of the edge of the correction section 44, improving the accuracy of the correction. Furthermore, since the opening 42a can be formed with good precision in the cover member 42, the correction section 44 can also be formed with good precision. This further improves the accuracy of the correction.

[0074] The calibration unit 44 includes a fluorescence component, namely a plate component 41. Therefore, calibration can be performed in the confocal microscope unit 1 for fluorescence observation.

[0075] Photodetectors 13a-13d detect the fluorescence generated by the object M in response to illumination light as observation light. Thus, fluorescence observation can be performed.

[0076] The correction unit 44 includes a first correction unit 44a disposed on the first side portion 40a and a second correction unit 44b disposed on the second side portion 40b. Thus, even if the configuration of each part is offset due to manufacturing errors, for example, the amplitude and phase of the MEMS mirror 4 can be reliably adjusted.

[0077] The width of the opening 43 of the frame member 40 is narrower than the width of the scanning area A1. As a result, the correction unit 44 can be positioned within the scanning area A1 to perform correction.

[0078] The MEMS mirror 4 is configured to oscillate around a first axis and a second axis. By resonating around the first axis, it scans illumination along the first direction D1 within the scanning area A1 of the imaging plane FS (along the plane of the frame member 40). Furthermore, by rotating around the second axis, the scanning position in the second direction D2, which intersects the first direction D1, changes. A correction unit 44 is provided on the first side 40a extending along the second direction D2. While the amplitude and phase of the oscillation around the first axis (resonance axis) are prone to change, the scan based on the oscillation around the first axis can be corrected within the confocal microscope unit 1.

[0079] The confocal microscope unit 1 includes: an accessory section 21 for mounting the microscope tube 3 to the connection port PT; and a movable section 22 for supporting the microscope tube 3 in a manner that allows changing the angle of the microscope tube 3 relative to the accessory section 21. Thus, by changing the angle of the microscope tube 3 relative to the accessory section 21, the optical axis of the scanning lens 7 can be aligned with the direction of the optical axis of the microscope optical system R. As a result, imaging that maintains both signal intensity and resolution can be achieved.

[0080] The frame component 40 is disposed on the imaging surface FS of the microscope optical system R. Thus, the MEMS mirror 4 can be used to scan the illumination light within the scanning area A1 of the imaging surface FS, and at least one of the amplitude and phase of the swing of the MEMS mirror 4 can be adjusted.

[0081] [Variation Example]

[0082] like Figure 10 As shown in the first variation (a), the first correction section 44a and the second correction section 44b may not be located on the same straight line parallel to the first direction D1. That is, the positions of the first correction section 44a and the second correction section 44b may also be different from each other in the second direction D2. According to this first variation, correction can be performed in the same way as in the above embodiment, and the generation of field of view shift can be suppressed.

[0083] like Figure 10 As shown in the second variation (b), the first correction portion 44a can also extend along the extending direction (second direction D2) of the first side portion 40a on which the first correction portion 44a is provided. Similarly, the second correction portion 44b can also extend along the extending direction (second direction D2) of the second side portion 40b on which the second correction portion 44b is provided. In this example, the first correction portion 44a and the second correction portion 44b extend straight along the second direction D2. In this example, the first correction portion 44a and the second correction portion 44b are each formed as a rectangle having a long side along the second direction. According to this second variation, correction can be performed in the same way as in the above embodiment, and the generation of field of view shift can be suppressed. In addition, the correctable area can be expanded.

[0084] exist Figure 11 In the third variation shown in (a), the correction unit 44 further includes a third correction unit 44c disposed on the third side portion 40c and a fourth correction unit 44d disposed on the fourth side portion 40d. The third correction unit 44c and the fourth correction unit 44d are located on the same straight line parallel to the second direction D2. According to this third variation, correction can be performed in the same way as in the above embodiment, and the generation of field of view shift can be suppressed. Furthermore, correction can be performed not only for scanning along one direction (first direction D1) but also for scanning along a direction intersecting that direction (second direction D2).

[0085] like Figure 11 As shown in the fourth variation of (b), the correction unit 44 may also include only the first correction unit 44a. According to such a first variation, at least one of the amplitude and phase of the swing of the MEMS mirror 4 can be adjusted, and the generation of field of view shift can be suppressed.

[0086] This invention is not limited to the embodiments and variations described above. For example, the materials and shapes of the various structures are not limited to those described above, and various materials and shapes can be used. In the above embodiments, the frame member 40 is disposed on the imaging surface FS of the microscope optical system R, but the frame member 40 only needs to be disposed along a predetermined plane perpendicular to the optical axis of the scanning lens 7, and it does not necessarily have to be disposed on the imaging surface FS.

[0087] In the above embodiment, the correction unit 44 generates fluorescence in response to the incident illumination light. However, the correction unit 44 only needs to generate correction light containing the sensitivity wavelength of at least one of the photodetectors 13a to 13d in a manner that allows the correction light to be detected using at least one of the photodetectors 13a to 13d. In other words, the correction unit 44 can also generate correction light containing the wavelength of the observation light.

[0088] The object M to be observed can also generate light other than fluorescence in response to the illumination light as the observation light. In this case, the correction unit can also generate light other than fluorescence in response to the illumination light. The observation light can also be the reflected light generated from the object M in response to the illumination light. The scanning microscope unit of the present invention is not limited to the confocal microscope A, and can be applied to any scanning microscope using a MEMS mirror, such as a general fluorescence microscope or a reflection microscope. The rocking adjustment mechanism 23 can also be omitted.

[0089] In the above embodiments, a pinhole plate is used as the aperture component to form a confocal optical system. However, the aperture component can be any optical element that confines the light beam, such as an iris aperture or an optical fiber core. When using a fiber optic output type light source, the position of the fiber core end face is set as the aperture position (the position where the light beam is confined). Solid-state lasers, diode lasers, and other laser sources can also be used. In this case, the beam waist position of these laser sources is set as the aperture position, and the light source itself acts as the aperture component.

[0090] The structures of the frame member 40 and the correction section 44 are not limited to the examples described above. For example, the correction section 44 may also be composed of a fluorescent component attached to the surface of the frame member, which is made of a metallic material. The frame member 40 may also be integrally formed with the housing that houses the scanning lens 7. That is, the frame member 40 and the housing that houses the scanning lens 7 may also be composed of a single component. For example, the correction section 44 may also be constructed by attaching a fluorescent component to a frame-shaped portion (frame member) provided in the housing and having an opening defined therein.

[0091] Explanation of symbols

[0092] 1…Confocal microscope unit (scanning microscope unit), 3…Tube (house), 4…MEMS mirror, 7…Scanning lens, 10a~10d…Light source, 13a~13d…Photodetector, 21…Accessories, 22…Modible part, 40…Frame part, 40a…First side, 40b…Second side, 40c…Third side, 41…Plate part, 42…Covering part, 42a…Opening (exposed opening), 43…Opening, 44…Correction part, 44a…First correction part, 44b…Second correction part, 44c…Third correction part, 50…Microscope, A…Confocal microscope (scanning microscope), A1…Scanning area, D1…First direction, D2…Second direction, FS…Imaging plane, M…Object under observation, PT…Connection port, R…Microscope optical system.

Claims

1. A scanning microscope unit, characterized in that, It is a scanning microscope unit that is installed at the connection port of a microscope with a microscope optical system to form a scanning microscope. have: A light source that outputs illumination light; A photodetector that detects observation light generated from the object being observed in response to the illumination of the illuminating light; MEMS mirrors enable the illumination light output from the light source to scan the object being observed, and guide the observation light generated from the object being observed in response to the illumination light toward the photodetector; A scanning lens 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; and The frame component is formed in a frame shape with defined openings, so that the illumination light and the observation light are positioned relative to the scanning lens on the microscope optical system side through the openings. The frame component has a correction section disposed on the edge defining the opening, and generates correction light containing the sensitivity wavelength of the photodetector in response to the incident illumination light. The frame member includes: a plate member that generates the correction light in response to the incident illumination light; and a cover member disposed on and covering the plate member. The correction section is composed of a portion of the plate component exposed from the exposed opening formed in the cover component.

2. The scanning microscope unit according to claim 1, characterized in that, The correction unit includes a fluorescent component.

3. The scanning microscope unit according to claim 1 or 2, characterized in that, The photodetector detects the fluorescence generated from the object under observation in response to the illumination light as the observation light.

4. The scanning microscope unit according to any one of claims 1 to 3, characterized in that, The frame component has a first side and a second side opposite the first side, separated by the opening. The correction section includes a first correction section disposed on the first side and a second correction section disposed on the second side.

5. The scanning microscope unit according to any one of claims 1 to 4, characterized in that, The correction section extends along the extension direction of the edge.

6. The scanning microscope unit according to any one of claims 1 to 5, characterized in that, The frame component has a first side and a third side extending in a direction intersecting the extending direction of the first side. The correction section includes a first correction section disposed on the first side and a third correction section disposed on the third side.

7. The scanning microscope unit according to any one of claims 1 to 6, characterized in that, The MEMS mirror scans the illumination light within a scanning area along the plane of the frame component. The width of the opening in the frame component is narrower than the width of the scanning area.

8. The scanning microscope unit according to any one of claims 1 to 7, characterized in that, The MEMS mirror is configured to oscillate around a first axis and a second axis. It resonates around the first axis to scan the illumination light along a first direction within a scanning area along the plane of the frame member, and rotates around the second axis to change the scanning position in a second direction intersecting the first direction. The correction section is disposed on the edge extending along the second direction.

9. The scanning microscope unit according to any one of claims 1 to 8, characterized in that, It also has: A housing in which the scanning lens is fixed; Accessory part for mounting the housing to the connection port; and A movable part that supports the housing in a manner that allows the angle of the housing relative to the accessory part to be changed.

10. The scanning microscope unit according to any one of claims 1 to 9, characterized in that, The frame component is disposed on the imaging surface of the microscope optical system.

11. A scanning microscope, characterized in that, have: The scanning microscope unit according to any one of claims 1 to 10; and A microscope having the microscope optical system and the connection port.

12. A method for calibrating a scanning microscope unit, characterized in that, The calibration method for the scanning microscope unit according to any one of claims 1 to 10 is as follows. include: In the first step, the MEMS mirror is used to scan the illumination light within a scanning area along the plane of the frame component, and at least a portion of the correction unit is located within the scanning area; The second step involves using the photodetector to detect the correction light generated from the correction unit in response to the incident illumination light; and The third step involves adjusting at least one of the amplitude and phase of the MEMS mirror's oscillation, based on the detection results of the photodetector.

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