JP6708289B2 - Microscope, observation method, and control program - Google Patents

Description

The present invention relates to a microscope, an observation method, and a control program.

A microscope that illuminates a sample obliquely is known (for example, Patent Document 1).

JP, 9-159922, A

It is desired to obtain a good cross-sectional image of a thick sample in a microscope that illuminates the sample obliquely.

According to the aspect of the present invention, an illumination optical system that illuminates the sample with illumination light, an observation optical system having an objective lens, an adjusting unit that adjusts an incident angle of the illumination light with respect to the sample, and a control unit. In the microscope provided with the control unit, the control unit controls at least one of the stage holding the sample and the objective lens to change the distance between the stage and the objective lens, and controls the adjusting unit to control the incident angle of the illumination light with respect to the sample. The first observation position of the sample and the first observation position of the sample based on the information about the incidence angle of the sample with respect to the first observation position and the information about the incidence angle of the second observation position different from the first observation position of the sample. A microscope is provided that determines information about the angle of incidence for a third viewing position that is different from the second viewing position.
According to the aspect of the present invention, in a microscope including an illumination optical system that illuminates a sample with illumination light and an observation optical system having an objective lens, the distance between the stage that holds the sample and the objective lens is changed. The incident angle of the illumination light with respect to the sample is changed, and based on the information regarding the incident angle of the sample with respect to the first observation position and the information regarding the incident angle of the sample with respect to the second observation position different from the first observation position, An observation method is provided for determining information regarding an incident angle of a sample with respect to a third observation position different from the first observation position and the second observation position.
According to the aspect of the present invention, the control of the microscope including the illumination optical system that illuminates the sample with the illumination light, the observation optical system having the objective lens, and the adjustment unit that adjusts the incident angle of the illumination light with respect to the sample is performed. A control program executed by a computer, wherein the control is to control at least one of a stage holding a sample and an objective lens to change a distance between the stage and the objective lens, and to control an adjusting unit to illuminate the sample with respect to the sample. The first observation of the sample is performed based on the information about the incident angle of the sample with respect to the first observation position and the information about the incident angle of the second observation position different from the first observation position of the sample by changing the incident angle. A control program is provided that determines information about the position and the angle of incidence for a third viewing position that is different from the second viewing position.
According to the aspect of the present invention, an illumination optical system that illuminates the sample with illumination light, an observation optical system having an objective lens, an adjusting unit that adjusts an incident angle of the illumination light with respect to the sample, and a control unit. In the microscope provided with the control unit, the control unit controls at least one of the stage holding the sample and the objective lens to change the distance between the stage and the objective lens, and controls the adjusting unit to control the incident angle of the illumination light with respect to the sample. A varying, microscope is provided.
According to an aspect of the present invention, there is provided a microscope including an illumination optical system that illuminates a sample with illumination light, an observation optical system having an objective lens, and a controller, the controller holding the sample. Provided is a microscope in which an incident angle of illumination light with respect to a sample is changed according to a distance between a stage and an objective lens.
According to an aspect of the present invention, there is provided a microscope including an illumination optical system that illuminates a sample with illumination light, an observation optical system having an objective lens, and a controller, the controller holding the sample. Provided is a microscope that controls at least one of a stage and an objective lens to change a distance between a stage holding a sample and the objective lens and change an incident position of illumination light on a pupil plane of the objective lens.
According to the aspect of the present invention, an illumination optical system that illuminates the sample with illumination light, an observation optical system having an objective lens, an imaging unit that captures an image formed in the observation optical system, and a controller. In the microscope, the control unit controls at least one of the stage holding the sample and the objective lens to change the distance between the stage holding the sample and the objective lens, and the brightness of the image captured by the imaging unit. A microscope is provided that changes the angle of incidence of illumination light on a sample such that the values are within a predetermined range.
According to the aspect of the present invention, an illumination optical system that illuminates the sample with illumination light, an observation optical system having an objective lens, an imaging unit that captures an image formed in the observation optical system, and a controller. In the microscope, the control unit controls at least one of the stage holding the sample and the objective lens to change the distance between the stage holding the sample and the objective lens, and the brightness of the image captured by the imaging unit. A microscope is provided that changes the angle of incidence of the illumination light on the sample based on the values.
According to the aspect of the present invention, an illumination optical system that obliquely illuminates the sample with illumination light, an observation optical system having an objective lens, and a control unit that moves at least one of a stage holding the sample and the objective lens. And an image pickup section for picking up an image formed in the observation optical system, wherein the incident angle of the illumination light with respect to the sample can be changed when the control section moves at least one of the stage and the objective lens. A microscope configured as described above is provided.
According to the aspect of the present invention, in a microscope including an illumination optical system that illuminates a sample with illumination light and an observation optical system having an objective lens, the distance between the stage that holds the sample and the objective lens is changed. An observation method is provided in which an incident angle of illumination light with respect to a sample is changed.
According to the aspect of the present invention, the control of the microscope including the illumination optical system that illuminates the sample with the illumination light, the observation optical system having the objective lens, and the adjustment unit that adjusts the incident angle of the illumination light with respect to the sample is performed. A control program executed by a computer, wherein the control is to control at least one of a stage holding a sample and an objective lens to change a distance between the stage and the objective lens, and to control an adjusting unit to illuminate the sample with respect to the sample. A control program is provided to change the angle of incidence.
According to the aspect of the present invention, the illumination optical system that obliquely illuminates the sample with the illumination light, the observation optical system having the objective lens, and at least one of the stage and the objective lens holding the sample are It is a microscope provided with a control part which moves in the same direction as an optical axis, and an image pick-up part which picks up an image formed in an observation optical system, Comprising: A control part is when moving at least one of a stage and an objective lens. A microscope is provided that changes the angle of incidence of illumination light on a sample.
According to the aspect of the present invention, a stage on which a sample is placed, an illumination optical system that obliquely illuminates the sample with illumination light, an observation optical system having an objective lens, a stage that holds the sample, and an objective A control unit that moves at least one of the lenses in the same direction as the optical axis of the objective lens, an imaging device that images the sample through the objective lens, and imaging conditions for each observation position in the optical axis direction of the objective lens are stored. And a storage unit for storing the illumination light, the imaging condition includes information about an incident angle of the illumination light on the sample, and the control unit, based on the imaging condition, moves the illumination light when at least one of the stage and the objective lens is moved. A microscope is provided that changes the angle of incidence of the light on the sample.
According to the aspect of the present invention, an illumination optical system that obliquely illuminates the sample with illumination light and an observation optical system having an objective lens are provided, and at least one of the stage and the objective lens that holds the sample is provided. An observation method using a microscope capable of moving in the same direction as the optical axis of an objective lens, which comprises changing an incident angle of illumination light with respect to a sample when at least one of a stage and an objective lens is moved. Will be provided.
According to the aspect of the present invention, an illumination optical system that obliquely illuminates the sample with illumination light and an observation optical system having an objective lens are provided, and at least one of the stage and the objective lens that holds the sample is provided. A control program that causes a computer to execute control of a microscope that can move in the same direction as the optical axis of the objective lens, the control being such that when at least one of the stage and the objective lens is moved, the incident angle of the illumination light to the sample A control program is provided that includes varying.
According to the aspect of the present invention, an illumination optical system that illuminates the sample with illumination light, an observation optical system having an objective lens, an adjusting unit that adjusts an incident angle of the illumination light with respect to the sample, and a control unit. A microscope provided with the control unit, wherein the control unit controls the adjustment unit to change the incident angle of the illumination light with respect to the sample based on the distance between the sample and the objective lens.
According to the aspect of the present invention, an illumination optical system that illuminates the sample with illumination light, an observation optical system having an objective lens, an imaging unit that captures an image formed in the observation optical system, and a controller. In the microscope, the control unit controls at least one of the stage holding the sample and the objective lens to change the distance between the stage holding the sample and the objective lens, and the contrast of the image captured by the imaging unit. Based on, a microscope is provided that changes the angle of incidence of illumination light on the sample.
According to an aspect of the present invention, in a microscope including an illumination optical system that illuminates a sample with illumination light and an observation optical system having an objective lens, the incident angle of the illumination light with respect to the sample is changed to An observation method is provided that corrects changes in the observation position of the.
According to the first aspect of the present invention, at least one of an illumination optical system that obliquely illuminates the sample with illumination light and an observation optical system that has an objective lens, and a stage that holds the sample and the objective lens are used. A control unit that moves the lens in the same direction as the optical axis of the lens, wherein the control unit changes the incident angle of the illumination light with respect to the sample when moving at least one of the stage and the objective lens. A featured microscope is provided.

According to the second aspect of the present invention, a stage on which a sample is placed, an illumination optical system that obliquely illuminates the sample with illumination light, an observation optical system having an objective lens, and a sample are held. A control unit that moves at least one of the stage and the objective lens in the same direction as the optical axis of the objective lens, an imaging device that images the sample through the objective lens, and an imaging for each observation position in the optical axis direction of the objective lens. And a storage unit that stores the condition, wherein the imaging condition includes information about an incident angle of the illumination light on the sample, and the control unit, based on the imaging condition, moves at least one of the stage and the objective lens. A microscope is provided, which is characterized in that an incident angle of illumination light with respect to a sample is changed.

According to the third aspect of the present invention, an illumination optical system that obliquely illuminates the sample with illumination light and an observation optical system having an objective lens are provided, and at least the stage for holding the sample and the objective lens are provided. An observation method using a microscope, one of which is movable in the same direction as the optical axis of an objective lens, including changing an incident angle of illumination light with respect to a sample when at least one of a stage and an objective lens is moved. An observation method is provided, which is characterized by the above.

According to the fourth aspect of the present invention, an illumination optical system that obliquely illuminates the sample with illumination light and an observation optical system having an objective lens are provided, and at least the stage for holding the sample and the objective lens are provided. One is a control program that causes a computer to execute control of a microscope capable of moving in the same direction as the optical axis of the objective lens, wherein the control is performed when at least one of the stage and the objective lens is moved, with respect to the sample of illumination light. A control program is provided, characterized in that it comprises varying the angle of incidence.

It is a figure which shows the microscope which concerns on 1st Embodiment. It is a figure which shows adjustment of the incident angle with respect to the sample of illumination light by an angle adjustment part. It is a figure which shows the illumination field of an illumination optical system, and the visual field of an observation optical system. It is a figure which shows the relationship of the observation position and incident angle which the calculation part which concerns on 1st Embodiment calculates. It is a flow chart which shows an example of the observation method concerning an embodiment. It is a figure which shows the relationship of the observation position and incident angle which the calculation part which concerns on 2nd Embodiment calculates. It is a figure which shows the microscope 1 which concerns on 4th Embodiment. It is a figure which shows an example of the sequence of illumination and imaging which concerns on 4th Embodiment. It is a figure which shows notionally the example of the image which an image processing part produces|generates.

[First Embodiment]
The first embodiment will be described. FIG. 1 is a diagram showing a microscope 1 according to this embodiment. The microscope 1 is a microscope capable of obliquely illuminating a sample S to be observed and observing an image of the sample S. The microscope 1 is configured to be able to change the irradiation angle of the illumination light with respect to the sample S, for example. Here, the microscope 1 is described as being a fluorescence microscope, but the microscope 1 may be a microscope other than the fluorescence microscope. The microscope 1 can observe the inside of the sample S and can generate an image (for example, Z-stack) in which cross-sections of the sample S in the thickness direction are stacked. The microscope 1 may be capable of observing only one cross section of the sample S in the thickness direction.

The microscope 1 includes a stage 2, a light source device 3, an illumination optical system 4, a first observation optical system 5, an image pickup unit 6, an angle adjustment unit 7, and a control device 8. The control device 8 includes a control unit 9 that comprehensively controls each unit of the microscope 1.

The stage 2 holds the sample S to be observed. The sample S can be mounted on the upper surface of the stage 2, for example. In the XYZ orthogonal coordinate system shown in FIG. 1 and the like, the X direction and the Y direction are directions parallel to the upper surface of the stage 2, and the Z direction is a direction perpendicular to the upper surface of the stage 2. The stage 2 may have a mechanism capable of moving in the X direction, the Y direction, and the Z direction, for example.

The light source device 3 includes a light source 11, a shutter 12, an acousto-optic device 13, and a lens 14. The light source 11 includes a light emitting element such as a laser diode (LD) or a light emitting diode (LED). When the microscope 1 is used for fluorescence observation, the light source 11 emits the illumination light L including the excitation light that excites the fluorescent substance contained in the sample S. The shutter 12 is controlled by the control unit 9 and can switch between a state of passing the illumination light L from the light source 11 and a state of blocking the illumination light L. The acousto-optic element 13 is provided on the light emission side of the shutter 12. The acousto-optic element 13 is, for example, an acousto-optic filter or the like. The acousto-optic element 13 is controlled by the controller 9 and can adjust the light intensity of the illumination light L. Further, the acousto-optic element 13 is controlled by the control unit 9, and the state in which the illumination light L passes through the acousto-optic element 13 (hereinafter, referred to as a light-transmitting state) and the state or intensity blocked by the acousto-optic element 13 are reduced. It is possible to switch between a state (hereinafter referred to as a light shielding state). The lens 14 is, for example, a coupler, and focuses the illumination light L from the acousto-optic element 13 on the light guide member 16.

The microscope 1 may not include at least a part of the light source device 3. For example, the light source device 3 may be unitized and provided in the microscope 1 so as to be replaceable (attachable or detachable). For example, the light source device 3 may be attached to the microscope 1 during observation with the microscope 1.

The illumination optical system 4 can obliquely illuminate the sample S with the illumination light L from the light source device 3. The illumination optical system 4 includes a light guide member 16, a lens 17, a lens 18, a filter 19, a dichroic mirror 20, and an objective lens 21. In FIG. 1 and the like, the optical axis of the illumination optical system 4 is represented by reference numeral 4a.

The light guide member 16 is, for example, an optical fiber, and guides the illumination light L to the lens 17. The lens 17 is, for example, a collimator, and converts the illumination light L into parallel light. The lens 18 condenses, for example, the illumination light L on a pupil plane (rear focal plane) of the objective lens 21 or a position in the vicinity thereof. Here, the vicinity of the pupil plane (rear focal plane) is, for example, within ±10 mm from the pupil plane (rear focal plane). The filter 19 has a characteristic of transmitting light in a wavelength band including a wavelength of excitation light (hereinafter, referred to as an excitation wavelength) that excites the fluorescent substance contained in the sample S, for example. The wavelength characteristic of the filter 19 is set so that at least a part of light having a wavelength other than the excitation wavelength is blocked. The dichroic mirror 20 has a characteristic that the illumination light L is reflected and the light (eg, fluorescence) in a predetermined wavelength band of the light from the sample S is transmitted. The light from the filter 19 is reflected by the dichroic mirror 20 and enters the objective lens 21. At the time of observation, a part of the sample S is arranged on the front focal plane of the objective lens 21.

The illumination optical system 4 described above is an example, and can be changed as appropriate. For example, a part of the illumination optical system 4 described above may be omitted or may be included in the light source device 3. Further, the illumination optical system 4 may include at least a part of the light source device 3. Further, the illumination optical system 4 may include an aperture stop, a field stop, and the like. The optical element closest to the sample S in the illumination optical system 4 is the objective lens 21 in FIG. 1, but may be a prism, a mirror, or the like.

The first observation optical system 5 forms an image by the light from the sample S. For example, the first observation optical system 5 forms an image of fluorescence from the fluorescent substance contained in the sample S. The first observation optical system 5 includes an objective lens 21, a dichroic mirror 20, a filter 24, a lens 25, an optical path switching member 26, a lens 27, and a lens 28. The first observation optical system 5 shares the objective lens 21 and the dichroic mirror 20 with the illumination optical system 4. The optical axis 21a of the objective lens 21 is coaxial with the optical axis 4a of the illumination optical system 4 and the optical axis 5a of the first observation optical system 5. In the example shown in FIG. 1, the optical axis 21a of the objective lens 21 is in the Z direction.

The fluorescence from the sample S enters the filter 24 through the objective lens 21 and the dichroic mirror 20. The filter 24 has a characteristic that light in a predetermined wavelength band of the light from the sample S is selectively transmitted. The predetermined wavelength band is set so as to include the wavelength of fluorescence from the sample S (hereinafter, referred to as fluorescence wavelength). The filter 24 blocks, for example, illumination light reflected by the sample S, external light, stray light, and the like. The filter 24 is unitized with, for example, the filter 19 and the dichroic mirror 20, and the filter unit 29 is replaceably provided. The filter unit 29 may be replaceable according to, for example, the wavelength of the illumination light L emitted from the light source device 3, the wavelength of fluorescence emitted from the sample S, or the like, or a single unit corresponding to a plurality of excitation wavelengths and fluorescence wavelengths. It is also possible to use one filter unit.

The light passing through the filter 24 enters the optical path switching member 26 via the lens 25. The light emitted from the lens 25 enters the optical path switching member 26. The optical path switching member 26 is, for example, a prism, and is provided so that it can be inserted into and removed from the optical path of the first observation optical system 5. The optical path switching member 26 is inserted into and removed from the optical path of the first observation optical system 5 by, for example, a drive unit (not shown) controlled by the control unit 9. The optical path switching member 26 guides the fluorescence from the sample S to the optical path toward the imaging unit 6 by internal reflection in a state of being inserted in the optical path of the first observation optical system 5. The fluorescence from the sample S passes through the optical path switching member 26 and then enters the imaging unit 6 via the lens 27 and the lens 28. In the first observation optical system 5, the objective lens 21 and the lens 25 form, for example, a primary image (eg, intermediate image) of the sample S. Further, in the first observation optical system 5, the lens 27 and the lens 28 form a secondary image (eg, final image) of the sample S.

The first observation optical system 5 described above is an example, and can be changed as appropriate. For example, a part of the first observation optical system 5 described above may be omitted. Moreover, the first observation optical system 5 may include an aperture stop, a field stop, and the like. The first observation optical system 5 may be provided independently of the illumination optical system 4, and may be provided, for example, on the opposite side of the objective lens 21 with respect to the sample S.

The microscope 1 according to this embodiment includes a second observation optical system 30 used for setting an observation range and the like. The second observation optical system 30 includes an objective lens 21, a dichroic mirror 20, a filter 24, a lens 25, a mirror 31, a lens 32, a mirror 33, a lens 34, and a lens 35 in order from the sample S toward the observer's viewpoint Vp. The mirror 36 and the lens 37 are provided. The second observation optical system 30 shares the configuration from the objective lens 21 to the lens 25 with the first observation optical system 5. After passing through the lens 25, the light from the sample S is incident on the mirror 31 with the optical path switching member 26 retracted from the optical path of the first observation optical system 5. The light reflected by the mirror 31 enters the mirror 33 through the lens 32, is reflected by the mirror 33, and then enters the mirror 36 through the lenses 34 and 35. The light reflected by the mirror 36 enters the viewpoint Vp via the lens 37. The second observation optical system 30 forms, for example, an intermediate image of the sample S on the optical path between the lens 35 and the lens 37. The lens 37 is, for example, an eyepiece lens, and an observer can set an observation range by observing the intermediate image.

The image capturing section 6 captures an image formed by the first observation optical system 5. The image pickup unit 6 includes an image pickup device 40 and a control unit 41. The image sensor 40 is, for example, a CMOS image sensor, but may be a CCD image sensor or the like. The image pickup device 40 has, for example, a plurality of pixels arranged two-dimensionally, and a photoelectric conversion device such as a photodiode is arranged in each pixel. The imaging element 40 reads the electric charge accumulated in the photoelectric conversion element by a reading circuit, for example. The image sensor 40 converts the read charges into digital data and outputs digital data (for example, image data) in which pixel positions and gradation values are associated with each other. The control unit 41 operates the image sensor 40 based on the control signal input from the control unit 9 of the control device 8 and outputs the data of the captured image to the control device 8. Further, the control unit 41 outputs the charge accumulation period and the charge reading period to the control device 8.

The microscope 1 includes an input/output device 42 and a storage device 43. The input/output device 42 is communicably connected to the control device 8 in a wired or wireless manner. The input/output device 42 includes a display device 44 (display unit, output unit) and an input device 45 (input unit). The display device 44 is, for example, a liquid crystal display or the like. The display device 44 displays various images such as an image showing various settings of the microscope 1, an image captured by the image capturing unit 6, and an image generated from the captured image. The control unit 9 controls the display device 44 to display various images on the display device 44. For example, the control unit 9 supplies the image data generated by the image processing unit 56 to the display device 44 and causes the display device 44 to display the image. The input device 45 is a user-operable input unit such as a keyboard, a mouse, or a trackball. The input device 45 accepts, for example, the user's input of various information such as an observation condition and an operation command for each unit of the microscope 1. The input device 45 supplies the information input to the own device to the control device 8. The input/output device 42 is not limited to the above configuration, and may be, for example, a touch panel in which a display unit and an input unit are integrated, or may include another output unit (eg, a printer) instead of the display device 44. Good.

In this embodiment, the microscope 1 can change the observation position on the sample S in the direction of the optical axis 4a of the illumination optical system 4. The observation position on the sample S is set at or near the position of the front focal plane of the objective lens 21. The observation position on the sample S can be changed, for example, by relatively moving the sample S and the objective lens 21 in the direction of the optical axis 21a of the objective lens 21 (optical axis 4a of the illumination optical system 4). For example, the observation position on the sample S can be changed by moving the stage 2 on which the sample S is placed in the direction of the optical axis 21a of the objective lens 21. The observation position on the sample S may be changed by moving the objective lens 21 in the optical axis direction. That is, to change the observation position on the sample S, the stage 2 may be moved, the objective lens 21 may be moved, or both of them may be moved.

The angle adjusting unit 7 adjusts the angle of the illumination light L emitted from the illumination optical system 4 according to the observation position of the sample S in the direction of the optical axis 4a of the illumination optical system 4. The angle adjusting unit 7 includes, for example, a holding member 51 and a driving unit 52. The holding member 51 holds the end of the light guide member 16 on the light emission side. The drive unit 52 can move the holding member 51 in a direction perpendicular to the optical axis 4 a of the illumination optical system 4. The holding member 51 is provided on the pupil conjugate plane of the objective lens 21 or in the vicinity thereof. The angle adjusting unit 7 will be described later in more detail with reference to FIGS. 2 and 3.

The control device 8 collectively controls each part of the microscope 1. The control device 8 includes a control unit 9, a calculation unit 55, and an image processing unit 56. The control unit 9 controls the driving unit 52 and moves the holding member 51 by the driving unit 52. Thereby, the incident angle of the illumination light L with respect to the sample S (eg, the angle between the illumination light L and the optical axis 4a) is adjusted. Note that the holding member 51 may be manually moved instead of the driving unit 52 moving the holding member 51.

In addition, the control unit 9 causes the acousto-optic element 13 to include the light source device based on a signal (image pickup timing information) that is supplied from the control unit 41 of the image pickup unit 6 and indicates the charge accumulation period and the charge read period. A control signal for switching between a light-transmitting state in which light from the light source 3 passes and a light-shielding state in which light from the light source device 3 is blocked is supplied. The acousto-optic element 13 switches between a light-transmitting state and a light-shielding state based on this control signal. The control unit 41, instead of the control unit 9, controls the acoustooptic device 13 to switch between a light blocking state and a light transmitting state based on a signal (image pickup timing information) indicating a charge accumulation period and a charge reading period. A signal may be supplied to control the acousto-optic device 13.

In addition, the control unit 9 controls the image pickup unit 6 to cause the image pickup device 40 to perform image pickup. The control unit 9 acquires the imaging result (data of the captured image) from the imaging unit 6. The image processing unit 56 performs image processing using the image pickup result of the image pickup unit 6. For example, the control unit 9 changes the observation position on the sample S by changing the position of the stage 2 in the direction of the optical axis 21a of the objective lens 21. Further, the control unit 9 causes the image pickup device 40 to perform image pickup at each observation position, and causes the pickup image to be acquired at a plurality of observation positions in the direction of the optical axis 21a of the objective lens 21. The image processing unit 56 can generate a three-dimensional image of the sample S using, for example, a plurality of acquired captured images.

Next, the angle adjusting unit 7 will be described. FIG. 2 is a diagram showing the adjustment of the incident angle of the illumination light L on the sample S by the angle adjusting unit 7. The angle adjusting unit 7 adjusts the incident angle of the illumination light L on the sample S. Here, the incident angle of the illumination light L with respect to the sample S is an angle formed by the optical axis 21a of the objective lens 21 (optical axis 4a of the illumination optical system 4) and the direction in which the illumination light L enters the sample S. (Α1, α2, etc. in FIG. 2). Further, when the incident angle of the illumination light L to the sample S (α1, α2, etc. in FIG. 2) changes, the optical axis of the objective lens 21 (optical axis 4a of the illumination optical system 4) and the sample S are illuminated (passed). The angle with the illumination light L (β1, β2, etc. in FIG. 2) also changes. Here, an example (α1 to α2) in which the angle adjusting unit 7 reduces the incident angle of the illumination light L on the sample S will be described, but it may be increased.

A light source image that can be regarded as a point light source is formed at the light exit side end 16a of the light guide member 16. The end 16a of the light guide member 16 is arranged by the holding member 51 at the position of the pupil conjugate plane PU2 optically conjugate with the pupil plane PU1 of the objective lens 21 or in the vicinity of the pupil conjugate plane PU2. Here, it is assumed that the holding member 51 is arranged at the position Q1 on the pupil conjugate plane PU2 before the angle of the illumination light L is adjusted. The illumination light L emitted from the light guide member 16 is incident on the incident position P1 on the pupil plane PU1 of the objective lens 21 and then is incident on the sample S at an angle α1. Here, the sample S is placed on a transparent member 2a such as a cover glass, and the transparent member 2a is supported by the stage 2. The space between the lower surface of the transparent member 2a and the end surface of the objective lens 21 is filled with the immersion liquid LQ. The refractive index of the immersion liquid LQ is substantially the same as the refractive index of the transparent member 2a, for example. When the refractive index of the sample S and the refractive index of the transparent member 2a on which the sample S is placed are different, the illumination light L is refracted at the interface Sa between the sample S and the transparent member 2a, as shown in FIG. .. Here, the angle between the illumination light L incident on the sample S at the angle α1 and refracted at the interface Sa between the sample S and the transparent member 2a and the optical axis 21a of the objective lens 21 (optical axis 4a of the illumination optical system 4) is defined as follows. It is represented by β1.

When reducing the angle of the illumination light L, the drive unit 52 moves the holding member 51 holding the end 16a of the light guide member 16 to the position Q2 closer to the optical axis 4a than the position Q1. As a result, the incident position of the illumination light L on the pupil plane PU1 of the objective lens 21 changes to the incident position P2 closer to the optical axis 4a than the incident position P1, and the angle of the illumination light changes to α2 smaller than α1. .. Here, the angle between the illumination light L incident on the sample S at the angle α2 and refracted at the interface Sa and the optical axis 4a is represented by β2. Since α2 is smaller than α1, β2 is smaller than β1.

FIG. 3 is a diagram showing the illumination field of the illumination optical system and the field of view of the observation optical system. Here, the observation position on the sample S is assumed to be the same position as the front focal plane of the objective lens 21. Note that, for convenience of description, a case where the illumination light L is condensed on the pupil plane PU1 of the objective lens 21 will be exemplified below, but the illumination light L does not necessarily need to be condensed on the pupil plane PU1 of the objective lens 21. .. For example, the illumination light L may be condensed near the pupil plane PU1. In FIG. 3A, the front focal plane FP1 of the objective lens 21 is set at the same position as the interface Sa between the sample S and the transparent member 2a. The illumination light L condensed at the incident position P1 on the pupil plane PU1 of the objective lens 21 becomes parallel light, illuminates the sample S, and forms an illumination field LF1 at the interface Sa between the sample S and the transparent member 2a. The illumination field LF1 is formed, for example, around the optical axis 21a (optical axis 4a) of the objective lens 21. The visual field VF (for example, the observation range on the sample S) of the first observation optical system 5 (see FIG. 1) is formed, for example, around the optical axis 21a (optical axis 4a) of the objective lens 21. The illumination field LF is formed, for example, in a region where the entire visual field VF fits.

FIG. 3B shows a state in which, for example, the stage 2 is brought closer to the objective lens 21 from the state of FIG. In FIG. 3B, the front focal plane FP2 of the objective lens 21 is located away from the interface Sa between the sample S and the transparent member 2a in the direction of the optical axis 21a (optical axis 4a) of the objective lens 21 (of the sample S). Internal). The illumination light L condensed on the pupil plane PU1 of the objective lens 21 becomes parallel light and forms an illumination field LF2 on the front focal plane FP2. The illumination light L that has entered the sample S has an angle β1 with the optical axis 21a (optical axis 4a) of the objective lens 21 due to refraction at the interface Sa between the sample S and the transparent member 2a, and is transparent to the sample S. Since there is a gap (ΔZ) between the interface Sa with the member 2a and the front focal plane FP2, the illumination field LF2 moves in the +X direction by the movement amount ΔX compared to the illumination field LF1. The amount of movement ΔX is an amount that depends on the distance ΔZ between the interface Sa between the sample S and the transparent member 2a and the front focal plane FP2, and the angle β1. The larger the distance ΔZ or the larger the angle β1, the larger the movement amount ΔX. As the movement amount ΔX increases, the overlapping area of the illumination field LF2 and the visual field VF decreases, and it becomes difficult to obtain a good image of the sample S. Therefore, the angle adjusting unit 7 shown in FIG. 2 adjusts the illumination light L according to the observation position of the sample S in the direction of the optical axis 21a (optical axis 4a) of the objective lens 21 (front focal plane of the objective lens 21). The incident angle with respect to the sample S is adjusted.

In FIG. 3C, the front focal plane FP2 of the objective lens 21 is set at the same position as in FIG. 3B. The angle adjusting unit 7 (see FIG. 2) makes the incident position (condensing position) of the illumination light L on the pupil plane PU1 of the objective lens 21 incident according to the position of the front focal plane FP2 of the objective lens 21, for example. Adjust to position P3. The incident position P3 is set closer to the optical axis 21a (optical axis 4a) of the objective lens 21 than the incident position P1 in FIG. The illumination light L condensed at the incident position P3 becomes parallel light and travels toward the sample S. The angle α3 of the illumination light L toward the sample S (the incident angle of the illumination light L with respect to the sample S) is smaller than the angle α1 in FIG. 3B, and the illumination light refracted at the interface Sa between the sample S and the transparent member 2a. The angle β3 (the angle between the illumination light L refracted at the interface Sa and the optical axis 21a (optical axis 4a) of the objective lens 21) β3 is smaller than the angle β1 in FIG. 3B. Therefore, the illumination field LF3 moves in the −X direction as compared with the illumination field LF2 in FIG. As a result, the overlapping area of the illumination field LF3 and the visual field VF is increased as compared with FIG. 3B, and the image of the sample S can be satisfactorily acquired.

The angle adjusting unit 7 shown in FIG. 2 is, for example, an observation position (the front focal plane FP2 of the objective lens 21 from the interface Sa between the sample S and the transparent member 2a in the direction of the optical axis 21a (optical axis 4a) of the objective lens 21). The incident position P is adjusted according to the distance ΔZ. For example, the angle adjusting unit 7 sets the incident position P of the illumination light L to the objective lens 21 as the front focal plane FP2 of the objective lens 21 is farther from the interface Sa between the sample S and the transparent member 2a (the larger ΔZ is). The optical axis 21a (optical axis 4a). That is, when the stage 2 is moved in the direction approaching the objective lens 21 along the optical axis 21a of the objective lens 21, the deep portion of the sample S (here, the portion away from the interface Sa in the direction of increasing the distance) is at the observation position. Becomes In this case, as described above, if the incident angle of the illumination light L with respect to the sample S remains constant, the overlapping area of the illumination field and the field of view becomes small, so that the stage 2 is moved along the optical axis and controlled. The unit 9 controls the driving unit 52 of the angle adjusting unit 7, and moves the holding member 51 by the driving unit 52 so that the incident position P approaches the optical axis 21a (optical axis 4a) of the objective lens 21. Similarly, even if the objective lens 21 is moved in the direction approaching the stage 2 along the optical axis, the deep portion of the sample S becomes the observation position. Also in this case, similarly, while moving the objective lens 21 along the optical axis, the control unit 9 controls the driving unit 52 of the angle adjusting unit 7, and the driving unit 52 causes the incident position P to be set to the objective lens 21. The holding member 51 is moved so as to approach the optical axis 21a (optical axis 4a).

In the present embodiment, the calculator 55 shown in FIG. 1 calculates the target value of the incident position (eg, the incident position P3 in FIG. 3) with respect to the position of the front focal plane FP of the objective lens 21 on the sample S. The control unit 9 controls the angle adjusting unit 7 based on the calculation result of the calculating unit 55. The calculator 55 calculates the target value of the incident position of the illumination light L on the pupil plane PU1 based on the information input via the input device 45, for example.

FIG. 4A is a diagram showing the relationship between the observation position and the incident angle calculated by the calculation unit 55 according to this embodiment. The calculation unit 55 uses, for example, two or more sets of an observation position (position of the front focal plane FP of the sample S of the objective lens 21 on the sample S of the objective lens 21) and the incident position P of the illumination light L to determine another observation position. The target value of the incident position P of the corresponding illumination light L is calculated. In FIG. 4A, the horizontal axis represents the observation position (unit is μm) with the inside of the sample S being positive with the interface Sa between the sample S and the transparent member 2a as a reference (0 μm). In FIG. 4A, the vertical axis is a value indicating the incident position P of the illumination light L on the pupil plane PU1, and its unit is an arbitrary unit (au). Here, an example is shown in which the movable range of the holding member 51 is standardized. Reference numeral U1 indicates a target value (about 0.897) of the incident position P corresponding to the first observation position (eg, about 0 μm) designated by the user. Reference numeral U2 indicates a target value of the incident position P (eg, about 0.841) corresponding to the second observation position (eg, about 4.5 μm) designated by the user. The calculator 55 calculates the target value of the incident position P with respect to the third observation position between the first observation position and the second observation position by, for example, interpolating between the plot U1 and the plot U2. Specifically, in FIG. 4, the calculation unit 55 uses linear interpolation to linearly interpolate U3 to U12. Further, in the above, the user specifies U1 and U2 and interpolates between U1 and U2. However, for example, the user specifies U5 and U10 and U1 to U4, U6 to U9 and U11. ~U12 may be calculated. Note that the number of observation positions is appropriately set according to the observation conditions and the like.

FIG. 4B is a diagram showing another example of the operation of the calculation unit 55 according to the present embodiment. In this example, the calculation unit 55 calculates the incident position P of the illumination light L on the pupil plane PU1 by a non-linear interpolation method. For example, the calculation unit 55 interpolates the section between the plots U1 and U2 such that the plots U1 and U2 are the end points and the plurality of plots U1 to U12 are arranged on the downward convex curve. For example, the calculation unit 55 calculates a plurality of plots U3 to U12 using an interpolation method (for example, γ interpolation) in which the target value of the incident position is expressed in a function form including the index of the observation position. The interpolation method used by the calculation unit 55 is not limited to the above example, and may be an interpolation method having a polygonal distribution. The controller 9 may output to the input/output device 42 selectable a plurality of calculation methods of information regarding the incident angle according to the position of the objective lens 21. For example, the control unit 9 causes the display device 44 to display a plurality of interpolation method candidates (eg, linear interpolation, γ interpolation, etc.), and the user operates the input device 45 to select a plurality of interpolation methods to be used. It may be selectable from. In addition, the calculation unit 55 calculates information regarding the incident angle according to the position of the objective lens 21 based on the calculation method input from the input/output device 42, and the control unit 9 uses the information calculated by the calculation unit 55. On the basis of this, the incident angle may be changed. Further, the control unit 9 selectively outputs a plurality of calculation methods of information regarding the incident angle according to the position of the stage 2 to the input/output device 42, and the calculation unit 55 outputs the calculation method input from the input/output device 42. Based on the above, information about the incident angle according to the position of the stage 2 may be calculated, and the control unit 9 may change the incident angle based on the information calculated by the calculation unit 55.

In FIG. 4, the horizontal axis is the observation position where the inside of the sample S is positive with the interface Sa between the sample S and the transparent member 2a as a reference (0 μm), but the position of the stage 2 or the objective lens 21 It may be the position. The user specifies the first observation position and the target value of the incident position P thereof, and the second observation position and the target value of the incident position P thereof, but the calculation unit 55 calculates the stage 2 of the stage 2 corresponding to the first observation position. The position (first stage position) and the position of the stage 2 (second stage position) corresponding to the second observation position are acquired, and the first stage position and the target value of its incident position P, the second stage position and its incident point are acquired. You may relate with the target value of the position P. In this case, the calculator 55 calculates the target value of the incident position P according to the position of the stage 2. At this time, in FIG. 4, the horizontal axis may be the absolute coordinate position of the stage 2 or may be the first stage position as a reference. Similarly, the user specifies the first observation position and the target value of the incident position P thereof and the second observation position and the target value of the incident position P thereof, but the calculation unit 55 corresponds to the first observation position. The position of the objective lens 21 (first objective lens position) and the position of the objective lens 21 corresponding to the second observation position (second objective lens position) are acquired, and the target values of the first objective lens position and its incident position P are acquired. , The second objective lens position may be associated with the target value of the incident position P thereof. In this case, the calculator 55 calculates the target value of the incident position P according to the position of the objective lens 21. At this time, in FIG. 4, the horizontal axis may be the absolute coordinate position of the objective lens 21 or may be the first objective lens position as a reference.

The control unit 9 controls the angle adjusting unit 7 based on the calculation result of the calculating unit 55. In this case, the control unit 9 controls the angle adjusting unit 7 using the position of the holding member 51 corresponding to the incident position P stored in the storage device 43 in advance.

In FIG. 4, the vertical axis is the incident position P of the illumination light L on the pupil plane PU1, but it may be the position of the holding member 51. In this case, the user specifies the first observation position and the position of the holding member 51, and the second observation position and the position of the holding member 51, and the calculator 55 determines the position of the holding member 51 according to the observation position. To calculate. In this case, the position of the holding member 51 corresponding to the incident position P does not need to be stored in the storage device 43 in advance.

The control unit 9 may change the incident angle based on information about the incident angle according to the position of the objective lens 21 (hereinafter, referred to as first reference information). The first reference information may be, for example, information about the incident angle associated with the absolute position information of the objective lens 21. The absolute position information is, for example, information on the position of the objective lens 21 with respect to a predetermined reference position, and is coordinates in the direction (Z direction) of the optical axis 21a of the objective lens 21. For example, the reference position is set by the control unit 9 when the power of the microscope 1 is turned on. In this case, as the first reference information, for example, a function (eg, mathematical expression, numerical table) indicating the relationship between the absolute position of the objective lens 21 and the incident angle can be used. Further, the first reference information may be information regarding the incident angle associated with the movement amount of the objective lens 21. In this case, as the first reference information, for example, a function (eg, mathematical expression, numerical table) indicating the relationship between the movement amount of the objective lens 21 and the incident angle can be used. Further, the first reference information may be information regarding an incident angle associated with relative position information of the objective lens 21 with respect to the stage 2. This relative position information is, for example, information on the difference between the coordinates of the stage 2 and the coordinates of the objective lens 21 in the direction (Z direction) of the optical axis 21a of the objective lens 21. In this case, as the first reference information, for example, a function (e.g., mathematical expression, numerical table) indicating the relationship between the absolute position of the objective lens 21 and the incident angle with respect to the position of the stage 2 can be used.

The control unit 9 may change the incident angle based on information on the incident angle according to the position of the stage 2 (hereinafter referred to as second reference information). The second reference information may be, for example, information about the incident angle associated with the absolute position information of the stage 2. The absolute position information is, for example, information on the position of the stage 2 with respect to a predetermined reference position, and is the coordinates of the stage 2 in the direction (Z direction) of the optical axis 21a of the objective lens 21. For example, the reference position is set by the control unit 9 when the power of the microscope 1 is turned on. In this case, as the second reference information, for example, a function (e.g., mathematical expression, numerical table) indicating the relationship between the absolute position of the stage 2 and the incident angle can be used. Further, the second reference information may be information on the incident angle associated with the movement amount of the stage 2. In this case, as the second reference information, for example, a function (eg, mathematical expression, numerical table) indicating the relationship between the movement amount of the stage 2 and the incident angle can be used. Further, the second reference information may be information regarding the incident angle associated with the relative position information of the stage 2 with respect to the objective lens 21. This relative position information is, for example, information on the difference between the coordinates of the stage 2 and the coordinates of the objective lens 21 in the direction (Z direction) of the optical axis 21a of the objective lens 21. In this case, as the second reference information, for example, a function (for example, a mathematical expression, a numerical value table) indicating the relationship between the absolute position of the stage 2 and the incident angle with respect to the position of the objective lens 21 can be used.

Hereinafter, the observation method according to the embodiment will be described based on the configuration of the microscope 1 described above. FIG. 5 is a flowchart showing an example of the observation method according to the embodiment. First, in step S1, the user sets the observation conditions using the input device 45. The observation conditions are, for example, the first observation position (lower limit of the observation position) and the incident position at that time, the second observation position (upper limit of the observation position) and the incident position at that time, and the observation in the optical axis direction of the objective lens 21. For example, the number of positions. Here, the user determines the optimum incident position at the first observation position and the second observation position based on the contrast, light intensity, etc. of a plurality of images captured by moving the holding member 51 to change the incident position. decide. The above observation conditions may be stored in the storage unit 43 in advance, for example.

In step S2, the calculation unit 55 calculates the relationship between the observation position and the incident position. For example, the calculation unit 55 uses the first observation position, the second observation position, and the number of observation positions in the optical axis direction of the objective lens 21 that are set in the observation conditions to determine the first observation position and the second observation position. Calculate the observation position between. Further, for example, as shown in FIG. 4, the calculation unit 55 uses the incident position in the case of the first observation position and the incident position in the case of the second observation position to calculate the incident position at each observation position therebetween. To do.

In step S3, the microscope 1 starts an operation related to observation in response to, for example, a user instruction. In step S4, the control unit 41 controls the stage 2 to move the stage 2 to the position corresponding to the first observation position. In step S5, the control unit 41 moves the holding member 51 using the relationship between the observation position and the incident angle. In step S6, the control unit 41 causes the image capturing unit 6 to capture the image formed by the first observation optical system 5 while the sample S is illuminated with the illumination light from the illumination optical system 4, and causes the image capturing unit 6 to acquire the image. In step S7, the control unit 41 determines whether acquisition of images has been completed for all the observation positions. When the control unit 41 determines that a part of the observation positions set in the observation condition in Step S1 is not completed (Step S7; No), in Step S8, the stage 2 is moved to a position corresponding to the next observation position. Then, the process from step S5 to step S7 is repeated. In addition, when the control unit 41 determines that the acquisition of the images for all the observation positions is completed (step S7; Yes), the control unit 41 ends the series of processes. In the above, in step S2, the calculation unit 55 calculates the relationship between the observation position and the incident position, but the relationship between the observation position and the incident position may be stored in the storage device 43 in advance. In this case, step S2 is omitted.

[Second Embodiment]
The second embodiment will be described. In the present embodiment, configurations similar to those of the above-described embodiments are designated by the same reference numerals, and description thereof will be omitted or simplified. In the present embodiment, the configuration of the microscope 1 is the same as that of FIG. 1, but the operation of the calculation unit 55 is different from that of the first embodiment.

FIG. 6 is a diagram showing the relationship between the observation position and the incident angle calculated by the calculation unit 55 according to the second embodiment. In the second embodiment, unlike the first embodiment, the user does not need to specify the set of the observation position (the position of the front focal plane FP of the sample S of the objective lens 21) and the incident position P of the illumination light L. The calculation unit 55 calculates the relationship between the observation position and the target value of the incident position P based on a predetermined observation condition, as indicated by reference numeral D1 in FIG. The above observation conditions are, for example, the refractive index between the sample S and the objective lens 21 (eg, the refractive index of the transparent member, the refractive index of the immersion liquid), the refractive index of the sample S, the type of the objective lens, and the temperature of the environment. Including the thickness of the cover glass 2a. When the user designates the observation position (the position of the front focal plane FP of the sample S of the objective lens 21) and the incident position P of the illumination light L, the user changes the incident angle P at the observation position and acquires a plurality of images. Therefore, it is necessary to determine the optimum incident position P, but in the second embodiment, the labor can be saved. Note that the calculation unit 55 calculates, for example, in the region near the interface Sa between the sample S and the transparent member 2a in consideration of the influence of the aberration, the evanescent field, and the like, as shown by reference numeral D2 in FIG. May be.

[Third Embodiment]
A third embodiment will be described. In the present embodiment, configurations similar to those of the above-described embodiments are designated by the same reference numerals, and description thereof will be omitted or simplified. In the present embodiment, the configuration of the microscope 1 is similar to that of FIG. 1, but the operation of the calculation unit 55 is different from that of the first embodiment.

In the first embodiment, the user specifies the set of the observation position (the position of the front focal plane FP of the sample S of the objective lens 21) and the incident position P of the illumination light L (reference symbols U1, U2, etc.). In the embodiment, it is not necessary to specify the incident position P. In other words, the calculation unit 55 calculates the relationship between the observation position and the incident position P by the user designating the observation position (the position of the front focal plane FP of the sample S of the objective lens 21). For example, when the user specifies the first observation position, the control unit 9 controls the drive unit 52 to move the holding member 51, and the sample S illuminated at a different incident position P at the specified observation position. The image pickup unit 6 is caused to pick up a plurality of images. The calculation unit 55 determines the target value of the incident position P corresponding to the first observation position input by the user, based on the information regarding the light from the sample S detected each time the incident position P is changed. For example, the calculation unit 55 sets the incident position P where the intensity or contrast of the light from the sample S is the largest as the target value of the incident position P at the first observation position. The calculation unit 55 similarly calculates the target value of the incident position P at the second observation position. Further, the calculation unit 55 similarly relates to the light from the sample S detected every time when the incident position P is changed at the observation positions (eg, U3 to U12, etc.) other than the first observation position and the second observation position. The target value of the incident position P may be calculated based on the information. In this case, the relationship between the observation position and the incident position P can be calculated more accurately than in the case of linearly interpolating U1 and U2 to U3 to U12. In the first embodiment, the user had to specify the target value of the incident position P corresponding to the observation position, but in the third embodiment, the calculation unit 55 causes the incident portion corresponding to the observation position specified by the user. Since the position P is calculated, the labor of the user can be saved.

[Fourth Embodiment]
A fourth embodiment will be described. In the present embodiment, configurations similar to those of the above-described embodiments are designated by the same reference numerals, and description thereof will be omitted or simplified. FIG. 7 is a diagram showing the microscope 1 according to the present embodiment. The microscope according to this embodiment is, for example, a microscope that uses a single-molecule localization microscopy method such as STORM and PALM. The microscope according to the embodiment can be used for both fluorescence observation of a sample labeled with one type of fluorescent substance and fluorescence observation of a sample labeled with two or more types of fluorescent substances. Further, the microscope according to the embodiment has, for example, a mode for generating a two-dimensional super-resolution image and a mode for generating a three-dimensional super-resolution image, and can switch between the two modes. It may have only one of the two modes. In the present embodiment, a mode in which there are two types of fluorescent dyes (eg, reporter dyes) used for labeling will be described, but the number of fluorescent dyes (eg, reporter dyes) may be one type, or may be three or more types. ..

The sample may contain living cells (live cells), may contain cells fixed using a tissue fixing solution such as formaldehyde solution, or may be tissue or the like. The fluorescent substance may be a fluorescent dye such as a cyanine dye or a fluorescent protein. The fluorescent dye includes a reporter dye that emits fluorescence when receiving excitation light in an activated state (hereinafter referred to as an activated state). In addition, the fluorescent dye may include an activator dye that receives activation light to activate the reporter dye. If the fluorescent dye does not contain an activator dye, the reporter dye is activated by receiving activating light. The fluorescent pigment is, for example, a dye pair (for example, Cy3-Cy5 dye pair (Cy3, Cy5 is a registered trademark), or a Cy2-Cy5 dye pair (Cy2 and Cy5 are registered trademarks) in which two types of cyanine dyes are combined. , Cy3-Alexa Fluor 647 dye pair (Cy 3, Alexa Fluor is a registered trademark), and one type of dye (eg, Alexa Fluor 647 (Alexa Fluor is a registered trademark)). The fluorescent protein is, for example, PA-GFP or Dronpa.

In the present embodiment, the illumination optical system 4 irradiates the sample S with excitation light of two types of wavelengths depending on the fluorescent substance (eg, reporter dye). The light source device 3 includes an activation light source 11a, an excitation light source 11b, an excitation light source 11c, a shutter 12a, a shutter 12b, a shutter 12c, a mirror 61, a dichroic mirror 62, and a dichroic mirror 63.

The activation light source 11a emits, as illumination light, activation light L0 that activates a part of the fluorescent substance contained in the sample S. Here, it is assumed that the fluorescent substance contains the reporter dye and does not contain the activator dye. The reporter dye of the fluorescent substance is brought into an activated state capable of emitting fluorescence by being irradiated with the activation light L0. The wavelength of the activation light L0 is 405 nm, for example. The fluorescent substance may include a reporter dye and an activator dye, and in this case, the activator dye activates the reporter dye when receiving the activation light L0. The fluorescent substance may be a fluorescent protein such as PA-GFP or Dronpa. The shutter 12a is arranged on the light emitting side of the activation light source 11a. The shutter 12a is controlled by the control unit 9 and can switch between a state of passing the activation light L0 from the activation light source 11a and a state of blocking the activation light L0.

The excitation light source 11b emits the first excitation light L1 having the first wavelength as the illumination light. The shutter 12b is arranged on the light emission side of the excitation light source 11b. The shutter 12b is controlled by the control unit 9 and can switch between a state in which the first excitation light L1 from the excitation light source 11b passes and a state in which the first excitation light L1 is blocked. The excitation light source 11c generates, as illumination light, the second excitation light L2 having a second wavelength different from the first wavelength. Here, it is assumed that the second wavelength is shorter than the first wavelength. For example, the first wavelength is 647 nm and the second wavelength is 561 nm. The shutter 12c is arranged on the light emission side of the excitation light source 11c. The shutter 12c is controlled by the control unit 9 and can switch between a state of passing the second excitation light L2 from the excitation light source 11c and a state of blocking the second excitation light L2.

The mirror 61 is arranged on the light emitting side of the shutter 12c. The second excitation light L2 that has passed through the shutter 12c is reflected by the mirror 61 and enters the dichroic mirror 62. The dichroic mirror 62 is arranged on the light emitting side of the shutter 12b. The dichroic mirror 62 has a characteristic that the second excitation light L2 is transmitted and the first excitation light L1 is reflected. The first excitation light L1 reflected by the dichroic mirror 62 and the second excitation light L2 transmitted through the dichroic mirror 62 enter the dichroic mirror 63 through the same optical path. The dichroic mirror 63 is arranged on the light emitting side of the shutter 12a. The dichroic mirror 63 has a characteristic of reflecting the first excitation light L1 and the second excitation light L2 and a characteristic of transmitting the activation light L0. The first excitation light L1 and the second excitation light L2 are applied to the sample S through the same optical path as the activation light L0.

The angle adjusting unit 7 may adjust the incident angle of the illumination light according to the wavelength of the illumination light, for example. For example, the incident angle may be the optimum incident angle for the activation light L0, the optimum incident angle for the first excitation light L1, the optimum incident angle for the second excitation light L2, or the minimum of these. The angle between the angle and the maximum angle may be the incident angle. When the first excitation light L1 is emitted, the incident angle is set to be the most suitable for the wavelength of the first excitation light L1, and when the second excitation light L2 is emitted, the second excitation light L2 The incident angle may be reset to the optimum wavelength. The activation light L0 does not necessarily have to be obliquely illuminated.

The image processing unit 56 uses the image pickup result of the image pickup unit 6 to perform image processing such as obtaining the barycentric position of each image. The control unit 9 causes the image capturing unit 6 to capture images in a plurality of frame periods, and the image processing unit 56 generates one image by using at least a part of the image capturing results obtained in the plurality of frame periods. For example, the image processing unit 56 calculates, for each of the plurality of captured images, the barycentric position of the fluorescence image (point image) included in each image. For example, the image processing unit 56 calculates the barycentric position by performing Gaussian fitting on the distribution of pixel values in the area corresponding to the point image in the captured image. The image processing unit 56 represents, for example, the barycentric position of the fluorescence image by a bright spot, and uses (at least, merges) at least a part of the plurality of bright spots corresponding to the multiple fluorescence images included in the plurality of captured images. ) Generate one image (eg, super-resolution image).

The first observation optical system 5 includes an astigmatism optical system (for example, a cylindrical lens 65). The cylindrical lens 65 acts on at least a part of the fluorescence from the sample S to generate astigmatism on at least a part of the fluorescence. That is, the astigmatism optical system such as the cylindrical lens 65 causes astigmatism to at least a part of the fluorescence to generate an astigmatic difference. The cylindrical lens 65 is provided so that it can be inserted into and removed from the optical path between the sample S and the imaging section 6 (eg, the imaging element 40). The image processing unit 56 uses, for example, astigmatism to determine the position of the fluorescent substance in the depth direction of the sample S (the optical axis 21a of the objective lens 21, that is, the optical axis 5a direction of the first observation optical system 5). calculate. The image processing unit 56 can calculate the position of the fluorescent substance in the sample S by performing elliptic Gaussian fitting, for example. Note that, as shown in FIG. 7, the cylindrical lens 65 is provided so that it can be inserted and removed.

FIG. 8 is a diagram showing an example of an illumination and imaging sequence according to the present embodiment. FIG. 9 is a diagram conceptually showing an example of an image generated by the image processing unit 56. Note that, hereinafter, the relationship between the observation position and the incident position P shown in FIGS. 4 and 6 will be described as being calculated in advance by the calculation unit 55. The controller 9 moves the stage 2 along the optical axis 21a of the objective lens 21 to set it at a predetermined observation position (Z _{0 in} FIG. 8). Further, the control unit 9 controls the drive unit 52 of the angle adjusting unit 7 by using the relationship between the observation position and the incident position P shown in FIGS. 4 and 6, etc., and the incident position in the first image generation period T1. Is moved to P _0-1 and the holding member 51 is moved so that the incident position is P _0-2 in the second image generation period T2. The control unit 9 executes the imaging sequence as follows at a predetermined observation position (Z _{0 in} FIG. 9).

The control unit 9 emits the first excitation light (first excitation light L1) and does not emit the second excitation light (second excitation light L2) in the image generation period T1. The control unit 9 also causes the activation light L0 to be emitted during the image generation period T1. Further, the control unit 9 causes the image capturing unit 6 to capture an image in each of the plurality of frame periods Tf of the image generation period T1 (first image capturing process in the figure; ON). The controller 9 emits the second excitation light and does not emit the first excitation light in the image generation period T2 subsequent to the image generation period T1. The control unit 9 also causes the activation light L0 to be emitted during the image generation period T2. Further, the control unit 9 causes the image capturing unit 6 to capture an image in each of the plurality of frame periods Tf of the image generation period T2 (second image capturing process in the figure; ON). The intensity of the activation light L0 is adjusted according to the fluorescent substance, for example. For example, the intensity of the activation light L0 is set to be higher when irradiating the second excitation light than when irradiating the first excitation light. For example, the control unit 9 controls the acoustooptic device 13 and adjusts the intensity of the activation light.

The image processing unit 56 generates the first image Pa ₀ using at least a part of the imaging result (eg, captured image) obtained by the plurality of first imaging processes in the image generation period T1. In addition, the image processing unit 56 generates the second image Pb ₀ by using at least a part of the imaging result (eg, captured image) obtained by the plurality of second imaging processes in the image generation period T2. Two types of fluorescent substances (eg, reporter dyes) are labeled on different organs in the sample S, and images of different organs are obtained between the first image Pa ₀ and the second image Pb _0. .. The image processing unit 56 may be, for example, by combining the first image Pa ₀ and the second image Pb _0, to generate an image Pt ₀ of one as shown in FIG. 9 (C).

The image processing unit 56 may generate one image Pt ₀ without using the first image Pa ₀ and the second image Pb ₀ . For example, the image processing unit 56 uses at least a part of the imaging results obtained by the plurality of first imaging processes and at least a part of the imaging results obtained by the second imaging processes to form one image Pt ₀ . May be generated. Further, the image processing unit 56 does not have to generate the image Pt ₀ .

Next, the control unit 9 moves the stage 2 along the optical axis 21a of the objective lens 21 to set it at the next observation position (Z _{1 in} FIG. 8). Further, the control unit 9 controls the drive unit 52 of the angle adjusting unit 7 by using the relationship between the observation position and the incident position P shown in FIGS. 4 and 6, etc., and the incident position in the first image generation period T1. Is moved to P _1-1 , and the holding member 51 is moved so that the incident position is P _1-2 in the second image generation period T2. The control unit 9 also executes the above-described imaging sequence at this observation position, and obtains the first image Pa ₁ , the second image Pb ₁ , and the image Pt ₁ . Thus, as shown in FIG. 8, in the observation position _Z 0 to Z _n respectively, the first image _Pa 0 _{~Pa n,} the second image _Pb 0 _{~Pb n} image Pt ₀ ~Pt _n obtained.

For example, the image processing unit 56, as shown in FIG. 9 (A), using the first image Pa ₀ ~Pa _n, it is possible to generate a three-dimensional picture Pa. The image processing unit 56, as shown in FIG. 9 (B), using the second image Pb ₀ ~Pb _n, it is possible to generate a three-dimensional picture Pb. The image processing unit 56 can generate a three-dimensional image (eg, the three-dimensional image Pt in FIG. 9C) by combining the three-dimensional image image Pa and the three-dimensional image image Pb, for example. Further, as shown in FIG. 9C, the image processing unit 56 can generate the image Pt ₀ using the first image Pa ₀ and the second image Pb ₀ , and similarly, the image Pt ₁ ~ Pt _n can be generated. The image processing unit 56 can also generate the three-dimensional image image Pt using the images Pt _{0 to} Pt _n . Note that the first image Pa ₀ ~Pa _n and the second image Pb ₀ ~Pb _n respectively, is a three-dimensional image, if it retracts the cylindrical lens 65 from the optical path in FIG. 7, a two-dimensional image Become.

In this embodiment, since the incident angle of the excitation light (first excitation light, second excitation light) to the sample is optimally set at each of the observation positions Z _{0 to} Z _n , it is possible to obtain a highly accurate image. it can.

In the present embodiment, by operating the stage 2, in each viewing position Z ₀ to Z _n, but executes the image generation period T1 and the image generation period T2, at each viewing position Z ₀ to Z _n, Only the image generation period T1 may be executed. In this case, the stage 2 may be operated again to execute only the image generation period T2 at each of the observation positions Z _{0 to} Z _n .

In single-molecule localization microscopy such as STORM and PALM, which can generate high-resolution images, the incident angle of the illumination light to the sample is changed when at least one of the stage and the objective lens of the illumination light is moved. Varying is particularly useful. If the incident angle of the illumination light with respect to the sample is not changed, the resolution may decrease, but by appropriately changing the incident angle of the illumination light with respect to the sample as in the present embodiment, single-molecule localization In Microscopy, for example, it is possible to suppress the distortion of the distribution of the light intensity corresponding to the fluorescence image. Therefore, for example, the barycentric position of the fluorescence image can be obtained with high accuracy, and a cross-sectional image of a thick sample can also be obtained with high resolution.

[Fifth Embodiment]
A fifth embodiment will be described. In the present embodiment, configurations similar to those of the above-described embodiments are designated by the same reference numerals, and description thereof will be omitted or simplified. In this embodiment, the configuration of the microscope 1 is the same as that of FIG. 1, but the operation of the control unit 9 is different from that of the first embodiment. In the present embodiment, the control unit 9 causes the imaging unit 6 to capture a plurality of images at different incident angles when moving at least one of the stage 2 and the objective lens 21. For example, the control unit 9 causes the image capturing unit 6 to capture one or two or more images for one incident angle in a state where the stage 2 and the objective lens 21 are arranged at the first relative position, and a plurality of images are acquired. The imaging unit 6 is caused to capture one or more images for each incident angle. The control unit 9 may determine the incident angle corresponding to the first relative position based on a plurality of images with different incident angles. For example, the control unit 9 may employ, as the incident angle at the first relative position, the incident angle corresponding to the image having the highest luminance value among the plurality of images having different incident angles. Further, the image processing unit 56 may select an image used for image processing from a plurality of images having different incident angles. For example, the image processing unit 56 selects an image having a luminance value within a predetermined range from a plurality of images having different incident angles, and performs image processing using only the selected image (for example, the first image shown in FIG. 8). Generation process of the image Pa ₀ of 1) may be performed.

The control unit 9 also captures one or more images for one incident angle in a state where the stage 2 and the objective lens 21 are arranged at a second relative position different from the first relative position. The image capturing unit 6 may capture an image, and the image capturing unit 6 may capture one or two or more images for each of a plurality of incident angles. The image processing unit 56 selects an image having a luminance value within a predetermined range from a plurality of images having different incident angles, and performs image processing using only the selected image (eg, the first image shown in FIG. 8). The process of generating the image Pa ₁ ) may be performed. Further, the control unit 9 may determine the incident angle corresponding to the second relative position as well as the first relative position with respect to the second relative position. Further, the calculation unit 55 uses the incident angle for the first relative position and the incident angle for the second relative position to determine the third angle between the first relative position and the second relative position. The incident angle for the relative position may be calculated.

In the above embodiment, the control unit 9 includes, for example, a computer system. The control unit 9 reads a program stored in the storage device 43 and executes various processes according to the program. This program includes, for example, an illumination optical system that obliquely illuminates the sample with illumination light, and an observation optical system having an objective lens. At least one of the stage and the objective lens that holds the sample is an objective lens. Is a control program that causes a computer to execute control of a microscope that can move in the same direction as the optical axis of the control, and the control is to change the incident angle of the illumination light with respect to the sample when at least one of the stage and the objective lens is moved. including. This program may be provided by being recorded in a computer-readable storage medium.

Note that the technical scope of the present invention is not limited to the modes described in the above embodiments and the like. One or more of the requirements described in the above embodiments and the like may be omitted. Further, the requirements described in the above-described embodiments and the like can be combined as appropriate. Further, as far as legally permitted, the disclosure of all documents cited in the above-described embodiments and the like is incorporated as a part of the description of the text.

DESCRIPTION OF SYMBOLS 1... Microscope, 4... Illumination optical system, 4a... Optical axis, 5... Imaging optical system, 7... Angle adjustment part, 9... Control part, 16... Guide Optical member, 21... Objective lens, 45... Input device, 52... Driving unit, 55... Calculation unit, FP1... Focal plane, FP2... Focal plane, L... Illumination Light, LF1... Teruno, LF2... Teruno, LF3... Teruno, PU1... Pupil plane, PU2... Pupillary conjugate plane, S... Sample

Claims (11)

An illumination optical system that illuminates the sample with illumination light,
An observation optical system having an objective lens,
An adjusting unit that adjusts an incident angle of the illumination light with respect to the sample,
A control unit,
A microscope comprising:
The control unit is
Controlling at least one of the stage holding the sample and the objective lens to change the distance between the stage and the objective lens,
Changing the incident angle of the illumination light with respect to the sample by controlling the adjustment unit,
The first observation position of the sample based on the information about the incident angle of the sample with respect to the first observation position and the information about the incident angle of the sample with respect to the second observation position different from the first observation position. And a microscope for determining information about an incident angle with respect to a third observation position different from the second observation position. The control unit interpolates information about an incident angle of the sample with respect to the first observation position and information about an incident angle of the sample with respect to the second observation position to the third observation position of the sample. The microscope according to claim 1, wherein information regarding an incident angle is determined. The microscope according to claim 2, wherein the interpolation is linear interpolation. The microscope according to claim 2, wherein the interpolation is non-linear interpolation. The illumination optical system includes the objective lens, and illuminates the illumination light to the sample via the objective lens.
The microscope according to any one of claims 1 to 4. The microscope according to claim 5, wherein the information regarding the incident angle is an incident position of the illumination light on a pupil plane of the objective lens. The control unit controls the adjusting unit to change the incident angle of the illumination light with respect to the sample based on information on the determined incident angle of the sample with respect to the third observation position. The microscope according to any one of 1. Further comprising an imaging unit that captures an image formed by the observation optical system to form an image,
The said control part determines the information regarding the incident angle with respect to the said 1st observation position of the said sample, and the information regarding the incident angle with respect to the said 2nd observation position of the said sample based on the said image. The microscope according to any one of items. The first observation position, the second observation position, and the third observation position of the sample are positions different from each other in the optical axis direction of the objective lens. Microscope. An illumination optical system that illuminates the sample with illumination light,
An observation optical system having an objective lens,
In a microscope equipped with
By changing the distance between the stage holding the sample and the objective lens,
Changing the incident angle of the illumination light with respect to the sample,
The first observation position of the sample based on the information about the incident angle of the sample with respect to the first observation position and the information about the incident angle of the sample with respect to the second observation position different from the first observation position. And an observation method for determining information about an incident angle with respect to a third observation position different from the second observation position. A control program that causes a computer to execute control of a microscope including an illumination optical system that illuminates a sample with illumination light, an observation optical system having an objective lens, and an adjusting unit that adjusts an incident angle of the illumination light with respect to the sample. And
The control controls at least one of a stage holding the sample and the objective lens to change a distance between the stage and the objective lens, and controls the adjusting unit to make an incident angle of the illumination light with respect to the sample. Change
The first observation position of the sample based on the information about the incident angle of the sample with respect to the first observation position and the information about the incident angle of the sample with respect to the second observation position different from the first observation position. And a control program for determining information about an incident angle with respect to a third observation position different from the second observation position.

Images