JP7105006B1 - scanning fluorescence microscope - Google Patents

Abstract

An object of the present invention is to scan a condensed region of excitation light accurately at high speed, and to obtain an image with no brightness unevenness. An illumination optical system (10) has a rotating shaft (21) and a rotating motor (23) that drives the rotating shaft (21) to rotate, and the rotation of the rotating shaft (21) causes excitation light (IL) that is condensed or diffused in the middle of an optical path to enter. A rotating mirror 20 having a reflecting portion 22 whose incident position changes in the rotating direction of a rotating shaft 21 is provided. The reflecting portion 22 has a reflecting surface that linearly changes the reflection position of the excitation light IL with respect to the traveling direction of the reflected excitation light IL from a first position to a second position with respect to a change in the rotation angle of the rotation shaft 21 . , and a step that changes the reflection position from the second position to the first position when rotated by the rotary shaft 21 . [Selection drawing] Fig. 1

Description

The present invention relates to scanning fluorescence microscopes.

As a fluorescence microscope, excitation light is applied to the specimen from a direction that intersects the optical axis of the optical system for observing the specimen, and a fluorescence image of the specimen obtained by scanning the focused region of the excitation light in a two-dimensional plane is captured. Scanning fluorescence microscopes have been developed. This scanning fluorescence microscope requires a mechanism for changing the relative position between the scanning region of the excitation light and the specimen. Such mechanisms include, for example, moving a sample with respect to excitation light by driving a piezo element (see Non-Patent Document 1), and changing the curvature of the lens with a fluid lens (see Non-Patent Document 2). Conceivable.

Non-Patent Documents 3 and 4 disclose a microscope that scans a condensed region of excitation light on a specimen. In this microscope, the position of the condensed region of the excitation light is changed sinusoidally, and the condensed region is scanned on the specimen, and the scanning frequency is fixed.

Kevin M Dean, Philippe Roudot, Erik S Welf, Gaudenz Danuser, Reto Fiolka,"Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy"; Biophys J, 16 June 2015;108(12):2807-15. Per Niklas Hedde, Enrico Gratton,"Selective plane illumination microscopy with a light sheet of uniform thickness formed by an electrically tunable lens"; Microscopy Research and Technique, 24 June 2016;Vol.81:924-928. Mitutoyo Corporation, "Press release", "World's fastest class variable focal length lens -TAGLENS? series release-", [online], [published on April 11, 2019], [searched on March 22, 2021] Internet <URL: https://www.mitutoyo.co.jp/new/news/2019/04/-taglens-_1.html> Weijian Zong, Jia Zhao, Xuanyang Chen, Yuan Lin, Huixia Ren, Yunfeng Zhang, Ming Fan, Zhuan Zhou, Heping Cheng, Yujie Sun & Liangyi Chen, "Large-field high-resolution two-photon digital scanned light-sheet microscopy" Cell Research; 26 September 2014, volume 25: pages 254-257(2015).

For accurate and high-speed imaging, the higher the scanning frequency of the condensing position of the excitation light, the better. However, when trying to move the sample by 1 mm with the piezo element, the scanning frequency is limited to about 10 Hz. Moreover, the liquid lens requires a settling time of 10 ms or more for the curvature of the lens, and the scanning frequency at which accurate scanning is possible is limited to about 20 to 30 Hz. In the microscopes disclosed in Non-Patent Documents 3 and 4 above, when the position of the condensed region of the excitation light is changed sinusoidally on the specimen, if the intensity of the excitation light is kept constant, Since the irradiation amount of the excitation light differs depending on the position, and uneven brightness occurs in the image of the specimen, it is necessary to change the intensity of the excitation light according to the positional change of the light collection region. In addition, since the scanning frequency is fixed in this microscope, the light source that irradiates the excitation light requires a control system for synchronizing the irradiation timing with the scanning timing, which complicates the device configuration.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a scanning fluorescence microscope capable of scanning a condensed region of excitation light accurately at high speed and obtaining an image with no brightness unevenness. and

In order to achieve the above object, the scanning fluorescence microscope according to the present invention comprises:
an illumination optical system for forming a focused area of the excitation light on the specimen by irradiating the specimen while condensing the excitation light that causes the specimen to generate fluorescence;
an imaging optical system that has an optical axis that intersects with the optical axis of the illumination optical system and that forms a fluorescent image of the specimen generated in the condensing area;
an imaging unit having an imaging surface in which imaging elements are arranged two-dimensionally, and wherein the imaging surface is arranged at a position where the fluorescent image is formed by the imaging optical system;
The illumination optical system is
A reflecting unit having a rotating shaft and a driving unit for rotationally driving the rotating shaft, wherein the incident position of the excitation light condensed in the optical path changes in the rotating direction of the rotating shaft due to the rotation of the rotating shaft. a rotating mirror having
In the reflecting part,
a reflecting surface that linearly changes a reflection position of the excitation light with respect to a traveling direction of the reflected excitation light from a first position to a second position with respect to a change in the rotation angle of the rotation shaft;
a step that changes the reflecting position from the second position to the first position by rotation of the rotating shaft;
is provided ,
In the reflecting section, the incident direction and the emitting direction of the excitation light are not opposite to each other,
The imaging unit captures a plurality of images while the rotating mirror rotates once, and synthesizes the plurality of images to generate a single captured image .

In this case, the driving unit rotates the rotating shaft to scan the condensed area on the specimen, and also receives an image signal from the image pickup element located in the imaged area on the imaging surface corresponding to the condensed area. A control unit that controls the imaging unit to read out,
The imaging unit is
Acquiring a fluorescent image of the specimen based on the imaging signal read from the imaging device in the imaging region;
You can do it.

A first detection unit that detects that the rotational position of the rotating shaft has become a reference position,
The control unit
Initializing the position of the imaging region on the imaging plane when the first detection unit detects that the rotational position has become the reference position;
You can do it.

A second detection unit that detects the rotation angle of the rotation shaft,
The control unit
controlling the position of the imaging region on the imaging plane based on the rotation angle detected by the second detection unit;
You can do it.

The reflection position of the excitation light changes by one cycle or by a plurality of cycles with respect to one rotation of the rotating shaft.
You can do it.

the center of gravity of the reflector is on the axis of rotation;
You can do it.

wherein the illumination optical system is capable of switching a direction in which the specimen is irradiated with the excitation light;
You can do it.

According to the present invention, the reflection position of the excitation light condensed or diffused in the optical path is changed linearly from the first position to the second position in the reflection direction of the excitation light, and from the second position to the first position. Since the position can be changed instantaneously by steps, the excitation light condensing region can be accurately scanned on the sample without delay, and the irradiation amount of the excitation light to the sample can be made uniform. . Therefore, it is possible to scan the condensed region of the excitation light accurately at high speed, and to obtain an image with no brightness unevenness.

1 is a block diagram showing the configuration of a scanning fluorescence microscope according to an embodiment of the present invention; FIG. It is a figure which shows the imaging surface of an imaging part. (A), (B) and (C) are optical path diagrams showing the configuration of the illumination optical system of the scanning fluorescence microscope of FIG. (A) is a perspective view of a rotating mirror. (B) is a trihedral view of a rotating mirror. (A) and (B) are diagrams showing the reflection state of the excitation light when the rotating mirror is rotated. (A) is a diagram showing a change in the reflection position of excitation light on a rotating mirror. (B) is a diagram showing how the focal area is scanned by driving the rotating mirror. (A) is a diagram showing a control system for rotating a rotating mirror and scanning an imaging area. (B) and (C) are diagrams showing the detection principle in the detection unit. (A) is a graph showing changes in the reflection position of excitation light. (B) is a graph showing changes in the x-position of the condensing position. (C) is a graph showing the x position of the readout line of the imaging unit. It is a schematic diagram of a three-dimensional image of a specimen. (A) and (B) are diagrams showing other configurations of the illumination optical system. It is a figure which shows the modification of the reflection part of a rotating mirror. It is a schematic diagram which shows a mode that a captured image is synthesize|combined and a final captured image is produced|generated.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In each drawing, the same reference numerals are given to the same or equivalent parts. In the drawings, an xyz three-axis orthogonal coordinate system is defined as necessary. The following description will be made with reference to this coordinate system.

As shown in FIG. 1, a scanning fluorescence microscope 1 according to the present embodiment is a microscope that observes a sample S using fluorescence EL emitted from the sample S, which is a biological sample or a non-biological sample. The scanning fluorescence microscope 1 includes an illumination optical system 10 , an imaging optical system 11 , an imaging section 12 , a control section 13 and a stage 14 .

The illumination optical system 10 has an optical axis AX1, and is an optical system that irradiates the specimen S with the excitation light IL along the optical axis AX1. The illumination optical system 10 irradiates the sample S while condensing the excitation light IL. As a result, a condensing area A1 of the excitation light IL is formed on the specimen S, and the molecules of the specimen S in the condensing area A1 are excited to emit fluorescence EL.

The imaging optical system 11 has an optical axis AX2 that intersects the optical axis AX1 of the illumination optical system 10 . In FIG. 1, the optical axis AX2 extends parallel to the z-axis. The imaging optical system 11 receives the fluorescence EL emitted from the specimen S, emits the fluorescence EL traveling along the optical axis AX2, and forms a fluorescence image (inverted image) of the specimen S generated in the light collection area A1. An image is formed on the image plane. The imaging optical system 11 is a telecentric optical system and projects a fluorescent image onto an imaging plane with an adjustable magnification.

The imaging unit 12 has an imaging surface 12s in which the imaging elements 12a are two-dimensionally arranged. In the imaging unit 12 , the imaging surface 12 s is arranged at the imaging position of the fluorescence image formed by the imaging optical system 11 . As the imaging unit 12, for example, a CMOS (Complementary Metal-Oxide Semiconductor) sensor is used. The imaging unit 12 can read, line by line, a signal indicating the imaging result of the two-dimensionally arranged imaging elements. In this embodiment, as shown in FIG. 2, the imaging signals of the imaging elements 12a arranged in the y-axis direction are sequentially output from the position X1 to the position X2 along the x-axis direction. A row in the y direction of the image sensor 12a to be read out is also called a readout line.

Returning to FIG. 1 , the control unit 13 is a controller that controls the illumination optical system 10 and the imaging unit 12 . As will be described later, the control unit 13 detects the timing of rotation of a rotation mirror 20 (described later) of the illumination optical system 10, and controls the output timing of the imaging signal of the imaging unit 12, that is, the readout line to be read.

A specimen S is mounted on the stage 14 so as to face the imaging optical system 11 . The stage 14 may be adjustable in position and orientation with six degrees of freedom of position in the x-axis, y-axis, and z-axis directions, and inclination of the stage 14 around the x-axis, around the y-axis, and around the z-axis. In this case, the control unit 13 controls the position and attitude of the stage.

The illumination optical system 10 has a rotating mirror 20 . The rotating mirror 20 receives and reflects the excitation light IL that is condensed or diffused in the optical path of the excitation light IL. The rotating mirror 20 includes a rotating shaft 21, a reflecting section 22, and a rotating motor 23 (see FIG. 3A) as a driving section. The rotating shaft 21 rotates the reflector 22 . As the rotating shaft 21 rotates, the incident position of the reflecting portion 22 where the excitation light IL condensed or diffused in the optical path changes in the rotating direction of the rotating shaft 21 . The rotary motor 23 rotates the rotating shaft 21 .

As indicated by the arrow in FIG. 1, the control unit 13 rotates the rotating shaft 21 by a rotary motor 23 (see FIG. 3A) to scan the condensing area A1 on the specimen S, and rotates the condensing area A1. The imaging area A2 corresponding to is synchronously scanned on the imaging surface 12s. That is, the control unit 13 controls the imaging unit 12 so as to read the imaging signal from the imaging element 12a in the imaging area A2 on the imaging surface 12s corresponding to the converging area A1. The imaging unit 12 acquires a fluorescent image of the specimen S based on the imaging signal output from the imaging element 12a in the imaging area A2. In this embodiment, the width of the imaging area A2 in the x-axis direction is defined to be equal to or less than the width of the imaging device 12a in the x-axis direction.

A more detailed configuration of the illumination optical system 10 will be described. As shown in FIG. 3A, the illumination optical system 10 includes a light source 30, lenses 31a and 31b, a reflecting mirror 32, a polarizing beam splitter 33, a quarter-wave plate 34, and a condenser lens 35. , provided.

The light source 30 emits excitation light IL. The excitation light IL emitted from the light source 30 is light with a wavelength that causes fluorescence EL to be generated from the observation target. This light can include, for example, light between 400 nm and 1500 nm. This light may be incoherent light or coherent light. A mercury lamp, a xenon lamp, a light emitting diode, or a laser light source is used as the light source 30 .

The lenses 31a and 31b relay the excitation light IL emitted from the light source 30 and shape the excitation light IL into parallel light. The reflecting mirror 32 reflects the excitation light IL relayed by the lenses 31a and 31b. The excitation light IL reflected by the reflecting mirror 32 reaches the polarizing beam splitter 33 as parallel light.

Here, the excitation light IL is P-polarized, and the polarization beam splitter 33 transmits the excitation light IL. The excitation light IL transmitted through the polarizing beam splitter 33 is condensed or diffused through the quarter-wave plate 34 and the condensing lens 35, and is reflected on the reflecting surfaces 22a and 22b of the reflecting portion 22 of the rotating mirror 20 (see FIG. 4A). ) and then reflected in the opposite direction. The excitation light IL reflected in the reverse direction by the rotating mirror 20 becomes parallel light by the condenser lens 35 , becomes S-polarized light by the quarter-wave plate 34 , and enters the polarization beam splitter 33 .

As shown in FIG. 3B, the excitation light IL as S-polarized light incident on the polarization beam splitter 33 is reflected by the polarization beam splitter 33 in the −z direction. The illumination optical system 10 includes a lens 36 , a galvanomirror 37 and a lens 38 . The y'-axis extends in a direction parallel to the xy-plane and obliquely crossing the x-axis and the y-axis. The excitation light IL reflected by the rotating mirror 20 and incident on the polarization beam splitter 33 passes through the lens 36, is reflected by the galvanomirror 37, travels in the -y' direction, and reaches the lens . A lens 36 is provided for adjusting the focus, and a galvanomirror 37 is provided for adjusting the z-position of the excitation light IL.

As shown in FIG. 3C, the illumination optical system 10 includes a galvanomirror 39 and a rectangular prism 40. As shown in FIG. The excitation light IL that has passed through the lens 38 is incident on the galvanomirror 39 . The excitation light IL reflected by the galvanomirror 39 enters the rectangular prism 40 . The galvanomirror 39 is provided for switching whether the surface of the rectangular prism 40 on which the excitation light IL is incident is on the -x side or on the +x side.

The illumination optical system 10 further includes a lens 41A, a reflecting mirror 42A, lenses 43Aa and 43Ab, a galvanomirror 44A, and an objective lens 45A. An optical system configured by these constituent elements is referred to as an optical system 50A. When the excitation light IL reflected by the galvanomirror 39 is incident on the −x side surface of the rectangular prism 40, the excitation light IL reflected by the rectangular prism 40 travels through the lens 41A to the reflection mirror 42A. After being reflected by the reflecting mirror 42A, the excitation light IL passes through the lenses 43Aa and 43Ab and is reflected by the galvanomirror 44A. The excitation light IL reflected by the galvanomirror 44A enters the specimen S in the +x direction from the -x side while being collected by the objective lens 45A, forming a condensed area A1 of the excitation light IL on the specimen S. Due to the vibration of the galvanomirror 44A, the condensing area A1 becomes a linear area extending in the y-axis direction. The signal frequency of the galvanomirror 44A is twice the frequency of the exposure time of each imaging device 12a of the imaging unit 12. FIG.

The illumination optical system 10 further includes a lens 41B, a reflecting mirror 42B, lenses 43Ba and 43Bb, a galvanomirror 44B, and an objective lens 45B. An optical system configured by these constituent elements is referred to as an optical system 50B. The components of optical system 50B are the same as those of optical system 50A. When the excitation light IL reflected by the galvanomirror 39 is incident on the +x side surface of the rectangular prism 40, these optical systems cause the excitation light IL to be incident on the sample S from the +x side in the -x direction, and the excitation light IL is formed on the specimen S. Due to the vibration of the galvanomirror 44B, the condensing area A1 becomes a linear area extending in the y-axis direction.

Thus, the illumination optical system 10 can switch the optical system for irradiating the specimen S with the excitation light IL to either the optical system 50A or the optical system 50B according to the direction of the galvanomirror 39. . By switching between the optical system 50A and the optical system 50B, the imaging unit 12 can capture two images irradiated with the excitation light IL from opposite directions. For example, if these two images are tiled and displayed, the effect of the artifact on the observation result of the specimen S can be reduced. The direction of rotation of the rotating mirror 20 is opposite between when the optical system 50A emits the excitation light IL and when the optical system 50B emits the excitation light IL.

A detailed configuration of the rotating mirror 20 will be described. As shown in FIGS. 4A and 4B, the reflecting portion 22 of the rotating mirror 20 has a disk shape as a whole. The reflector 22 is made of phosphor bronze, for example. The reflecting portion 22 is provided with reflecting surfaces 22a and 22b. Reflective surfaces 22a and 22b are coated with aluminum. The reflecting surface 22a and the reflecting surface 22b form an annular reflecting surface around the central axis AX3. However, the material of the rotating mirror 20 is not limited to this, and various materials can be applied.

As shown in FIG. 4B, a rotary shaft 21 is attached along the central axis AX3 of this cylinder. The direction of the central axis AX3 of this cylinder is the axial direction of the rotating shaft 21 . By rotating the rotating mirror 20, the incident position of the excitation light IL alternates between the reflecting surface 22a and the reflecting surface 22b.

As shown in FIGS. 4A and 4B, the reflecting surfaces 22a and 22b are inclined with respect to a plane orthogonal to the central axis AX3. In particular, the reflecting surfaces 22a and 22b are inclined at a constant angle of inclination with respect to the plane along the rotation direction of the central axis AX3. That is, each of the reflecting surfaces 22a and 22b has a spiral shape around the central axis AX3. Level differences 22c and 22d are provided at the two boundaries between the reflecting surfaces 22a and 22b. In the present embodiment, it is assumed that the reflector 22 rotates in the direction of the arrow in FIG. 4(B).

Further, as shown in FIG. 4B, the reflecting surfaces 22a and 22b form a chevron as a whole on a cross section passing through the center of the central axis AX3 with respect to a plane orthogonal to the central axis AX3. is inclined radially outward at an angle θ so as to

The excitation light IL is incident on the reflecting surface 22a or the reflecting surface 22b of the reflecting section 22 along the x-axis direction, as shown in FIGS. 5A and 5B. The rotary motor 23, the rotary shaft 21, and the reflector 22 are installed at an angle θ with respect to the direction parallel to the x-axis. Due to this inclination, linear portions orthogonal to the x-axis direction along the radial direction around the central axis AX3 are formed on the reflecting surfaces 22a and 22b. The excitation light IL is incident on this linear portion. Therefore, the excitation light IL is reflected and propagates in the direction opposite to the incident direction.

As shown in FIGS. 5A and 5B, the rotation of the rotating mirror 20 changes the condensing position of the excitation light IL with respect to the traveling direction of the reflected excitation light IL. Here, it is assumed that the reflector 22 rotates clockwise when viewed from the front side in the paper surface direction of FIG. 4(B). FIG. 5A shows a state in which the excitation light IL is reflected from the reflecting surface 22a over the step 22d from the reflecting surface 22b. In this state, the reflection position of the excitation light IL with respect to the traveling direction of the reflected excitation light IL is the first position P1.

As the rotating mirror 20 rotates further, the reflected position of the excitation light IL advances on the reflecting surface 22a according to the rotation speed of the rotating mirror 20, and linearly changes from the first position P1 to the second position P2. FIG. 5B shows a state in which the rotary mirror 20 has rotated 180 degrees and the reflection position of the excitation light IL is just before the step 22c on the reflection surface 22a. In this state, the reflection position of the excitation light IL in the traveling direction of the reflected excitation light IL is the second position P2. When the reflection position of the excitation light IL exceeds the step 22c, the excitation light IL is incident on the reflection surface 22b, and the reflection position changes from the second position P2 to the first position P1.

Similarly, when the excitation light IL passes over the step 22d from the reflection surface 22b and enters the reflection surface 22a, the reflection position of the excitation light IL becomes the first position P1. , the reflected position of the excitation light IL linearly changes from the first position P1 to the second position P2. When the pumping light IL reaches just before the step 22c, the reflection position of the pumping light IL becomes the second position P2. When the reflection position of the excitation light IL exceeds the step 22c, the excitation light IL is incident on the reflection surface 22b, and the reflection position changes from the second position P2 to the first position P1.

In this way, when the reflecting section 22 is rotated by driving the rotary motor 23, the reflection position of the excitation light IL in the reflecting section 22 in the traveling direction of the reflected excitation light IL changes from the first position P1 to the second position P2. changes linearly with respect to the change in the rotation angle of the rotating shaft 21. As the rotating shaft 21 rotates, the steps 22c and 22d change the reflection position of the excitation light IL in the axial direction of the rotating shaft 21 from the second position P2 to the first position P1.

As shown in FIG. 6A, it is assumed that the excitation light IL is condensed at an intermediate position PN between the first position P1 and the second position P2, and this condensing position is FN. . In this case, when the reflection position of the excitation light IL is at the first position P1, the focal position of the reflected excitation light IL is F1. Further, when the reflection position of the excitation light IL is at the second position P2, the focal position of the reflected light of the excitation light IL is F2.

The focal positions F1 and F2 near the rotating mirror 20 and the focal positions F1' and F2' on the specimen S have a conjugate relationship. Therefore, due to the above-described change in the focal position of the excitation light IL, the converging area A1 on the sample S moves from the focal position F1' to the focal position F2' as shown in FIG. 6B. That is, by rotating the rotating mirror 20 to move the reflection position of the excitation light IL from the first position P1 to the second position P2, the converging area A1 on the sample S is moved from the focal position F1' to the focal position. It can be scanned to F2'.

As shown in FIG. 3A, the illumination optical system 10 further includes a detector 25 as a first detector. A drive shaft of a rotary motor 23 is connected to the rotary shaft 21 of the rotary mirror 20, and the drive of the rotary motor 23 rotates the rotary shaft 21 and the reflector 22 at a constant speed. The detector 25 includes a cam 26 and a photosensor 27 .

The cam 26 is a disc-shaped member fixed to the rotating shaft 21 . The cam 26 rotates as the rotating shaft 21 rotates. As shown in FIG. 7A, the cam 26 is formed with radially extending slits 26a at positions corresponding to the steps 22c and 22d along the rotational direction.

As shown in FIGS. 7B and 7C, the photosensor 27 irradiates the beam 24 onto the outer edge of the cam 26 . The photosensor 27 receives the beam 24 only when the slit 26a is positioned at the irradiation position of the beam 24, as shown in FIG. 7(C). The rotational position of the rotating mirror 20 when the beam 24 is received is the reference position.

The detector 25 is provided to detect that the rotational position of the rotary shaft 21 has reached the reference position. The fact that the reference position has been reached is sent from the detection section 25 to the control section 13, as shown in FIG. 7(A). When the detection unit 25 detects that the rotational position has become the reference position, the control unit 13 initializes the position of the imaging area A2 on the imaging surface 12s to the position X1. At this timing, the controller 13 synchronously scans the condensing area A1 and the imaging area A2.

Thus, in this rotating mirror 20, as shown in FIG. 8A, each time the rotating shaft 21 makes a half rotation (time 0 to T), the reflection position of the excitation light IL shifts from the first position P1 to the second position. 2 to position P2. As a result, as shown in FIG. 8B, the converging area A1 linearly changes on the sample S from the focal position F1' to the focal position F2'. The control unit 13 linearly changes the readout line of the imaging area A2 from the position X1 to the position X2 on the imaging surface 12s of the imaging unit 12, as shown in FIG. 8(C). As a result, the imaging signal of the imaging area A2 corresponding to the condensing area A1 on the sample S is read out. The control unit 13 generates a captured image Im of the specimen S based on the imaging signal output from the imaging element 12a within the imaging region A2. Note that when the rotating mirror 20 rotates once, the reflection position of the excitation light IL, the condensing area A1, and the imaging area A2 are each scanned twice, that is, scanned for two cycles. That is, two captured images Im are obtained for one rotation of the rotating mirror 20 .

The illumination optical system 10 changes the angle of the galvanomirror 37 to change the z-position of the excitation light IL each time scanning is performed. Im can be imaged. A three-dimensional image 60 of the specimen S can be obtained as shown in FIG. 9 by superimposing the captured images Im of the specimen S at different z positions.

In addition, it is desirable that the center of gravity of the reflecting portion 22 is on the rotation axis 21, that is, on the central axis AX3. In the present embodiment, the reflecting portion 22 has a portion of the reflecting surface 22a and a portion of the reflecting surface 22b. there is Therefore, the center of gravity of the reflector 22 is on the rotation axis 21 . As a result, it is possible to prevent unnecessary vibrations due to eccentricity from occurring in the reflecting portion 22 that is rotationally driven by the rotary motor 23 . As a result, stable rotation of the reflecting section 22 becomes possible.

In the scanning fluorescence microscope 1 according to the present embodiment, the illumination optical system 10 includes a cylindrical lens 55 as shown in FIGS. 10A and 10B instead of the objective lens 45A shown in FIG. , instead of the galvanomirror 44A, a reflecting mirror 44A' whose position is fixed may be provided. In this case, the excitation light IL reflected by the reflection mirror 44A' is incident on the cylindrical lens 55 and then condensed in the z-axis direction to form a linear condensing area A1 extending in the y-axis direction. This condensing area A1 is scanned in the x-axis direction by the rotation of the rotating mirror 20, as in the first embodiment.

As explained in detail above, according to the scanning fluorescence microscope 1 according to the above-described embodiment, the reflection position of the excitation light IL condensed or diffused in the optical path is set to the first position in the reflection direction of the excitation light IL. Since it is possible to linearly change from the position P1 to the second position P2 and to instantaneously change from the second position P2 to the first position P1 by the steps 22c and 22d, the condensing area A1 of the excitation light IL can be changed to the specimen. In addition to being able to accurately scan the sample S without delay, the irradiation amount of the excitation light IL to the sample S can be made uniform. Therefore, it is possible to scan the condensed region of the excitation light IL accurately at high speed and to obtain an image with no brightness unevenness.

That is, since the condensing area A1 is scanned by the rotation of the rotating mirror 20, it is not necessary to consider the settling time when performing the position control, and the position of the condensing area A1 can be instantaneously and accurately controlled. Become.

Further, in the above-described embodiment, in the reflecting portion 22 of the rotating mirror 20, the reflection position of the excitation light IL with respect to the axial direction of the rotating shaft 21 corresponds to the rotation angle of the rotating shaft 21 from the first position P1 to the second position P2. It is supposed to change linearly according to the In this way, the light collection area A1 on the sample S can be changed at a uniform speed, and the image captured by the fluorescence emitted from the sample S can be made by the excitation light IL of uniform intensity.

In the above-described embodiment, the reflecting surface of the reflecting portion 22 of the rotating mirror 20 for the excitation light IL has the reflection position of the excitation light IL with respect to the traveling direction of the incident excitation light IL from the first position P1 to the second position. It is assumed that steps 22c and 22d for changing to the position P2 are provided. By providing the steps 22c and 22d, the condensing area A1 can be instantaneously moved from the focal position F2' to the focal position F1'.

Further, in the above-described embodiment, the detector 25 is provided to detect that the rotational position of the rotating shaft 21 of the rotating mirror 20 has reached the reference position. When the detection unit 25 detects that the rotation position of the rotating mirror 20 has become the reference position, the control unit 13 initializes the position of the imaging area A2 on the imaging surface 12s, thereby forming the converging area A1 and the imaging area. Synchronization with area A2 is assumed. In this way, the sample S can be imaged without accumulating synchronization deviations between the light collecting area A1 and the imaging area A2.

It should be noted that the rotary motor 23 may be provided with an encoder for detecting its rotational position, and the encoder may be used as a second detector for detecting the rotational position of the rotary shaft. In this case, the control unit 13 controls the imaging unit 12 so that the position of the imaging area A2 on the imaging surface 12s is synchronized with the rotational position detected by the encoder. By doing so, the position of the imaging area A2 can always be synchronized with the rotational position detected by the encoder, so that the synchronization deviation between the condensing area A1 and the imaging area A2 can be reduced.

In this embodiment, the condensing area A1 is scanned twice each time the rotating shaft 21 rotates once. However, it is not limited to this. For example, as shown in FIG. 11, a rotating mirror 20 may be used in which the condensing area A1 is scanned once each time the rotating shaft 21 rotates once, that is, the rotating mirror 20 changes by one cycle. In this case, the reflecting surface 22e of the rotary shaft 21 is annular and spiral, and a step 22f is provided between the starting point and the ending point. Further, the condensing area A1 may be scanned three times or more each time the rotating shaft 21 rotates once. The rotational speed of the rotating mirror 20 can be reduced as the number of times of scanning the condensing area A1 when the rotating shaft 21 makes one rotation is increased.

In the above-described embodiment, the center of gravity of the reflector 22 is assumed to be on the rotation shaft 21 . By doing so, it is possible to prevent unnecessary vibration from occurring due to the rotation of the rotating mirror 20 . In addition, in the rotating mirror 20 shown in FIG. 11, the shape on the opposite side of the reflecting surface 22e is the same shape as the reflecting surface 22e, and has two-fold rotational symmetry with the reflecting surface 22e about the central axis AX3. Therefore, the center of gravity is positioned on the central axis AX3.

In this way, the reflecting section 22 can be shaped to allow one-time scanning, two-time scanning, or three-time scanning or more, and its center of gravity can be on the central axis AX3. However, the center of gravity of the reflector 22 does not have to be on the rotating shaft 21 as long as eccentricity does not occur.

Further, in the above embodiment, the rotation axis 21 of the rotation mirror 20 is inclined by the angle θ with respect to the optical axis AX1 of the illumination optical system 10 . However, the invention is not limited to this. The excitation light IL may be made incident on a reflecting surface perpendicular to the direction of incidence.

In the above embodiment, the illumination optical system 10 can switch the direction of irradiating the specimen with the excitation light IL from two directions. However, the invention is not limited to this. The excitation light IL may be applied in one direction, or may be applied in three or more directions. If the excitation light IL is incident from multiple directions and the fluorescent image is obtained, the influence of fading and absorption can be reduced, and a fluorescent image free from uneven brightness can be obtained.

In the above embodiment, the rotating mirror 20 reflects the excitation light IL in the direction opposite to the incident direction. However, the invention is not limited to this. In the rotating mirror 20, the direction of incidence and the direction of emission of the excitation light IL may be different. The rotating mirror 20 may be one that periodically and linearly changes the reflection position of the excitation light IL with respect to the traveling direction of the reflected excitation light IL according to the rotation of the rotating shaft 21 .

In the above-described embodiment, the readout line extending in the y-axis direction in the imaging unit 12 is synchronously scanned in the x-axis direction, and the captured image Im is obtained from the signal from the readout line. However, the invention is not so limited. The rotation speed of the rotating mirror 20 is made slower than the time taken by the imaging unit 12 to capture one captured image Im. For example, as shown in FIG. ˜Im6, and among the captured images Im1 to Im6, image regions M1 to M6 corresponding to the imaging region A2 conjugate with the converging region A1 are extracted from the captured images Im1 to Im6, and the captured images Im1 to Im6 are extracted. may be combined to generate a single captured image Im. Here, six captured images Im1 to Im6 are used for synthesis, but the number of captured images to be synthesized may be five or less, or may be seven or more.

Also, a shutter may be provided on the imaging surface 12s of the imaging unit 12. FIG. In the shutter, the control unit 13 may move the opening portion of the shutter from position X1 to position X2 along with the movement of the condensing area A1 to perform synchronous scanning of the condensing area A1 and the imaging area A2. . Such a shutter may be provided at a position conjugate with the imaging surface 12s in the imaging optical system 11. FIG.

Further, in the above-described embodiment, the imaging unit 12 is composed of a CMOS sensor. However, it is not limited to this. The imaging unit 12 may be configured with a CCD (Charge-Coupled Device) sensor.

The present invention is capable of various embodiments and modifications without departing from the broader spirit and scope of the invention. Moreover, the embodiment described above is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of equivalent inventions are considered to be within the scope of the present invention.

INDUSTRIAL APPLICABILITY The present invention can be applied to observe a sample that emits fluorescence due to excitation light.

1 scanning fluorescence microscope, 10 illumination optical system, 11 imaging optical system, 12 imaging unit, 12a imaging element, 12s imaging plane, 13 control unit, 14 stage, 20 rotating mirror, 21 rotating shaft, 22 reflecting unit, 22a, 22b reflection surface 22c, 22d step 22e reflection surface 22f step 23 rotary motor 24 beam 25 detector 26 cam 26a slit 27 photosensor 30 light source 31a, 31b lens 32 reflection mirror 33 Polarization beam splitter 34 1/4 wavelength plate 35 condenser lens 36 lens 37 galvanomirror 38 lens 39 galvanomirror 40 rectangular prism 41A, 41B lens 42A, 42B reflection mirror 43Aa, 43Ab, 43Ba , 43Bb lens, 44A, 44B galvanomirror, 44A' reflecting mirror, 45A, 45B objective lens, 50A, 50B optical system, 55 cylindrical lens, 60 three-dimensional image, A1 condensing area, A2 imaging area, AX1 optical axis, AX2 Optical axis, AX3 Central axis, EL Fluorescence, IL Excitation light, Im, Im1-Im6 Captured image, M1-M6 Image area, S Specimen

Claims (7)

an illumination optical system for forming a focused area of the excitation light on the specimen by irradiating the specimen while condensing the excitation light that causes the specimen to generate fluorescence;
an imaging optical system that has an optical axis that intersects with the optical axis of the illumination optical system and that forms a fluorescent image of the specimen generated in the condensing area;
an imaging unit having an imaging surface in which imaging elements are arranged two-dimensionally, and wherein the imaging surface is arranged at a position where the fluorescent image is formed by the imaging optical system;
The illumination optical system is
A reflecting unit having a rotating shaft and a driving unit for rotationally driving the rotating shaft, wherein the incident position of the excitation light condensed in the optical path changes in the rotating direction of the rotating shaft due to the rotation of the rotating shaft. a rotating mirror having
In the reflecting part,
a reflecting surface that linearly changes a reflection position of the excitation light with respect to a traveling direction of the reflected excitation light from a first position to a second position with respect to a change in the rotation angle of the rotation shaft;
a step that changes the reflecting position from the second position to the first position by rotation of the rotating shaft;
is provided ,
In the reflecting section, the incident direction and the emitting direction of the excitation light are not opposite to each other,
The imaging unit captures a plurality of images while the rotating mirror rotates once, and combines the plurality of images to generate a single captured image.
Scanning fluorescence microscopy. The rotating shaft is rotationally driven by the drive unit to scan the condensed area on the specimen, and an imaging signal is read out from the imaging element located in the imaging area on the imaging plane corresponding to the condensed area. A control unit that controls the imaging unit,
The imaging unit is
Acquiring a fluorescent image of the specimen based on the imaging signal read from the imaging device in the imaging region;
A scanning fluorescence microscope according to claim 1 . A first detection unit that detects that the rotational position of the rotating shaft has become a reference position,
The control unit
Initializing the position of the imaging region on the imaging plane when the first detection unit detects that the rotational position has become the reference position;
A scanning fluorescence microscope according to claim 2 . A second detection unit that detects the rotation angle of the rotation shaft,
The control unit
controlling the position of the imaging region on the imaging plane based on the rotation angle detected by the second detection unit;
A scanning fluorescence microscope according to claim 2 . The reflection position of the excitation light changes by one cycle or by a plurality of cycles with respect to one rotation of the rotating shaft.
A scanning fluorescence microscope according to any one of claims 1 to 4. the center of gravity of the reflector is on the axis of rotation;
A scanning fluorescence microscope according to any one of claims 1 to 5. wherein the illumination optical system is capable of switching a direction in which the specimen is irradiated with the excitation light;
A scanning fluorescence microscope according to any one of claims 1 to 6.

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