JP5603761B2 - Illumination optics for fluorescent microscopes - Google Patents

Description

  The present invention relates to an illumination optical system for a fluorescence microscope, and more particularly to an illumination optical system for a fluorescence microscope provided with an optical integrator.

Critical illumination and Koehler illumination are known as typical illumination methods for microscopes.
Critical illumination is an illumination method for forming a light source image on a sample. In the critical illumination, bright illumination can be realized, but since the illuminance distribution of the light source image becomes the illuminance distribution on the sample as it is, it is difficult to uniformly illuminate the sample. On the other hand, Koehler illumination is an illumination method that forms a light source image at the pupil position of an objective lens. In Koehler illumination, the light source image is not projected onto the sample surface, and the illuminance distribution on the sample does not depend on the luminance distribution of the light source, so that the sample can be illuminated more uniformly.

  In the microscope, since uniformity of illumination is generally more important, an illumination optical system that realizes Koehler illumination or an illumination optical system that realizes by arbitrarily switching between Koehler illumination and critical illumination is widely adopted.

  The illumination optical system that realizes Koehler illumination adjusts the range of the illumination area (hereinafter referred to as the illumination range) with a field stop provided at a position conjugate with the sample surface, and is located at a position conjugate with the pupil of the objective lens. With the aperture stop provided, the illumination intensity per unit area in the illumination area (hereinafter simply referred to as illumination intensity) can be adjusted without changing the illumination range. However, adjustment of the illumination range and illumination intensity by the field stop or aperture stop interrupts a part of the luminous flux of the illumination light, and thus a loss of light quantity is inevitable.

  As an effective technique for such a problem, for example, as disclosed in Patent Document 1, a technique for changing a light beam diameter between a light source unit and a projector tube, that is, a light source to the pupil of an objective lens. A technique for changing the projection magnification is known.

  FIG. 7A and FIG. 7B are diagrams for explaining the prior art for changing the beam diameter between the light source unit and the light projection tube. The illumination optical system 100 illustrated in FIGS. 7A and 7B includes a light source unit 110 including a light source 111 and a collector lens 112, a light projection tube unit 120, and an objective lens 130. The zoom lens unit 140 includes a variable power lens unit 140 that is inserted into an optical path between the light source unit 110 and the light projecting tube unit 120 to change the diameter of the light beam, as necessary. In FIG. 7A and FIG. 7B, the light beam with the variable power lens unit 140 inserted is represented by a broken line, and the light beam with the variable power lens unit 140 removed is represented by a solid line. Has been.

  The illumination optical system 100 is configured such that the aperture stop AS and the pupil PL of the objective lens 130 are conjugate, and the front focal position of the field stop FS and the objective lens 130 is conjugate. The illumination optical system 100 can change the position where the image of the light source 111 is formed by moving the collector lens 112 within the light source unit 110. FIG. 7A is a view showing a state in which an image of the light source 111 is formed on the aperture stop AS and Koehler illumination is realized. FIG. 7B is a diagram showing a state where an image of the light source 111 is formed on the field stop FS and critical illumination is realized.

  In the illumination optical system 100, the variable power lens unit 140 is configured as an afocal optical system. For this reason, as illustrated in FIG. 7A, in the Koehler illumination in which a parallel light beam is emitted from the light source unit 110, the conjugate relationship between the light source 111 and the aperture stop AS is independent of the presence or absence of the variable power lens unit 140. Maintained. For this reason, insertion of the variable power lens unit 140 changes the light beam diameter and changes the illumination range and the illumination intensity associated therewith, but almost no loss of light quantity occurs.

  Further, in the illumination optical system 100, as illustrated in FIG. 7B, the illumination light emitted from the light source unit 110 is a substantially parallel light beam even in the critical state. The conjugate relationship between the light source 111 and the field stop FS is substantially maintained. For this reason, insertion of the variable power lens unit 140 changes the light beam diameter and changes the illumination range and the illumination intensity associated therewith, but almost no loss of light quantity occurs.

  As described above, according to the technique for changing the beam diameter between the light source unit and the light projecting tube unit as disclosed in Patent Document 1, the amount of light can be adjusted by adjusting the illumination range and the illumination intensity associated therewith. It is possible to achieve efficient illumination with reduced loss.

JP 2002-250867 A

  Meanwhile, in the field of microscopes, further improvement in illumination uniformity is required, and in recent years, an illumination optical system that further improves the illumination uniformity in Koehler illumination using an optical integrator has been put into practical use.

  However, for an illumination optical system using an optical integrator, a technique for changing the beam diameter between the light source unit and the light projection tube unit is not effective, and the illumination range and the illumination intensity associated therewith cannot be adjusted. .

  When the above technique is applied to an illumination optical system using, for example, a fly-eye lens as an optical integrator, the diameter of the light beam incident on the fly-eye lens is changed by the variable power lens, so the number of light source images formed by the fly-eye lens changes. Will do. However, since the illumination lights emitted from each of these light source images overlap each other on the sample surface, the illumination range itself does not change depending on the number of light source images.

  Therefore, the technology for changing the beam diameter between the light source unit and the light projecting tube unit simultaneously achieves adjustment of the illumination range and the associated illumination intensity per unit area and high uniformity of illumination by the optical integrator. It is not possible.

  Based on the above circumstances, the present invention realizes efficient illumination with reduced light loss by adjusting the illumination range and the associated illumination intensity while realizing high uniformity of illumination. It is an object of the present invention to provide an illumination optical system technique.

A first aspect of the present invention is an illumination optical system for a fluorescence microscope, and in order from the light source side, a light source, an optical integrator for improving illumination uniformity, a relay lens, a variable power lens, an objective lens and the lens, between the relay lens and the objective lens, comprising: a dichroic mirror, a zoom lens, a lens with variable magnification, or Ri lens and switchable lens der having different magnifications, a relay lens and the dichroic Provided is an illumination optical system for a fluorescence microscope disposed between mirrors .

According to a second aspect of the present invention, in the illumination optical system for a fluorescence microscope according to the first aspect, the relay lens has an exit surface of the optical integrator and a pupil plane of the objective lens in a state where there is no variable power lens. Provided is an illumination optical system for a fluorescence microscope that is conjugated .

According to a third aspect of the present invention, there is provided the fluorescent microscope illumination optical system according to the first or second aspect, wherein the zoom lens is an afocal optical system .

According to a fourth aspect of the present invention, the illumination optical system for a fluorescence microscope according to any one of the first to third aspects further includes a field stop that is conjugate with the front focal position of the objective lens. Provided is an illumination optical system for a fluorescence microscope.

According to a fifth aspect of the present invention, in the illumination optical system for a fluorescence microscope according to any one of the first to fourth aspects, the collector lens further includes a collector lens between the light source and the optical integrator. Depending on the position, the optical integrator provides an illumination optical system for a fluorescence microscope that forms images of a plurality of light sources on the exit surface or emits parallel light from the exit surface.
According to a sixth aspect of the present invention, in the illumination optical system for a fluorescence microscope according to any one of the first to fourth aspects, further includes a collector lens movable between the light source and the optical integrator. Provided is an illumination optical system for a fluorescence microscope that can be changed between Kohler illumination and critical illumination by moving a collector lens.
According to a seventh aspect of the present invention, in the illumination optical system for a fluorescence microscope according to any one of the first to sixth aspects, the optical integrator is a fly-eye lens. I will provide a.
According to an eighth aspect of the present invention, in the illumination optical system for a fluorescence microscope according to any one of the first aspect to the seventh aspect, the variable power lens is illuminated by selecting a projection variable power on the sample surface. Provided is an illumination optical system for a fluorescence microscope that can arbitrarily adjust the range.

  According to the present invention, an illumination optical system capable of realizing efficient illumination with reduced loss of light quantity by adjusting the illumination range and the associated illumination intensity while realizing high uniformity of illumination. Technology can be provided.

It is the figure which illustrated the structure of the illumination optical system which concerns on one Embodiment of this invention. It is the figure which illustrated the structure of the fluorescence microscope containing the illumination optical system illustrated by FIG. It is a figure for demonstrating the insertion position of a variable magnification lens unit. FIG. 6 is a diagram illustrating states of an entrance surface and an exit surface of a fly-eye lens, a pupil surface of an objective lens, and a sample surface in an illumination optical system in which a variable power lens unit is not inserted on the optical path. FIG. 5 is a diagram illustrating states of an entrance surface and an exit surface of a fly-eye lens, a pupil plane of an objective lens, and a sample surface in an illumination optical system in which a variable power lens unit is inserted between a light source unit and a light projection tube unit. is there. FIG. 4 is a diagram illustrating states of an entrance surface and an exit surface of a fly-eye lens, a pupil plane of an objective lens, and a sample surface in an illumination optical system in which a variable power lens unit is inserted between a light projection tube unit and an objective lens. is there. It is a figure for demonstrating the prior art which changes the light beam diameter between a light source unit and a light projection tube.

  FIG. 1A and FIG. 1B are diagrams illustrating the configuration of an illumination optical system according to an embodiment of the present invention. An illumination optical system 1 illustrated in FIGS. 1A and 1B is an illumination optical system for a fluorescence microscope, and sequentially from the light source side along the light source 11 that emits illumination light and the optical axis AX. The light source unit 10 including the movable collector lens 12, the light projecting tube unit 20, the variable power lens unit 40 including the variable power lens 43, and the objective lens 30 are included.

  In the illumination optical system 1, the aperture stop AS and the pupil PL of the objective lens 30 are conjugated while the variable magnification lens 43 is not on the optical path, and the field stop FS and the front focal position of the objective lens 30 are conjugated. It is configured. In the illumination optical system 1, when the collector lens 12 moves in the light source unit 10, the position where the image of the light source 11 is formed changes. FIG. 1A is a diagram illustrating a state in which an image of the light source 11 is formed on the aperture stop AS and Koehler illumination is realized. FIG. 1B is a diagram showing a state in which an image of the light source 11 is formed on the field stop FS and critical illumination is realized.

  The light projecting tube unit 20 includes, in order from the light source side, an optical integrator 21 that forms images of a plurality of light sources 11, and a relay lens 24 including a lens 22 and a lens 23. Furthermore, the light projection tube unit 20 includes an aperture stop AS and a field stop FS.

  The relay lens 24 is configured to conjugate the images of the plurality of light sources 11 formed by the optical integrator 21 with the pupil PL of the objective lens 30 in a state where the zoom lens 43 is not on the optical path. More specifically, as illustrated in FIG. 1A, in the lens 22 constituting the relay lens 24, the images of the plurality of light sources 11 formed by the optical integrator 21 and the aperture stop AS are conjugate. Similarly, the lens 23 constituting the relay lens 24 is configured such that the aperture stop AS and the pupil PL of the objective lens 30 are conjugate. Further, as illustrated in FIG. 1B, the lens 23 constituting the relay lens 24 converts the light condensed on the field stop FS into parallel light, and the field stop is formed by the lens 23 and the objective lens 30. The FS is configured to be conjugate with the front focal position of the objective lens 30.

  The optical integrator 21 is an optical element for improving the uniformity of illumination on the sample surface, and examples thereof include a fly-eye lens and a rod lens. 1A and 1B illustrate a fly-eye lens that forms an image of the light source 11 on the exit surface. For this reason, the relay lens 24 conjugates the exit surface of the fly-eye lens (optical integrator 21) to the pupil PL of the objective lens 30 in the absence of the zoom lens 43.

  The variable power lens unit 40 includes a variable power lens 43 including a lens 41 and a lens 42. The variable magnification lens 43 may be configured as a lens having a variable magnification, for example, a zoom lens. The variable power lens 43 may be a lens that can be switched to a lens having a different magnification by exchanging the variable power lens unit 40. The lens switching may be performed by a switching mechanism (not shown), for example, a slider, a drum, a turret, and the like. In FIGS. 1A and 1B, the light beam in a state where the variable power lens 43 (the variable power lens unit 40) is inserted is represented by a broken line, and the variable power lens 43 (the variable power lens unit 40). The light beam with the) removed is represented by a solid line.

  According to the illumination optical system 1 configured as described above, each of the images of the plurality of light sources 11 formed by the optical integrator 21 is applied to the pupil PL of the objective lens 30 as illustrated in FIG. The magnification to be projected can be changed by the variable power lens 43. Further, as illustrated in FIG. 1B, the magnification at which the light source 11 is projected onto the front focal position of the objective lens 30 can be varied by the variable power lens 43. Therefore, the illumination optical system 1 can adjust the illumination range and the associated illumination intensity per unit area while realizing high uniformity of illumination by the optical integrator 21. As a result, the illumination optical system 1 can efficiently illuminate a necessary area while suppressing a loss of light amount.

  However, in the illumination optical system 1, the conjugate relationship between the aperture stop AS and the pupil PL of the objective lens 30 is not strictly maintained in the state where the variable power lens 43 is added. As a result, as illustrated in FIG. 1A, the image of the light source 11 is formed at a position shifted from the pupil PL of the objective lens 30. For this reason, exact Koehler illumination cannot be realized. This is a cause of deterioration in observation performance in bright field observation in which illumination light emitted from a light source detects light reflected by a sample surface or transmitted light.

  However, strict Koehler illumination is not necessarily required for fluorescence observation. First, in fluorescence observation, illumination light itself as excitation light is not a detection target, but fluorescence emitted from a fluorescent dye or fluorescent protein (hereinafter referred to as a fluorescent substance) introduced into a sample. The main reason is that it is a detection target and secondly, fluorescence is emitted isotropically from a fluorescent material that functions as a new light source by irradiation of illumination light. For this reason, the illumination optical system 1 is particularly suitable as an illumination light optical system for a fluorescence microscope.

  Further, in the illumination optical system 1, not only the conjugate relationship between the aperture stop AS and the pupil PL of the objective lens 30 but also the conjugate relationship between the field stop FS and the front focal position of the objective lens 30 in the state where the variable magnification lens 43 is added. May not be maintained. When the conjugate relationship between the field stop FS and the front focal position of the objective lens 30 is broken, the front focal position of the objective lens 30, that is, the image of the field stop FS projected on the sample surface is blurred. For this reason, the observer cannot clearly observe the field stop FS, and cannot accurately recognize the range in which the field of view is narrowed by the field stop FS.

  Therefore, it is desirable that the variable power lens 43 is configured as an afocal optical system. Since the variable power lens 43 is configured as an afocal optical system, the conjugate relationship between the field stop FS and the front focal position of the objective lens 30 can be maintained, so the observer clearly observes the field stop FS. be able to. In the fluorescence microscope using the illumination optical system 1, various optical elements, for example, a confocal stop for obtaining a confocal effect, are arranged at the position of the field stop FS that is conjugated with the sample surface. Sometimes. By configuring the variable power lens 43 as an afocal optical system, the illumination optical system 1 can be controlled by an optical element disposed at the position of the field stop FS even when the variable power lens 43 is inserted in the optical path. The effect obtained can be maintained.

  FIG. 2 is a diagram illustrating the configuration of a fluorescence microscope including the illumination optical system illustrated in FIG. As illustrated in FIG. 2, the illumination optical system 2 included in the fluorescence microscope 50 separates the projection light unit 20 (relay lens 24) in order to separate the illumination light that is excitation light and the fluorescence that is detection light. 1 and the objective lens 30 is different from the illumination optical system 1 illustrated in FIG. 1 in that a dichroic mirror 51 is included.

  In the illumination optical system 2 including the dichroic mirror 51, the zoom lens 43 is preferably disposed between the relay lens 24 and the dichroic mirror 51. This is because by arranging the zoom lens 43 between the relay lens 24 and the dichroic mirror 51, the zoom lens 43 acts only on the illumination light that is the excitation light and does not act on the fluorescence. This makes it possible to adjust only the illumination range and the illumination intensity associated therewith without changing the observation magnification.

  The difference in action depending on the insertion position of the variable power lens unit (variable power lens) will be specifically described below by taking an illumination optical system using a fly-eye lens as an optical integrator as an example.

  FIG. 3 is a diagram for explaining the insertion position of the variable magnification lens unit. The illumination optical system 3 illustrated in FIG. 3 includes a light source unit 60, a light projecting tube unit 70 including a fly-eye lens 71, and an objective lens 80. The light source unit 60, the light projecting tube unit 70, and the objective lens 80 are configured by the same components as the light source unit 10, the light projecting tube unit 20, and the objective lens 30 of the illumination optical system 1 illustrated in FIG. The same applies to the optical positional relationship. For this reason, the same components are denoted by the same reference numerals and description thereof is omitted. However, the light projecting tube unit 70 includes a fly-eye lens 71 instead of the optical integrator 21.

  Further, the illumination optical system 3 can insert the variable magnification lens unit 90 on the optical path. The variable power lens unit 90 includes the same components as the variable power lens unit 40 of the illumination optical system 1 illustrated in FIG. 1, and is between the light source unit 60 and the light projecting tube unit 70 or the light projecting tube unit 70. And the objective lens 80.

  The configuration in which the variable power lens unit 90 is inserted between the light source unit 60 and the light projecting tube unit 70 is disclosed in Patent Document 1 described above except that the light projecting tube unit 70 includes a fly-eye lens 71. This is a configuration close to the configuration to be performed. Further, the configuration in which the variable power lens unit 90 is inserted between the light projecting tube unit 70 and the objective lens 80 is the same as the configuration of the illumination optical system 1 illustrated in FIG.

  FIG. 4 is a diagram showing states of the entrance surface and exit surface of the fly-eye lens, the pupil surface of the objective lens, and the sample surface in the illumination optical system in which the variable power lens unit is not inserted on the optical path. FIG. 5 shows the states of the entrance surface and exit surface of the fly-eye lens, the pupil surface of the objective lens, and the sample surface in the illumination optical system in which the variable power lens unit is inserted between the light source unit and the light projection tube unit. FIG. FIG. 6 shows the states of the entrance surface and exit surface of the fly-eye lens, the pupil surface of the objective lens, and the sample surface in the illumination optical system in which the variable power lens unit is inserted between the projection tube unit and the objective lens. FIG.

  4, 5, and 6, the image IM of the light source 11 is indicated by a black circle, and the range R of the region through which the illumination light beam passes is indicated by a hatching line. Further, the diameter D1 shown in FIGS. 4C, 5C, and 6C indicates the pupil diameter of the objective lens 80. FIG.

With reference to FIGS. 4A to 4D, the state of each surface in the Koehler illumination in a state where the variable power lens unit 90 is not inserted on the optical path will be described.
As illustrated in FIG. 4A, illumination light having a light beam diameter substantially equal to the outer diameter of the fly-eye lens 71 is incident on the incident surface 71 a of the fly-eye lens 71 as parallel light.

  As illustrated in FIG. 4B, the light incident on the fly-eye lens 71 is collected by each element E of the fly-eye lens 71, and a light source for each element E on the exit surface 71 b of the fly-eye lens 71. Eleven images IM are formed.

  The exit surface 71b of the fly-eye lens 71 is projected at a predetermined magnification determined by the relay lens 24 onto the pupil plane of the objective lens 80 having a conjugate relationship. Here, since an example in which the projection magnification is 1.18 times is shown, the size of the image IM and the element E of the light source 11 on the pupil plane of the objective lens 80 as illustrated in FIG. Is 1.18 times the size of the image IM and the element E of the light source 11 on the exit surface 71 b of the fly-eye lens 71.

  As illustrated in FIG. 4C, the images IM of the plurality of light sources 11 are adequately filled without protruding the area indicated by the pupil diameter D <b> 1 of the objective lens 80. For this reason, the Koehler illumination shown in FIGS. 4A to 4D can illuminate the sample surface efficiently without losing the amount of light, and bright illumination is realized.

  In addition, since the illumination light emitted from each light source image formed on the pupil plane of the objective lens 80 is irradiated onto the sample surface so as to overlap each other, as illustrated in FIG. Illuminate the same area above. For this reason, uniform illumination is realized.

A state of each surface in the critical illumination in a state where the variable power lens unit 90 is not inserted on the optical path will be described with reference to FIGS. 4 (e) to 4 (f).
As illustrated in FIG. 4E, the image IM of the single light source 11 is formed on the incident surface 71 a of the fly-eye lens 71. Since the image IM of the light source 11 is formed across the plurality of elements E, the image IM of the single light source 11 is divided by the plurality of elements E.

As illustrated in FIG. 4F, the light from the divided image IM of the light source 11 is converted into parallel light by each element E and is emitted from the exit surface 71 b of the fly-eye lens 71.
As described above, the exit surface 71b of the fly-eye lens 71 is projected at 1.18 times on the pupil plane of the objective lens 80 having a conjugate relationship. For this reason, as illustrated in FIG. 4G, the illumination light has a region on the pupil plane of the objective lens 80 having a size 1.18 times larger than the region R of the exit surface 71 b of the fly-eye lens 71. Incident to R. The region R on the pupil plane of the objective lens 80 is within the region indicated by the pupil diameter D1 of the objective lens 80. Therefore, even with the critical illumination shown in FIGS. 4E to 4H, it is possible to efficiently illuminate the sample surface without losing the amount of light, and bright illumination is realized.

  The illumination light incident on the pupil surface of the objective lens 80 is condensed on the sample surface, and an image of the light source 11 is formed on the sample surface. The illumination range is determined by the size of the element E of the fly-eye lens and the projection magnification of the element E onto the sample surface. In this regard, there is no difference between Kohler lighting and critical lighting. For this reason, the illumination range shown in FIG. 4 (h) is the same as the illumination range shown in FIG. 4 (d).

  In the illumination optical system that does not include the zoom lens unit 90, the projection magnification of the element E onto the sample surface is fixed in both the Koehler illumination and the critical illumination, so that the illumination range can be arbitrarily adjusted. It is not possible to adjust the illumination intensity per unit area associated therewith. Therefore, when the illumination range by the illumination optical system 3 and the range of the area that actually needs illumination do not match, only the necessary area cannot be efficiently illuminated.

  With reference to FIGS. 5A to 5D, the state of each surface in Kohler illumination in a state where the variable power lens unit 90 is inserted on the optical path between the light source unit 60 and the light projection tube unit 70. Will be described.

  As illustrated in FIG. 5A, illumination light whose beam diameter is changed by the variable power lens 43 is incident on the incident surface 71 a of the fly-eye lens 71 as parallel light. FIG. 5A illustrates the case where the beam diameter is smaller than that in FIG.

  As illustrated in FIG. 5B, the illumination light incident on the fly-eye lens 71 is collected by each element E on which the illumination light is incident, and is incident on the exit surface 71 b of the fly-eye lens 71 for each element E. An image IM of the light source 11 is formed. In other words, the number of images of the light source 11 formed on the exit surface 71b of the fly-eye lens 71 changes due to the insertion of the variable power lens unit 90.

  The exit surface 71b of the fly-eye lens 71 is projected at 1.18 times on the pupil plane of the objective lens 80 having a conjugate relationship. For this reason, as illustrated in FIG. 5C, the size of the image IM and the element E of the light source 11 on the pupil plane of the objective lens 80 is the image of the light source 11 on the exit surface 71 b of the fly-eye lens 71. 1.18 times the size of IM and element E.

  As illustrated in FIG. 5C, the images IM of the plurality of light sources 11 are within the region indicated by the pupil diameter D <b> 1 of the objective lens 80. For this reason, the Koehler illumination shown in FIGS. 5A to 5D can illuminate the sample surface efficiently without losing the amount of light, and bright illumination is realized.

The illumination light emitted from each light source image IM formed on the pupil plane of the objective lens 80 is irradiated on the sample surface so as to overlap each other, and therefore, as illustrated in FIG. Illuminate the same area on the surface. For this reason, uniform illumination is realized.
In addition, since the illumination range is determined by the size of the element E of the fly-eye lens and the projection magnification of the element E onto the sample surface, the illumination range shown in FIG. 5D is shown in FIGS. This is the same as the illumination range indicated by h).

The state of each surface in the critical illumination in a state where the variable power lens unit 90 is not inserted on the optical path will be described with reference to FIGS. 5 (e) to 5 (f).
As illustrated in FIG. 5E, the image IM of the single light source 11 is formed on the incident surface 71 a of the fly-eye lens 71. Since the magnification at which the light source 11 is projected onto the incident surface of the fly-eye lens 71 due to the presence of the zoom lens 43 is different from that in FIG. 4E, the size of the image IM of the light source 11 is also the same as that in FIG. Is different. Since the image IM of the light source 11 is formed across the plurality of elements E, the image IM of the single light source 11 is divided by the plurality of elements E.

  As illustrated in FIG. 5F, the light from the divided image IM of the light source 11 is converted into parallel light by each element E and is emitted from the emission surface 71 b of the fly-eye lens 71. That is, the number of elements on the emission surface 71b from which the illumination light is emitted as parallel light is changed by the insertion of the zoom lens unit 90.

  As described above, the exit surface 71b of the fly-eye lens 71 is projected at 1.18 times on the pupil plane of the objective lens 80 having a conjugate relationship. Therefore, as illustrated in FIG. 5G, the illumination light has a region on the pupil plane of the objective lens 80 having a size 1.18 times larger than the region R of the exit surface 71 b of the fly-eye lens 71. Incident to R.

  As illustrated in FIG. 5G, the region R on the pupil plane of the objective lens 80 is within the region indicated by the pupil diameter D <b> 1 of the objective lens 80. Therefore, even with the critical illumination shown in FIGS. 5E to 5H, it is possible to efficiently illuminate the sample surface without losing the amount of light, and bright illumination is realized.

  The illumination light incident on the pupil surface of the objective lens 80 is condensed on the sample surface, and an image of the light source 11 is formed on the sample surface. Since the illumination range is determined by the size of the element E of the fly-eye lens and the projection magnification of the element E onto the sample surface, the illumination range shown in FIG. 5 (h) is shown in FIGS. h) and the illumination range shown in FIG. 5 (d).

  In the illumination optical system including the variable power lens unit 90 on the optical path between the light source unit 60 and the light projecting tube unit 70, the projection magnification of the element E onto the sample surface changes even if the magnification of the variable power lens 43 is changed. do not do. That is, the projection magnification of the element E onto the sample surface is fixed in both the Kohler illumination and the critical illumination. For this reason, an illumination range cannot be adjusted arbitrarily and the illumination intensity per unit area accompanying it cannot be adjusted. Therefore, similarly to the illumination optical system illustrated in FIG. 4, when the illumination range by the illumination optical system 3 does not coincide with the range of the area that actually needs illumination, only the necessary area is efficiently illuminated. I can't. In addition, since the projection magnification cannot be changed according to the pupil diameter of the objective lens, when using an objective lens with a small pupil diameter, part of the light beam cannot reach the sample surface, resulting in poor illumination efficiency. End up.

  6A to 6D, the state of each surface in Koehler illumination with the variable power lens unit 90 inserted on the optical path between the light projecting tube unit 70 and the objective lens 80. Will be described.

As illustrated in FIG. 6A, illumination light having a light beam diameter substantially equal to the outer diameter of the fly-eye lens 71 enters the incident surface 71 a of the fly-eye lens 71 as parallel light.
As illustrated in FIG. 6B, the light incident on the fly-eye lens 71 is collected by each element E of the fly-eye lens 71, and a light source for each element E on the exit surface 71 b of the fly-eye lens 71. Eleven images IM are formed.

  The exit surface of the fly-eye lens 71 is projected at an arbitrary magnification on the pupil plane of the objective lens 80 having a conjugate relationship depending on the magnification of the zoom lens 43. Here, as in FIG. 4, an example of projection at 1.18 times is shown. Therefore, as illustrated in FIG. 6C, the size of the image IM and the element E of the light source 11 on the pupil plane of the objective lens 80 is the image of the light source 11 on the exit surface 71 b of the fly-eye lens 71. 1.18 times the size of IM and element E.

  As illustrated in FIG. 6C, the images IM of the plurality of light sources 11 are adequately filled without protruding the region indicated by the diameter D <b> 1 on the pupil plane of the objective lens 80. For this reason, the Koehler illumination shown in FIGS. 6A to 6D realizes bright illumination with no light loss.

  Note that the illumination light emitted from each light source image formed on the pupil plane of the objective lens 80 is irradiated on the sample surface so as to overlap each other, and therefore, as illustrated in FIG. Illuminate the same area above. For this reason, uniform illumination is realized.

  With reference to FIGS. 6E to 6F, states of each surface in the critical illumination in a state where the variable power lens unit 90 is inserted on the optical path between the light projecting tube unit 70 and the objective lens 80. Will be described.

  As illustrated in FIG. 6E, an image IM of the single light source 11 is formed on the incident surface 71 a of the fly-eye lens 71. Since the image IM of the light source 11 is formed across the plurality of elements E, the image IM of the single light source 11 is divided by the plurality of elements E.

As illustrated in FIG. 6F, the light from the divided image IM of the light source 11 is converted into parallel light by each element E and is emitted from the emission surface 71 b of the fly-eye lens 71.
The exit surface 71b of the fly-eye lens 71 is projected at an arbitrary magnification on the pupil plane of the objective lens 80 having a conjugate relationship according to the magnification of the variable power lens 43.

  The illumination light incident on the pupil plane of the objective lens 80 is condensed on the sample surface and forms an image of the light source 11 on the sample surface. The illumination range is determined by the size of the element E of the fly-eye lens and the projection magnification of the element E onto the sample surface. Therefore, by adjusting the magnification of the zoom lens 43, the illumination range and the illumination intensity associated therewith can be arbitrarily adjusted. In FIG. 6, since the magnification of the zoom lens 43 is different between Koehler illumination and critical illumination, the illumination range shown in FIG. 6 (h) is different from the illumination range shown in FIG. 6 (d).

  As described above, the illumination optical system including the variable power lens unit 90 on the optical path between the light projecting tube unit 70 and the objective lens 80 changes the magnification of the variable power lens 43 to thereby apply the element E to the sample surface. Since the projection magnification can be changed, the illumination range can be arbitrarily adjusted and the illumination intensity associated therewith can be adjusted.

  Further, when the region R in which the illumination light passes through the pupil plane of the objective lens 80 becomes larger than the region indicated by the pupil diameter D1 of the objective lens 80, a light amount loss occurs. For this reason, it is desirable to adjust the magnification of the zoom lens 43 within a range in which the region R in which the illumination light passes through the pupil plane of the objective lens 80 does not become larger than the region indicated by the pupil diameter D1 of the objective lens 80. As a result, it is possible to realize efficient illumination in which loss of light amount is suppressed.

  Although FIG. 6 shows an example in which the illumination range is adjusted by changing the magnification of the variable magnification lens 43 in critical illumination, the present invention is not limited to this. Similarly, in the Koehler illumination, the illumination range can be adjusted by changing the magnification of the zoom lens 43.

  Such adjustment of the illumination range is particularly suitable when the number of fields of view of the illumination optical system with the variable power lens unit 90 removed is different from the number of fields required for observation. For example, if the illumination optical system having the field number 22 is used with a camera corresponding to the field number 11 with the variable power lens unit 90 removed, the illumination range by the illumination optical system may be adjusted in half. As a result, the amount of light per unit area can be increased fourfold, and brighter illumination can be realized.

  1, 2, 100 ... Illumination optical system 10, 60, 110 ... Light source unit, 11, 111 ... Light source, 12, 112 ... Collector lens, 20, 70, 120 ... Light projection Tube unit, 21 ... optical integrator, 22, 23, 41, 42 ... lens, 24 ... relay lens, 71 ... fly-eye lens, 71a ... entrance surface, 71b ... exit surface , 30, 80, 130 ... objective lens, 40, 90, 140 ... variable power lens unit, 43 ... variable power lens, 50 ... fluorescent microscope, 51 ... dichroic mirror, AS ... Aperture stop, FS ... field stop, PL ... pupil, AX ... optical axis, E ... element, R ... range, D1 ... diameter, IM ... image, S. ..Sample surface

Claims (8)

An illumination optical system for a fluorescence microscope, in order from the light source side,
The light source;
An optical integrator to improve lighting uniformity,
A relay lens,
A variable power lens,
An objective lens;
A dichroic mirror between the relay lens and the objective lens ;
The zoom lens is a lens with variable magnification, or Ri lens and switchable lens der with different magnifications, and wherein the <br/> disposed between the said relay lens dichroic mirror Illumination optical system for fluorescent microscope. The illumination optical system for a fluorescence microscope according to claim 1,
An illumination optical system for a fluorescence microscope, wherein the relay lens conjugates the exit surface of the optical integrator and the pupil surface of the objective lens without the zoom lens. The illumination optical system for a fluorescence microscope according to claim 1 or 2,
An illumination optical system for a fluorescence microscope, wherein the variable magnification lens is an afocal optical system. The illumination optical system for a fluorescence microscope according to any one of claims 1 to 3, further comprising:
An illumination optical system for a fluorescence microscope, comprising a field stop that is conjugate with the front focal position of the objective lens. The illumination optical system for a fluorescence microscope according to any one of claims 1 to 4, further comprising:
Including a collector lens between the light source and the optical integrator;
An illumination optical system for a fluorescence microscope, wherein the optical integrator forms a plurality of images of the light source on an emission surface or emits parallel light from the emission surface depending on the position of the collector lens. The illumination optical system for a fluorescence microscope according to any one of claims 1 to 4, further comprising:
A collector lens movable between the light source and the optical integrator,
It can be changed between Kohler illumination and critical illumination by moving the collector lens
An illumination optical system for a fluorescence microscope. The illumination optical system for a fluorescence microscope according to any one of claims 1 to 6 ,
An illumination optical system for a fluorescence microscope, wherein the optical integrator is a fly-eye lens. The illumination optical system for a fluorescence microscope according to any one of claims 1 to 7 ,
The illuminating optical system for a fluorescence microscope, wherein the magnifying lens arbitrarily adjusts an illumination range by selecting a magnification of projection on a sample surface.