JP2016109731A - Illumination optical system, illumination optical device, and microscope having the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope capable of quickly and appropriately adjusting light irradiated on a specimen.SOLUTION: An illumination optical system includes reflection means including a reflective surface having a shape of a part of an elliptic cylinder surface, and splitting means configured to split light at a first focal position of the elliptic cylinder surface. The reflection means reflects the light split by the splitting means toward a second focal position different from the first focal position of the elliptic cylinder surface.SELECTED DRAWING: Figure 2

Description

  The present invention relates to an illumination optical system, an illumination optical apparatus, and a microscope using the same.

  Patent Document 1 proposes a three-dimensional microscope (digital microscope) of an asymmetric lattice illumination (ALI). This microscope illuminates the entire field of view of the sample in two different illumination modes, and extracts only the fluorescence information emitted by the sample located near the desired focal plane by processing the image data acquired for each illumination mode. To do. In order to obtain a three-dimensional stereoscopic image, the focus position of the objective lens is changed by moving the microscope stage up and down.

  In one illumination mode, the fluorescent sample is illuminated with excitation light having a uniform intensity distribution, and in another illumination mode, the sample is illuminated with excitation light having a periodic intensity distribution such as interference fringes. In ALI, the intensity distribution of excitation light is not uniform along the optical axis direction and has a finite period in the optical axis direction, thereby improving the resolution in the optical axis direction.

JP2014-44254A

  In order for the intensity distribution generated in the two-beam interference to have a finite period in the optical axis direction, the values of the optical axis direction components of the wave number vectors of the two parallel lights may be different. The wave vector of two parallel lights suitable for improving the resolution in the optical axis direction differs depending on the sample in addition to the specifications of the imaging optical system such as an objective lens and a digital camera. Therefore, there is a demand for a simple illumination optical system configuration that can freely and quickly adjust the wave vector of two parallel lights. Further, the illumination optical system needs to be able to smoothly switch to a mode in which the fluorescent sample is illuminated with excitation light having a uniform intensity distribution.

  By adjusting the mirror and half mirror of the Mach-Zehnder interference system, the wave vector of two parallel lights can be set to a suitable one. However, at the time of the adjustment, the overlapping position of the two plane waves is shifted in the direction perpendicular to the axis of the light to be irradiated onto the microscope (the optical axis from the aperture stop to the microscope main body) and deviates from the camera port of the microscope. was there.

  In the Mach-Zehnder interference system, half of the total amount of light is lost when the final multiplexing is performed by the half mirror. Furthermore, if the mode in which the fluorescent sample is illuminated with excitation light having a uniform intensity distribution is realized by shielding one of the optical paths of the Mach-Zehnder interference system, the amount of excitation light is halved.

  Therefore, an object of the present invention is to provide an illumination optical system, an illumination device, and a microscope using the illumination optical system that can quickly and appropriately adjust the irradiation light to the sample.

  An illumination optical system according to one aspect of the present invention includes a reflecting unit including a reflecting surface having a shape of a part of an elliptic cylindrical surface, and a dividing unit that splits light at a first focal position of the elliptic cylindrical surface. . The reflecting means reflects the light divided by the dividing means toward a second focal position different from the first focal position on the elliptical cylindrical surface.

  The present invention can provide an illumination optical system, an illumination device, and a microscope using the illumination optical system capable of quickly and appropriately adjusting the light irradiated to the sample.

It is a figure which shows the light source arrangement | positioning in an objective-lens pupil conjugate surface. It is the schematic of the illumination optical system in this embodiment. 2 is an optical path diagram of the illumination optical system in Embodiment 1. FIG. 6 is an optical path diagram of the illumination optical system in Embodiment 2. FIG. 6 is an optical path diagram of an illumination optical system in Embodiment 3. FIG.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  The present invention can be applied to a purpose of three-dimensionally observing a thick sample fluorescently stained using a digital slide scanner or the like with a microscope. A digital slide scanner is a device that scans a preparation used in biology, pathological examination, and the like at high speed and converts it into high-resolution digital data. The present invention can be applied to an asymmetric grating illumination system (ALI).

  ALI includes an illumination mode in which a fluorescently stained sample is illuminated by an interference pattern created by two parallel light beams coherent with each other. In order to increase the contrast of the interference pattern by aligning the direction of the linearly polarized light (polarization direction) of the two parallel lights, the two parallel lights are set to s waves or TE (Transverse Electric) waves. Inevitably, then, the two wave number vectors representing these parallel lights are represented as two points on the diameter on the pupil conjugate plane of the objective lens (objective optical system) (FIG. 1). Here, FIG. 1 is a diagram showing a light source position on the pupil plane of the objective lens or a plane optically conjugate with the pupil plane (hereinafter sometimes simply referred to as the pupil plane).

  In FIG. 1, two black dots (light sources) 1 and 2 correspond to two wave number vectors representing two coherent parallel lights. The horizontal axis and the vertical axis can be taken arbitrarily, and the radius of the pupil represented by a circle is 1. When the wave number vector of the excitation light is k = 2π / λ using the wavelength λ of the excitation light and the numerical aperture of the objective lens is expressed as NA, the radius of the pupil corresponds to kNA.

In FIG. 1, if the wave number vector representing the excitation parallel light indicated by the point 1 is (ksin θ ₁ , 0, kcos θ ₁ ), the coordinates of the point 1 are (sin θ ₁ / NA, 0). Similarly, the coordinates of the point 2 are (sin θ ₂ / NA, 0). When such two equal intensity light source points are arranged on the pupil conjugate plane of the objective lens, the fluorescently stained sample is illuminated with the intensity distribution I (x, y, z) expressed by Equation 1.

In Equation (1), the horizontal coordinate is x and the optical axis coordinate is z. I (x, y, z) does not depend on the vertical coordinate y. In ALI, the intensity distribution of excitation light is not uniform along the optical axis direction and has a finite period in the optical axis direction. For this purpose, the absolute value of θ _{1 and} the absolute value of θ ₂ are different. There is a need. At this time, in FIG. 1, the points 1 and 2 are arranged asymmetrically with respect to the vertical axis, and the direction of the vertical axis is the electric field oscillation direction common to the two parallel lights. The set of values of θ suitable for performing ALI differs depending on the specifications of the optical system, which is the main factor, as well as the sample and other experimental conditions. In any case, it is necessary to make an asymmetric light source arrangement as shown in FIG. 1 on a plane optically conjugate with the pupil of the objective lens of the microscope.

  Next, the configuration of the illumination optical apparatus 100 in the present embodiment will be described.

  FIG. 2 is a schematic diagram of the illumination optical apparatus 100 in the present embodiment, and the dotted line indicates the optical path of the excitation light beam. The light source unit 400 includes a coherent light source body 401, a collimation lens 402, and a beam expander 403. The coherent light source body 401 is, for example, various lasers that emit light having an excitation wavelength, and emits linearly polarized light in a direction perpendicular to the paper surface. The collimation lens 402 can be adjusted so that parallel light is formed at the position of the stop 408 by changing the focus position. The beam expander 403 changes the magnification to minimize the aberration when the excitation light beam emitted from the coherent light source body 401 is reflected by the inner surface (elliptical cylinder surface) of the elliptic cylinder mirror 404. The diameter can be kept thin. The collimation lens 402 and the beam expander 403 are electrically controlled by the light source adjustment mechanism 413 according to an instruction from the control unit 410.

  The elliptical cylindrical mirror 404 has an elliptical cylindrical shape that extends in the direction perpendicular to the plane of the paper, similar to the linear polarization of the excitation light beam. Therefore, the elliptical cylindrical mirror 404 has a focal axis (focus group) consisting of focal points that are continuous in the direction perpendicular to the paper surface. Since the ellipse has two focal points, the elliptical cylindrical mirror 404 also has two focal axes.

  The half mirror 405 (light splitting means) is disposed on the first focal axis of the elliptical cylindrical mirror 408 so that it can be inserted and removed in the optical path, and the first focal axis (first of the reflecting surface (split surface)). Can be rotated around the focal point). The light source unit 400 has an arc-shaped rail centered on the first focal axis, and the coherent light source body 401 can be driven along with the light source unit 400 along this rail. In the present embodiment, the half mirror 405 or the light source unit according to an instruction from the control unit 410 so that the incident angle of the excitation light beam from the coherent light source body 401 to the half mirror 405 is always 45 degrees (predetermined angle). 400 is electrically driven. The half mirror 405 is driven by a mirror driver 412 (rotation control means) according to an instruction from the control unit 410, and the light source unit 400 is driven by a light source adjustment mechanism 413 (light source driving mechanism) according to an instruction from the control unit 410. The Thereby, the branching ratio of the excitation light beam at the first focal axis can be kept one-to-one. It should be noted that the branched excitation light beam only needs to have a uniform intensity when irradiating the sample, and is not limited to these configurations as long as the branching ratio of the excitation light beam at the first focal axis can be kept constant. . Further, the control unit 410 may control the mirror driver 412 and the light source adjustment mechanism 413 in conjunction with each other by feeding back information on the rotational position of the half mirror 405 and the position of the light source unit 400.

  The excitation light beam branched (divided) by the half mirror 405 is reflected inside the elliptical cylindrical mirror 404 and intersects at the second focal point of the elliptical cylindrical mirror 404. At this time, the two wave number vectors of the two branched excitation light beams (parallel light) are asymmetric with respect to a straight line connecting the first focus and the second focus. The two beams that have passed through the second focal point are guided to the opening of the stop 408 via the lens 406 (optical member) and the variable magnification beam expander 407. The lens 406 can be moved on a straight line connecting the first focus and the second focus, thereby changing the angle at which the two excitation light beams (parallel light) intersect at the aperture 408 opening. . The variable magnification beam expander 407 adjusts the beam diameter so that the interference pattern covers the entire field of view of the sample to be observed. The variable magnification beam expander 407 and the lens 406 are electrically driven by a lens driver 411 (incident angle adjusting means) in response to an instruction from the control unit 410. The positions of the lens 406 and the variable magnification beam expander 407 may be manually adjusted.

  According to the illumination optical system of the present embodiment, it is possible to form a state in which two wave vectors of two parallel lights coherent with each other at a position of the stop 408 in FIG.

  In the present embodiment, a surface optically conjugate with the sample (object surface) in the microscope (for example, the position of the diaphragm 211 in FIG. 3, the image sensor 209 in FIG. 4, the position of the diaphragm 306 in FIG. 5) and the position of the diaphragm 408 coincide. As described above, a part of the configuration of a general fluorescence microscope is replaced with the illumination optical device 100 of FIG.

  In the conventional method using the Mach-Zehnder interference system, when the angle of the mirror or the half mirror is changed, the crossing position of the two parallel lights is the axis of light to be irradiated onto the microscope (the optical axis from the stop 408 to the microscope main body). It shifts to the perpendicular direction to. For this reason, it has been difficult to adjust the mirror or the like so that the two parallel lights are guided to the aperture opening. On the other hand, if the illumination optical system in the present embodiment is used, the two parallel lights intersect on the axis of the light to be irradiated on the above-described microscope regardless of the inclination of the half mirror 405, so It will be much easier to adjust. Further, the Mach-Zehnder interference system has problems such that the intensity of the two excitation light beams becomes non-uniform through the half mirror, and most of the light amount is lost by the half mirror. On the other hand, the illumination optical system of the present embodiment overcomes these problems by minimizing the use of half mirrors and keeping the branching ratio of the excitation light beam at the first focal axis constant.

  In addition to the illumination mode using an interference pattern, ALI also requires an illumination mode having a uniform intensity distribution. Considering that the incident angle of illumination is not a problem because the fluorescently stained sample is a self-luminous object, it is only necessary to block one of the two excitation light beams. However, in this case, the amount of light is half that of the illumination mode using the interference pattern, and the image becomes dark. When the image is dark, the image quality of the digital image deteriorates due to the occurrence of shot noise. Therefore, if the half mirror 405 is vertically lifted and pulled out of the optical path, the amount of light from the light source can be used effectively even in the illumination mode having a uniform intensity distribution. The half mirror 405 may be inserted into and removed from the optical path by the mirror driver 412.

  As described above, the illumination optical system according to the present embodiment satisfies the requirements of ALI and is easy to adjust. Therefore, when used as an illumination optical system of a normal fluorescent microscope, the image quality and operation speed of ALI are reduced. It can be greatly improved.

  Hereinafter, specific examples in the case where the illumination optical system in the present embodiment is applied to a general fluorescence microscope will be described using Examples 1 to 3.

  Hereinafter, a specific configuration in which the above-described illumination optical apparatus 100 is applied to the epi-fluorescent microscope will be described in detail with reference to FIG. Example 1 shows a configuration in which a general illumination optical device in an epi-illumination fluorescence microscope is replaced with the illumination optical device 100 shown in FIG. In FIG. 3, the control unit 410, the lens driver 411, the mirror driver 412, and the light source adjustment mechanism 413 are omitted.

  Fluorescence microscopes often have two locations that can serve as entrances for excitation light. The sample 202 includes a fixing liquid, a cover glass, etc. (object surface) in addition to the sample main body, and is disposed on the slide glass 201. Since the diaphragm 211 is an optically conjugate surface with the sample 202 (object surface), two coherent excitation light beams are incident on this position so as to intersect at a desired angle. The excitation light beam whose beam diameter is determined by the variable magnification beam expander 407 is bent by the lens 210 and then passes through the filter cube 207. The filter cube 207 selects and reflects only the light having the excitation wavelength. The excitation light beam reflected by the filter cube 207 changes the traveling direction by 90 degrees, and is then irradiated on the sample 202 as parallel light by the objective lens 203 (objective optical system). The objective lens 203 is set to NA 0.95, which is a typical specification in biological observation, and a magnification of 40 times.

  Upon receiving the excitation light beam, the sample 202 emits fluorescence (including phosphorescence, the same applies hereinafter). The fluorescence becomes a parallel light flux after passing through the objective lens 203. Here, the light from the sample 202 includes not only fluorescence but also excitation light reflected by a cover glass or the like, but these lights are reflected by the filter cube 207, and only the fluorescence goes straight. The reflected return excitation light returns to the light source by changing the traveling direction by 90 degrees. The parallel fluorescent light beam is divided into two hands by the half mirror 200. One of them is changed in the traveling direction by the mirror 204 and then imaged on the first image sensor 206 by the imaging lens 205. For example, a CCD or a CMOS sensor can be used as the imaging element. In a normal fluorescent microscope, this location is the first camera port. If a half mirror is inserted between the imaging lens 205 and the image sensor 206 and the optical path is bent 90 degrees and then converted into parallel light by an eyepiece, it becomes a so-called binocular portion for visual observation. The other of the fluorescence branched by the half mirror 200 is imaged on the second image sensor 209 by the imaging lens 208. This location is the second camera port.

As described above, the objective lens 203 is set to NA 0.95 and magnification 40 times. When using this aperture, the theoretical considerations of ALI, conditions resolution of the optical axis direction is maximum, coordinates (sinθ ₁ / NA, 0) of point 1 in FIG. 1, a coordinate point 2 (sin [theta ₂ / NA, 0), sin θ ₁ = −0.19 and sin θ ₂ = 0.76.

The objective lens of a microscope is usually designed so as to satisfy the sine condition expressed by Equation (2) in order to correct aberrations well. In Equation (2), M is the magnification of the objective lens, θ _i represents the maximum incident angle on the light source side, and θ _o represents the maximum incident angle on the object side.

That is, when a light beam is to be incident on the object plane at an angle θ _o , it is necessary that the light source be incident at a shallow angle by a factor of the objective lens magnification. In the first embodiment, the ratio is 1/40, and the above-described conditions of sin θ ₁ = −0.19 and sin θ ₂ = 0.76 are values on the object side, so that sin θ ₁ = −0.00475 on the light source side. , Sin θ ₂ = 0.0190, a shallow incident angle must be realized. In the following, it is shown that the illumination optical system of the present invention can realize such a shallow angle without any practical trouble.

  In FIG. 3, the straight line connecting the first focal point and the second focal point of the elliptical cylindrical mirror 404 on which the half mirror 405 is installed is the axis of light to be irradiated onto the microscope (the optical axis from the stop 211 to the microscope main body). Match the direction.

  In Example 1, when the first focal point is the origin and the positive distance on the optical axis is defined as the direction approaching the microscope main body, the surface vertex closer to the first focal point of the elliptical cylindrical mirror 404 is −5 mm. And When the distance variable in the optical axis direction is z, the distance from the optical axis in the paper is y, and the unit of the distance is millimeter, a mathematical expression representing this elliptical cylindrical mirror can be expressed as, for example, mathematical expression (3). .

  At this time, the distance between the focal points of the ellipse is 200 mm. Since the elliptical cylindrical mirror 404 is uniform in the direction perpendicular to the paper surface (assumed to be the x direction), the variable x does not appear in Equation 3. The elliptic cylindrical mirror 404 is configured so as not to prevent the excitation light beam from the light source unit 400 from entering and the excitation light beam from the lens 406 to exit.

  In FIG. 3, the light source unit 400 emits light having an excitation wavelength suitable for a fluorescent dye, and irradiates the half mirror 405 with parallel light whose angle formed by incident light with respect to the optical axis is 70.4 degrees. The half mirror 405 is inclined 25.4 degrees with respect to the optical axis, and parallel light from the light source is incident on the half mirror 405 at an angle of 45 degrees. The excitation light beam branched into two is collected at the second focal point of the elliptical cylindrical mirror 404.

In the first embodiment, two lenses for converting the angular magnification are inserted in order to achieve a shallow incident angle of sin θ ₁ = −0.00475 and sin θ ₂ = 0.0190 on the light source side. For example, a lens having a focal length of 306.4 mm and a focal length of 436.4 mm may be disposed 30 mm from the second focal point of the elliptical cylindrical mirror 404.

  The target interference pattern can be formed on the object plane by the illumination optical system. Although only the ratio of the focal lengths of the two lenses is related to the angular magnification conversion, the above-described values are shown as examples in the first embodiment in order to have the role of a collimator. In this case, the distance from the top focal point of the elliptical cylindrical mirror 404 to the stop 211 is about 1.2 m. By inserting a folding mirror or the like into the optical path, for example, the illumination unit of the confocal microscope It is possible to fit in a similar space.

  In the first embodiment, only numerical examples in the case where the resolution in the optical axis direction is maximized are shown. However, even if the values are other values, it is possible to easily provide a target illumination optical system by adjusting each optical element. Can do.

  Hereinafter, a specific configuration in which the above-described illumination optical apparatus 100 is applied to the epi-fluorescent microscope will be described in detail with reference to FIG. The description of the same components as those in the first embodiment is omitted. In FIG. 4, the control unit 410, the lens driver 411, the mirror driver 412 and the light source adjustment mechanism 413 are omitted.

  The second embodiment shows a configuration in which the imaging unit arranged in the second camera port is replaced with the illumination optical device 100 shown in FIG. 2 while maintaining a general illumination optical device in the epi-fluorescence microscope. . In addition, it is good also as replacing the imaging unit in another camera port with the illumination optical apparatus 100 instead of the imaging unit in a 2nd camera port.

  In FIG. 4, as a general illumination optical device, an excitation light source 215, a concave mirror 216 that reflects the excitation light from the excitation light source 215, a condenser lens 214 that condenses the excitation light, and an aperture stop that adjusts the illumination-side numerical aperture NA. Reference numeral 213 denotes a lens 212 that uses excitation light as parallel light.

  Since the position where the image sensor 209 is disposed is optically conjugate with the object surface, a diaphragm 408 is provided at this position, and two coherent parallel lights are made to cross and enter at a desired angle. Note that when the illumination optical apparatus 100 is operating, the above-described general illumination optical apparatus is not used. In FIG. 4, the image sensor 309 is shown, but this is only shown for easy understanding of the arrangement of the diaphragm 408, and is actually removed.

  The excitation light beam from the illumination optical system of the present invention is reflected by the half mirror 200 and travels toward the sample 202. In the second embodiment, the filter cube 207 is removed, and the excitation light beam reaches the sample 202 directly. This is because, in general, the filter cube 207 is optimized so as to transmit the fluorescence as much as possible, so that the excitation light beam is not necessarily transmitted to the sample side to the maximum.

  However, in this case, the light from the sample 202 reaches the image sensor 206 without removing the light reflected by the cover glass or the like. Therefore, a barrier filter 600 that cuts off light having an excitation wavelength and transmits only fluorescence is inserted between the imaging lens 205 and the image sensor 206. Thereby, the contrast of weak fluorescence can also be reproduced.

  According to the second embodiment, the ALI can be realized without removing the illumination optical device provided in the microscope main body. Therefore, even when it is desired to use it as a normal fluorescent microscope instead of the ALI, it is easier to restore the original state than the first embodiment. Less.

  Hereinafter, a specific configuration in which the illumination optical device 100 described above is applied to a transmission fluorescent microscope will be described in detail with reference to FIG. Example 3 shows a configuration in which a general illumination optical device in a transmission fluorescent microscope is replaced with the illumination optical device 100 shown in FIG. The description of the same configuration as in the first and second embodiments is omitted. In FIG. 5, the controller 410, the lens driver 411, the mirror driver 412, and the light source adjustment mechanism 413 are omitted.

  The sample 305 includes a fixing liquid, a cover glass, and the like (object surface) in addition to the sample main body, and is disposed on the slide glass 304. Since the stop 306 is an optically conjugate surface with the object, two coherent excitation light beams are incident on this position so as to intersect at a desired angle. The excitation light that has passed through the diaphragm 306 irradiates the sample 305. The sample 305 that has been irradiated with the excitation light beam emits fluorescence. The fluorescent light passes through the slide glass 304 and the objective lens 303 (objective optical system), becomes a parallel light beam, and is imaged on the image sensor 301 by the imaging lens 302. A barrier filter 700 is inserted between the imaging lens 302 and the image sensor 301 to transmit only fluorescence. If a half mirror is inserted between the imaging lens 302 and the barrier filter 700 and the optical path is bent 90 degrees and then converted into parallel light by an eyepiece lens, a so-called binocular portion for visual observation is obtained.

  According to the third embodiment, since the sample 305 may be directly irradiated without passing through the objective lens, the restriction of Expression (2) is not imposed. Therefore, since the incident angle can be kept relatively large, the lens can be reduced and a compact configuration can be realized.

  The preferred embodiments of the present invention have been described above, but the present invention is not limited to these embodiments, and various modifications and changes can be made within the scope of the gist.

  The illumination optical system of the present invention can be suitably used in a fluorescence microscope or a digital slide scanner, and is very useful in the field of biological / pathological observation.

100 Illumination optical device 404 Elliptical cylinder mirror 405 Half mirror

Claims (11)

A reflecting means including a reflecting surface having a shape of a part of an elliptic cylinder surface;
Splitting means for splitting light at the first focal position of the elliptical cylindrical surface,
The illumination optical system characterized in that the reflecting means reflects the light divided by the dividing means toward a second focal position different from the first focal position on the elliptic cylindrical surface.   2. The illumination optical system according to claim 1, wherein the reflection unit is arranged such that the second focal position is optically conjugate with an object plane of a microscope. The dividing means has a dividing surface that reflects and transmits light,
3. The illumination optical system according to claim 1, wherein the dividing surface is rotatable about a focal axis of the elliptical cylinder surface including the first focal position.   4. The illumination optical system according to claim 3, further comprising a rotation control unit that rotates the dividing surface of the dividing unit so that an incident angle of light from a light source with respect to the dividing unit maintains a predetermined angle. system.   5. The light source driving mechanism according to claim 1, further comprising a light source driving mechanism that drives the light source so that an incident angle of light from the light source with respect to the dividing unit is maintained at a predetermined angle. Lighting optics.   The illumination optical system according to any one of claims 1 to 5, wherein the dividing unit is insertable into and removable from an optical path of light from a light source.   The illumination according to any one of claims 1 to 6, further comprising an optical member that irradiates light passing through the second focal position to a position optically conjugate with an object plane of the microscope. Optical system.   The illumination optics according to claim 7, further comprising incident angle adjusting means for adjusting an incident angle of the light passing through the second focal position with respect to the conjugate position by driving the optical member. system.   9. The light according to claim 1, wherein the light from the light source is linearly polarized light whose polarization direction is parallel to a focal axis of the elliptic cylindrical surface including the first focal position. Illumination optical system. The illumination optical system according to any one of claims 1 to 9,
An illumination optical device comprising: a light source unit disposed so that a light source is positioned at a position optically conjugate with an object plane of a microscope. The illumination optical system according to any one of claims 1 to 10,
An objective optical system for observing a sample placed on the object surface irradiated by the illumination optical system.