JP4790248B2 - Observation device and fluorescence observation device - Google Patents

Description

  The present invention relates to an observation apparatus such as a microscope observation apparatus and a fluorescence observation apparatus.

  Conventionally, what is shown to patent document 1 is known as a microscope observation apparatus. This microscope observation apparatus is disposed so as to be detachable between an objective lens disposed opposite to a sample, an imaging lens that forms an enlarged image on an imaging means such as a CCD camera, and the objective lens and the imaging lens. And a variable magnification relay lens capable of continuously changing the magnification over a predetermined magnification range.

According to this microscope observation apparatus, even if an objective lens and an imaging lens are fixed and an afocal variable magnification relay lens is inserted / removed between them, there is no change in the focal position of the imaging surface, and the image due to the magnification change. It is possible to improve operability and performance.
JP-A-7-104192 (FIG. 1 etc.)

  However, since the conventional microscope observation apparatus changes the observation magnification by changing the magnification of the afocal variable magnification relay lens, it is difficult to change the magnification over a wide magnification range. That is, since the same objective lens and imaging lens are used from a small magnification to a large magnification, there is a disadvantage that the numerical aperture becomes excessively small and the resolution is lowered at a small magnification.

  The present invention has been made in view of the above-described circumstances, and even if the magnification is reduced, an image can be acquired with a high resolution without excessively reducing the numerical aperture, and the observation accuracy can be improved. An object of the present invention is to provide an observation device and a fluorescence observation device.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a light source for irradiating excitation light or illumination light to the sample placed on the stage, is opposed to the stage, and the fluorescent or different objective magnifications of the reflected light for focusing light from the sample, the be used in combination with the objective lens, and a plurality of imaging lenses having different magnifications for forming an image on a sample of each of the objective lens, imaging means for imaging an image on the sample that are imaged by the imaging lens, an objective lens switching mechanism you arranged on the optical axis by switching the objective lens, and Ruyuizo lens switching mechanism be disposed on the optical axis by switching each imaging lens, the objective lens and the high magnification of the high magnification When the imaging lens is combined and disposed on the optical axis, the excitation light or illumination light from the light source is switched to epi-axial illumination that irradiates the sample along the optical axis from above the sample. Magnification of said When the object lens and the low-magnification imaging lens are combined and disposed on the optical axis, the sample is irradiated with excitation light or illumination light from the light source from a direction inclined with respect to the optical axis. An observation device is provided that includes illumination switching means for switching to oblique illumination .

According to the present invention, when the excitation light or illumination light emitted from the light source is irradiated toward the sample, the fluorescence or reflected light emitted from the sample is incident on the objective lens to be condensed and imaged. An image is formed by being incident on the lens and is imaged by the imaging means. When it is desired to observe the image of the sample while changing the magnification, the objective lens is switched by operating the objective lens switching mechanism. Then, by operating the imaging lens switching mechanism, it is possible to select an imaging lens suitable for the objective lens. As a result, even at a low magnification, the numerical aperture is not excessively reduced, and an image can be acquired with a high resolution.
Then, when the high-magnification objective lens and the high-magnification imaging lens are combined and placed on the optical axis, the illumination switching means switches to epi-axial illumination, so that high-definition and high-magnification observation is possible. When the low-magnification objective lens and the low-magnification imaging lens are combined and placed on the optical axis, the illumination switching unit switches to the oblique illumination, thereby changing the path of the illumination light. It is possible to perform observation at a low magnification by separating from the return light path returning from the sample. In this case, it is not necessary to pass the illumination light through the objective lens, and the generation of autofluorescence in the objective lens can be reduced. As a result, an image with good contrast can be acquired.

In the above invention, the illumination switching means is a relay optical system that relays the illumination light that illuminates the sample, and a reflection that is held by the imaging lens and deflects the illumination light from the light source toward the relay optical system. It is good also as having a member.
In this way, the illumination light path can be separated from the return light path returning from the sample. Therefore, it is not necessary to pass illumination light through the objective lens, and generation of autofluorescence in the objective lens can be reduced. As a result, an image with good contrast can be acquired. For example, when the illumination light is excitation light and the return light is fluorescence, a dichroic mirror that separates the excitation light and fluorescence can be small, and an inexpensive illumination system can be provided.

In the above invention, the illumination switching means relays the illumination light that illuminates the sample, and a reflecting member that deflects the illumination light from the plurality of dichroic mirrors and the light source toward the relay optical system. It is good also as having a rotation turret which hold | maintains and selectively arrange | positions facing a light source.
By doing in this way, the illumination system which can freely switch between coaxial illumination and incident illumination according to the observation method can be provided.

In the above invention, the relay optical system may be held by an objective lens or an objective lens switching mechanism.
By doing in this way, the observation range of each objective lens can be illuminated without waste, and an efficient illumination system can be realized.

Furthermore, in the said invention, it is preferable that the said relay optical system divides the illumination light from a light source into 2 or more, and irradiates the divided 2 or more illumination light with respect to a sample from a separate direction.
By doing so, it is possible to realize an illumination system that can reduce the generation of shadows on the sample and can acquire an observation image with good contrast.

In the above invention, when a high-magnification objective lens and a high-magnification imaging lens are selected, they are inserted on the optical axis between the high-magnification objective lens and the high-magnification imaging lens. A zoom mechanism may be provided.
In the above invention, it is preferable that the zoom mechanism is detachable from the optical axis when a low-magnification objective lens and a low-magnification imaging lens are selected.

  When a high-magnification objective lens and a high-magnification imaging lens are selected, a relatively large numerical aperture can be secured, so that a zoom mechanism can be inserted to continuously change the magnification. In this case, when a low-magnification objective lens and a low-magnification imaging lens are selected, a combination of the objective lens and the imaging lens that secures the numerical aperture by removing the zoom mechanism from the optical axis Can be adopted. When the magnification is changed from a high magnification to a low magnification only by the zoom mechanism, the numerical aperture on the low magnification side becomes excessively small. Therefore, such an adverse effect can be prevented by removing the zoom mechanism.

Moreover, in the said invention, it is preferable to provide the focusing adjustment mechanism which adjusts the image formation position of the said image formation lens.
When the imaging position of the imaging lens fluctuates due to individual differences between the combined imaging lens and objective lens, or zoom mechanism, this is corrected by the operation of the focusing adjustment mechanism to obtain a clearer image. Can do.

In the present invention, the high-magnification imaging lens bypasses the optical path between the imaging lens and the imaging unit, and the linear distance from the high-magnification imaging lens to the image position of the imaging unit is reduced. It is preferable that an optical path detour means is provided to match that of the magnification imaging lens.
Optical performance such as aberration can be improved by making the imaging lens and the objective lens have a positional relationship in which the rear focal positions are substantially coincident, that is, a telecentric positional relationship. If the imaging lenses having different magnifications are switched while maintaining the positional relationship as in the present invention, the optical path length becomes longer at high magnifications due to the difference in focal length. Therefore, by operating the optical path detouring means and matching the linear distance from the high magnification imaging lens to the image position of the imaging means with that of the low magnification imaging lens, all the imaging means can be moved without moving them. A clear image can be obtained at a magnification.

In the above invention, the optical path detouring means may be provided with optical path length adjusting means capable of adjusting the optical path length.
Moreover, in the said invention, it is preferable that the said optical path detour means is provided with the angle adjustment means which can adjust the inclination angle of the optical axis.
When different imaging lenses are used, the optical path length and optical axis may vary due to individual lens differences, so the optical path length is matched with that of the imaging lens used as a reference by operating the optical path length adjustment means. Alternatively, the angle can be adjusted so that the optical axis of the imaging lens is accurately directed to the imaging means by the operation of the angle adjusting means.

Moreover, in the said invention, it is good also as providing the objective focusing adjustment mechanism which adjusts the optical axis direction position of the said objective lens.
By operating the objective confocal adjustment mechanism, the individual difference between the lenses can be corrected by adjusting the position of the objective lens that is conjugate with the imaging position of the imaging lens.

  In the above invention, the objective lens, the zoom mechanism, and the imaging lens may be attached so as to be rotatable about the same axis line arranged along the vertical direction. By doing in this way, a switching mechanism can be constituted compactly.

In the above invention, the objective lens, the zoom mechanism, and the imaging lens are attached to be rotatable about two or more axes arranged along the vertical direction, and the objective lens and the zoom mechanism are different. It is good also as being attached so that rotation around an axis line is possible.
Objective lenses having different magnifications are arranged at positions shifted in the optical axis direction due to differences in focal position. The objective lens on the high magnification side can be arranged close to the sample, but the objective lens on the low magnification side is arranged away from the sample. By rotating the objective lens and zoom mechanism around different axes, it is possible to set the low-magnification side objective lens and zoom mechanism that are not used at the same time to interfere with each other in the optical axis direction, making it compact in the height direction. It becomes possible to comprise.

Further, in the above invention, a base installed horizontally, two or more struts extending from the base in the vertical direction along the axis, and a beam member spanned over the upper ends of these struts, Imaging means may be fixed to the beam member.
By doing in this way, an imaging means can be stably supported by the beam member supported by two or more support | pillars, the vibration of an imaging means can be suppressed, and observation accuracy can be improved.

  Furthermore, in the said invention, it is preferable that the said optical axis is arrange | positioned in the position away from the plane containing the axis line of the said 2 or more support | pillar. By doing in this way, two or more support | pillars can be arrange | positioned mutually close, and it becomes possible to comprise compactly in the width direction.

Further, in the above invention, the objective lens switching mechanism, the zoom mechanism, and the imaging lens switching mechanism are fitted to the support column from above and fixed to the support column, and the objective lens switching mechanism, The zoom mechanism or the imaging lens switching mechanism is fixedly mounted around the column axis by an assembly composed of a movable bracket for fixing the zoom mechanism or the imaging lens switching mechanism and a bearing for mounting the movable bracket to the fixed bracket so as to be horizontally rotatable. Is preferred.
By comprising in this way, the movable bracket can be rotatably supported by the support | pillar by fitting and fixing the fixed bracket of the assembly assembled outside to the support | pillar. Therefore, assembly is easy, and manufacture, maintenance, adjustment, and the like can be easily performed.

Furthermore, in the above invention, the base includes a first base that fixes the stage, and a second base that is spaced above the first base, and the first base and the second base. Are fixed by a spacing member, and the column is preferably fixed to the second base.
By comprising in this way, the space | interval dimension of a space | interval member can be determined irrespective of the space | interval of the support | pillar fixed to the 2nd base. As a result, the operability when handling the sample can be improved by increasing the interval dimension of the interval member to ensure a space around the stage.
In addition, by making the spacing member replaceable, it is possible to replace the spacing member with a different length in accordance with the size of the sample and to secure a space around the stage.

Furthermore, in the said invention, it is good also as fixing the tray member which fixed the sample to the said stage in the positioning state.
By doing in this way, a sample can be fixed on a tray member in places other than a stage, and a tray member which fixed a sample can be fixed to a stage. The space around the stage tends to be relatively narrow because the objective lenses are close to each other. Therefore, there are cases where the operability is poor for performing a work of fixing a sample such as an experimental small animal alive. Therefore, preparation for observation can be easily performed by performing such work outside and performing only the work of attaching the tray member to the stage under the objective lens.

Furthermore, in the said invention, it is preferable that the said tray member consists of a material which is transparent or absorbs light.
By doing in this way, among the illumination light irradiated from above the sample, the illumination light that comes off the sample and strikes the tray member passes through the tray member or is absorbed by the tray member, so that it returns to the objective lens side as stray light. Can be prevented.

Furthermore, in the said invention, it is preferable that the said imaging means is provided so that replacement | exchange is possible.
By doing so, it is possible to select and use an imaging means suitable for the type of sample and the observation method, and an image suitable for the purpose of observation can be obtained.

In the above invention. It is preferable that the imaging means is provided so as to be rotatable around the optical axis.
By rotating the imaging means around the optical axis, the orientation of the obtained image can be arbitrarily selected. Further, when observing in real time by connecting the imaging means to a monitor, the direction of the image shown on the monitor can be arbitrarily selected, and observation from an easy-to-see direction can be performed.

In addition, the present invention is a combination of a laser light source that irradiates a sample placed on a stage with excitation light, a plurality of objective lenses that are arranged opposite to the stage and that expands fluorescence from the sample, and has different magnifications, and each objective lens. A plurality of lens groups including an imaging lens that forms an image of fluorescence from the sample magnified by each objective lens, and an imaging means for imaging fluorescence from the sample imaged by the imaging lens; When the lens group switching mechanism for switching the lens group, the high-magnification objective lens, and the high-magnification imaging lens are combined and arranged on the optical axis, excitation light or illumination light from the light source Is switched to epi-axial illumination that irradiates from above the sample along the optical axis, and the low-magnification objective lens and the low-magnification imaging lens are combined on the optical axis. When it is location, to provide a fluorescence observation apparatus comprising an illumination switching means for switching the excitation light or illumination light from the light source biased illumination irradiating the sample from a direction inclined with respect to the optical axis.

According to this invention, when changing the magnification and performing observation, the lens group including the objective lens and the imaging lens is switched by the operation of the lens group switching mechanism. A bright fluorescent image can be obtained without excessively reducing the number.
Then, when the high-magnification objective lens and the high-magnification imaging lens are combined and placed on the optical axis, the illumination switching means switches to epi-axial illumination, so that high-definition and high-magnification observation is possible. When the low-magnification objective lens and the low-magnification imaging lens are combined and placed on the optical axis, the illumination switching unit switches to the oblique illumination, thereby changing the path of the illumination light. It is possible to perform observation at a low magnification by separating from the return light path returning from the sample. In this case, it is not necessary to pass the illumination light through the objective lens, and the generation of autofluorescence in the objective lens can be reduced. As a result, an image with good contrast can be acquired.

  In the said invention, it is good also as providing the process means which performs the spectral deconvolution process to the imaged fluorescence, and the said process means is good also as performing a spectral blind deconvolution process.

  According to the present invention, the imaging lens can be switched simultaneously with the switching of the objective lens according to the change in magnification. In particular, when observing a sample at a low magnification, the objective lens is switched to a low-magnification objective lens and the imaging lens is also switched to a low-magnification imaging lens to ensure that the numerical aperture is not excessively reduced. In addition, there is an effect that it is possible to improve the observation accuracy by suppressing the decrease in the resolution of the obtained image.

A microscope observation apparatus according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the microscope observation apparatus 1 according to the present embodiment includes a light source 2 that generates light to irradiate an experimental small animal such as a mouse or other sample A, a stage 3 on which the sample A is placed, and a sample A. Lens units 4a to 4d for enlarging the return light, imaging lens units 5a and 5b for enlarging and forming an image on the sample A magnified by the objective lens units 4a to 4d, and an imaging lens unit 5a , 5b and a camera (imaging means) 6 for capturing an image on the sample A.

  The stage 3 is provided on a base 7 installed horizontally. The base 7 includes a first base 7a installed on a horizontal installation surface and a second base 7b arranged horizontally above the first base 7a with a space therebetween. Between the 1st base 7a and the 2nd base 7b, the some space | interval member 8 which determines the space | interval dimension between both is arrange | positioned so that replacement | exchange is possible.

  The stage 3 is installed on the first base 7a, and the mounted sample A can be moved in two horizontal directions and in a vertical direction. Further, as shown in FIG. 2, the stage 3 is provided with a through hole 3a so that the tray 9 on which the sample A is placed is fitted in a positioning state. In the present embodiment, the tray 9 is made of a transparent material or a black member that absorbs light.

  The second base 7 b is disposed above the stage 3 and is cut out so as not to block the upper space over the entire operation range of the stage 3. Further, the spacing member 8 is arranged around the stage 3 with a sufficiently wider interval than the operation range of the stage 3. Thereby, when the operator places the sample A on the stage 3 or operates the sample on the stage 3, a wide space is formed so as not to get in the way.

  From the upper surface of the second base 7b, two support columns 10 and 11 extend in the vertical direction. The upper ends of the two pillars 10 and 11 are connected by an upper plate (beam member) 12 so as to span the pillars 10 and 11. Thus, a gate-shaped frame including the two support columns 10 and 11 and the upper plate 12 is configured on the second base 7b.

The objective lens units 4 a to 4 d are attached to a turret 13 attached to the first support column 10 so as to be rotatable around the vertical axis of the support column 10.
The objective lens units 4a to 4d are fixed to the turret 13 at intervals in the circumferential direction. As shown in FIGS. 3 to 6, these objective lens units 4 a to 4 d have different magnifications. For example, in order from the shortest focal length , the focal points of 50 mm, 90 mm, 180 mm, and 300 mm are used. Have a distance. The operator can select the objective lens units 4a to 4d having a desired focal length by rotating the turret 13 as necessary. In these drawings, the lens is omitted.

  The imaging lens units 5a and 5b are respectively provided with low magnification imaging lenses at the tips of two first arms 14 attached to the second support column 11 so as to be rotatable about the vertical axis of the support column 11. A unit 5a and an imaging lens unit 5b for high magnification are attached. The low-magnification imaging lens unit 5a has a focal length of 75 mm, and the high-magnification imaging lens unit 5b has a focal length of 210 mm.

  Further, the second support column 11 includes a zoom mechanism 15 for continuously changing the magnification on the high magnification side and an illumination device 16 for performing epi-illumination at the time of high magnification observation around the vertical axis of the support column 11, respectively. It is attached to be separately rotatable. In the example shown in the figure, the zoom mechanism 15 is attached to the tip of a second arm 17 that is rotatably attached to the second support column 11. The lighting device 16 is fixed to a bracket 18 that is rotatably attached to the second support column 11. The total magnification when the objective lens units 4a to 4d, the coupling lens units 5a and 5b, and the zoom mechanism 15 in the case of high magnification are combined is 1.26 times to 16.2 times.

  The turret 13, the first arm 14, the bracket 18 and the second arm 17 are respectively rotated through a bearing 20 on a cylindrical fixed bracket 19 fitted to the columns 10 and 11, as shown in FIG. The assembly 21 is configured by being attached as possible. FIG. 7 illustrates the second arm 17 as an example. These assemblies 21 are inserted from the upper end of the first support column 10 or the second support column 12 with the upper plate 12 removed, and are respectively disposed at predetermined positions. By fastening, the fixing bracket 19 is fixed to the support columns 10 and 11, and can be horizontally rotated at each position.

  Also, each assembly 21 is inserted from the upper end of each support column 10 and 11 and adjusted directly or on the upper surface of the sleeve 23 positioned by being abutted against the upper surface of the second base 7b, the upper surface of the assembly 21 or the like. It is positioned in the vertical direction by being abutted through a spacer (not shown). That is, the assembly 21 and the sleeve 23 are inserted and stacked from the upper ends of the columns 10 and 11 with the upper plate 12 removed, and can be easily assembled in a positioning state.

  As shown in FIG. 8, the camera 6 is fixed to the upper plate 12 with the optical axis C arranged vertically downward. An absorption filter 24 is arranged between the upper plate 12 and the camera 6. A plurality of types of absorption filters 24 are attached to a turret 25 rotated about a vertical axis so that only light to be imaged is allowed to pass through.

  As shown in FIGS. 8 and 9, the casing 26 of the absorption filter 24 is provided with a mounting hole 27 having a female ant-shaped portion 27a. The camera 6 is provided with an ant-shaped boss portion 29 that is engaged with the female ant-shaped portion 27 a by being inserted into the mounting hole 27 and pressed in the horizontal direction with a set screw 28. The boss portion 29 is formed in a tapered shape whose outer diameter increases toward the tip, and the engaged state with the female ant-shaped portion 27a is maintained by loosening the set screw 28 as indicated by an arrow in FIG. The camera 6 can be rotated around the optical axis C as it is.

The optical axis C of the camera 6 is disposed between the two support columns 10 and 11. The positions of the optical axes of the objective lens units 4a to 4d with the distance between the axes of the two columns 10 and 11 as L and the axis of the first column 10 as the center are indicated by hatching in FIG. The rotation radius of the optical axis is A, the radius of the arc drawn by the outermost shapes of the objective lens units 4 a to 4 d is B, and the optical axes of the imaging lens units 5 a and 5 b and the zoom mechanism 15 centering on the axis of the second support column 11. the rotation radius C, these imaging lens unit 5a, the radius of the circular arc drawn by the outermost shape D and 5b and the zoom mechanism 15, the radius of the first strut r _1, the radius of the second support column 11 as r ₂ of It is arranged at a point where a circle with a radius A and a circle with a radius C set in a range satisfying the following expression intersect.
C <D <Lr ₁ (1)
L−C <A <B <L−r ₂ (2)

  In the present embodiment, the optical axis C of the camera 6 is not arranged in a plane including the axes of the two support columns 10 and 11 in this region, and as shown in FIG. It is located at a distance. The turret 13 is rotated around the axis of the first support column 10, the arbitrarily selected objective lens units 4 a to 4 d are arranged at positions corresponding to the optical axis C of the camera 6, and the axis of the second support column 11 is rotated around the axis of the second support column 11. The first arm 14 is rotated to select the imaging lens units 5a and 5b suitable for the objective lens units 4a to 4d, and can be arranged at a position that coincides with the optical axis C of the camera 6. .

  When the high-magnification objective lens unit 4d and the imaging lens unit 5b are selected, the second arm 17 is rotated around the axis of the second support column 11 so that the zoom mechanism 15 is set to the optical axis C of the camera 6. It can be arranged at the matching position. At this time, the objective lens unit 4d, the imaging lens unit 5b, and the zoom mechanism 15 can be rotated without interfering with the support columns 10 and 11, and without reducing the dimensions of the turret 13 and the arms 14 and 17, The distance L between the support columns 10 and 11 can be reduced.

  In general, it is preferable to arrange the objective lenses 4a to 4d and the imaging lenses 5a and 5b in a positional relationship in which their rear focal positions substantially coincide with each other in order to improve optical performance. In the microscope observation apparatus 1 according to the present embodiment, when such a positional relationship is achieved, as shown in FIG. 12A, when the high-magnification objective lens unit 4a and the imaging lens unit 5a are selected. When the low-magnification objective lens unit 4d and the imaging lens unit 5b are selected as shown in FIG. 5B, the objective lens units 4a to 4d have different focal lengths. The distances L1 and L2 from the imaging position to the imaging positions of the imaging lens units 5a and 5b are different.

  Therefore, as shown in FIG. 12C, the microscope observation apparatus 1 according to the present embodiment causes the high-magnification imaging lens unit 5a having a long distance L1 between the imaging positions to bend the optical path and make a detour. Thus, a prism (optical path detouring means) 30 is provided that matches the linear distance L2 between the imaging positions with the distance L2 on the low magnification side. As a result, a clear image can be taken from a low magnification to a high magnification without moving the camera 6 fixed to the upper plate 12.

Note that the tilt of the optical axis due to individual differences such as manufacturing errors of the prism 30 may be corrected by providing a rotation mechanism that rotates the prism 30 around a horizontal axis, for example, a motor or an adjustment knob.
Further, as shown in FIG. 13, an optical path detouring unit that employs a combination of two or more prisms 31 and 32 is adopted, and an adjustment mechanism that adjusts the distance between the prisms 31 and 32 in the direction of the arrow ( It is also possible to correct fluctuations in the optical path length due to individual differences between the prisms 31 and 32 by providing a not-shown). Further, as shown in FIG. 14, three prisms 31, 33, and 34 are used as the optical path detouring means, and the final prism 34 that deflects the detoured optical path back to the vertical optical path toward the camera 6 is horizontal. The inclination of the optical axis C described above may be corrected by providing a rotation mechanism (not shown) that rotates around the axis. In this way, since only the inclination of the last optical path toward the camera 6 can be adjusted, it is possible to easily perform accurate correction without changing the optical path in a complicated manner.

In the microscope observation apparatus 1 according to the present embodiment, the objective lens units 4a to 4d and the imaging lens units 5a and 5b are provided with an objective focusing mechanism 35 and an imaging focusing mechanism that adjust the focal positions of these lens units. 36 is provided.
In the case of the objective lens units 4 a to 4 c shown in FIGS. 3 to 5, the objective focusing mechanism 35 is fixed by being fastened to a screw hole 13 a provided in the turret 13 and has a fixed bracket 37 having a female screw 37 a. The movable bracket 38 has a male screw 38a fixed to the objective lens units 4a to 4c and fastened to the female screw 37a, and a set screw 39 for fixing the relative displacement of the brackets 37 and 38.

  Note that the objective focusing unit 35 is not provided in the high-magnification objective lens unit 4d shown in FIG. In the case of the present embodiment, since there is only one combination of the objective lens unit 4d for high magnification and the imaging lens unit 5b, it is not necessary to make the objective lenses different. However, an objective focusing mechanism may be provided in such an objective lens unit 4d, and when a plurality of objective lens units are used as the high-magnification objective lens unit 4d, the same as in the case of low magnification is used. An objective focusing mechanism 35 is preferably provided.

  As shown in FIG. 15, the imaging focusing mechanism 36 includes a fixed holder 40 fixed to the first arm 14, and a horizontal adjustment holder 41 fixed to the fixed holder 40 so as to be movable in the horizontal direction. And a vertical adjustment holder 42 fixed to the horizontal adjustment holder 41 so as to be movable in the vertical direction and fixing the imaging lens units 5a and 5b. The horizontal adjustment holder 41 is attached to the lower surface of the fixed holder 40, and by loosening the fixing screw 43, the imaging lens unit is horizontally aligned by a gap between the hole 44 provided in the horizontal adjustment holder 41 and the fixing screw 43. By moving the 5a and 5b and fastening the fixing screw 43, the imaging lens units 5a and 5b can be positioned at the adjusted horizontal positions. The vertical adjustment holder 42 has a male screw 42a that is screwed into a female screw 41a provided on the horizontal adjustment holder 41. The imaging lens units 5a and 5b are rotated by rotating the male screw 42a with respect to the female screw 41a. Is moved in the vertical direction and the set screw 45 is fastened to fix the imaging lens units 5a and 5b at the adjusted vertical position.

  The illumination device 16 is connected to the light source 2 disposed outside by an optical fiber 46. A dichroic mirror 47 is provided at the upper end of the zoom mechanism 15 to reflect light emitted from the illumination device 16 vertically downward and irradiate the sample A via the zoom mechanism 15 and the objective lens unit 4d. In the light source 2, a second illumination device 50 for illuminating the entire sample A via the switch 48 and the optical fiber 49 is disposed near the stage 3. The switch 48 may be any device such as a galvanometer mirror or a shutter. When the low-magnification objective lens unit 4a and the imaging lens unit 5a are selected, light is sent to the second illumination device 50 by the switch 48 so that the entire sample A is irradiated.

The operation of the microscope observation apparatus 1 according to this embodiment configured as described above will be described below.
First, in order to observe the sample A such as an experimental small animal using the microscope observation apparatus 1 according to this embodiment, the sample A is fixed to the tray member 9 outside the microscope observation apparatus 1, and the sample A is fixed. The tray member 9 is fitted into a through hole 3 a provided in the stage 3 and positioned. In this case, since a sufficient space is ensured between the first base 7a and the second base 7b, the installation work of the tray member 9 can be easily performed. In addition, since the operation range of the sample A due to the operation of the stage 3 is sufficiently ensured, the observer can perform observation by changing the observation position of the sample A freely.

Next, when observing the sample A thus positioned at a low magnification, the turret 13 attached to the first support column 10 is rotated so that the low-magnification objective lens unit 4a is light of the camera 6. Move to a position that coincides with axis C. In low-magnification observation, the zoom mechanism 15 is not used. Therefore, as shown in FIG. 3, the objective lens unit 4a that protrudes greatly above the turret 13 can be adopted, and thereby the numerical aperture of the objective lens is excessively increased. It can be prevented from becoming smaller.
Further, the first arm 14 attached to the second support column 11 is rotated to move the low-magnification imaging lens unit 5 a to a position coinciding with the optical axis C of the camera 6. Thereby, a combination of the objective lens unit 4a and the imaging lens unit 5a suitable for observation at a low magnification is selected.

  Then, the switch 48 is switched to the second illumination device 50 side, the light emitted from the light source 2 is irradiated to the entire sample A, and the return light from the sample A is passed through the objective lens unit 4a and the imaging lens unit 5a. To form an image on the camera 6. In this case, since the imaging lens unit 5a is provided with the prism 30 as an optical path detour means, the light transmitted through the imaging lens unit 5a is imaged on the camera 6 attached to the upper plate 12. Will be.

  In order to increase the observation magnification of the sample A, the turret 13 is rotated and the other objective lens units 4b to 4d are selected. In this case, in the microscope observation apparatus 1 according to the present embodiment, since three types of low-magnification objective lens units 4a to 4c are prepared, the objective lens units 4b and 4c are not changed without changing the imaging lens unit 5a. The magnification can be changed by changing only. When the objective lens units 4a to 4c are changed in this way, the position of the objective lens conjugate with the imaging position of the imaging lens may vary due to individual differences between the lens units 4a to 4d and 5a. According to the present embodiment, fine adjustment is performed by the objective confocal adjustment mechanism 35 and the imaging confocal adjustment mechanism 36, so that the adjustment is performed with high accuracy. Therefore, it is possible to obtain a clear image accurately focused at any magnification.

When observing the sample A at a high magnification, first, the turret 13 is rotated, and the high-power objective lens unit 4d is moved to a position coinciding with the optical axis C of the camera 6. Thereby, the low-magnification objective lens units 4 a to 4 c protruding above the turret 13 are arranged at positions removed from the optical axis C of the camera 6. Next, zooming is performed by rotating the second arm 17 into a space formed above the high-magnification objective lens unit 4d of the optical axis C of the camera 6 by removing the low-magnification objective lens units 4a to 4c. The mechanism 15 is inserted. Further, by rotating the first arm 14, the high-magnification imaging lens unit 5 b can be disposed at a position that coincides with the optical axis C of the camera 6.
Thus, a combination of the objective lens unit 4d, the zoom mechanism 15 and the imaging lens unit 5b suitable for high-magnification observation is configured.

  In order to observe the sample A using the high-magnification lens units 4d, 5b, and 15 configured as described above, the observation position of the sample A placed on the stage 3 is set by operating the stage 3 in the same manner as described above. It is made to coincide with the optical axis C of the camera 6. Next, the light emitted from the light source 2 by the operation of the switch 48 is sent to the first illumination device 16 side, deflected by the dichroic mirror 47 provided at the upper end of the zoom mechanism 15, and directed toward the sample A. The return light emitted from the sample A by irradiating the sample A through the zoom mechanism 15 and the objective lens unit 4d is condensed by the objective lens unit 4d, and the image of the sample A is further enlarged by the zoom mechanism 15. Then, the light passes through the dichroic mirror 47 and is imaged on the camera 6 by the imaging lens unit 5b. The observer can perform observation by operating the zoom mechanism 15 as necessary and setting the magnification to an arbitrary magnification. At this time, only the light having a desired wavelength is picked up by the camera 6 by passing through an arbitrarily selected absorption filter 24.

  As described above, according to the microscope observation apparatus 1 according to the present embodiment, the objective lens units 4a to 4d can be easily switched by the rotation of the turret 13, and the objective lens unit 4a by the rotation of the first arm 14. The imaging lens units 5a and 5b conforming to 4d can be selectively switched. Thereby, the magnification can be changed not only by the objective lens units 4a to 4d but also by the imaging lens units 5a and 5b. Further, the numerical aperture is not excessively reduced in the case of low magnification.

In this case, since the combination of the objective lens units 4a to 4d and the imaging lens units 5a and 5b is changed, it is conceivable that defocusing occurs due to individual differences of the lens units. Since the lens units 4a to 4d and the imaging lens units 5a and 5b are respectively provided with the in-focus adjusting mechanisms 35 and 36, the objective lens and the imaging lens are prevented from being out of focus when the objective lens and the imaging lens are changed. The arrangement of the imaging lens can be easily corrected.
Further, the imaging lens unit 5a having a long focal length, that is, the imaging lens unit 5a on the high magnification side is provided with the optical path detouring means 30, so that the position of the camera 6 can be changed from low magnification to high A clear image can be obtained up to the magnification, and the linear distance from the sample A to the camera 6 can be reduced. Further, by performing the in-focus adjustment in the optical path detour means 30, there is an advantage that the image forming lens unit 5b need not be moved and can be adjusted easily.

In addition, since the zoom mechanism 15 that can be disposed between the objective lens unit 4d on the high magnification side and the imaging lens unit 5b is provided, observation can be performed by continuously changing the magnification. When selecting the low-magnification objective lens units 4a to 4c and the imaging lens unit 5a, the zoom mechanism 15 can be removed from the optical axis C of the camera 6, and an observation image is obtained during low-magnification observation with a small numerical aperture. Can be brightened.
In this case, the zoom mechanism 15 can be downsized by reducing the lens of the zoom mechanism 15.

Moreover, according to the microscope observation apparatus 1 according to the present embodiment, the zoom mechanism 15 and the objective lens units 4a to 4d are provided to be separately rotatable around the two support columns 10 and 11 provided on the second base 7b. Therefore, the low-magnification objective lens units 4a to 4c and the zoom mechanism 15 that are not used at the same time can be arranged at positions overlapping in the height direction. As a result, there is an advantage that the size in the height direction can be shortened to reduce the size. In this case, by arranging the optical axis C of the camera 6 at a position away from the plane including the axes of the two columns 10 and 11, the two columns 10 and 11 can be brought close to each other, and the width direction compact. Can be achieved.
In addition, the support | pillars 10 and 11 are not limited to two, Three or more may be sufficient.

  Since the low-magnification objective lens units 4a to 4c and the zoom mechanism 15 are arranged at positions overlapping in the height direction, it is considered that the objective lens units 4a to 4c and the zoom mechanism 15 interfere when the lens unit is switched. However, such an inconvenience may be solved by mechanically or electrically interlocking the insertion and removal of the zoom mechanism 15 and the insertion and removal of the objective lens units 4a to 4c. Further, as in the case of the microscope observation apparatus 1 according to the present embodiment, when the high-magnification objective lens unit 4d and the zoom mechanism 15 have a one-to-one correspondence, the high-magnification objective lens unit 4d The zoom mechanism 15 may be fixed to the upper part.

  Furthermore, according to the microscope observation apparatus 1 according to the present embodiment, like the turret 13, the first arm 14, the second arm 17, and the first illumination device bracket 18, the support columns 10 and 11 can be rotated around the axis. Since the member to be attached to the outside is configured as an assembly 21 outside and is assembled in a stacking manner from the upper ends of the columns 10 and 11, the assemblability is good, and other lens units There is an advantage that addition and change can be easily performed.

  Further, according to the microscope observation apparatus 1 according to the present embodiment, the base 7 is configured in a two-stage structure, the stage 3 is provided on the lower first base 7a, and the columns 10 and 11 are provided on the upper second base 7b. Since it is attached, the spacing member 8 between the two bases 7a and 7b can be arranged at a wide interval regardless of the spacing dimension of the support columns 10 and 11. As a result, a wide space around the stage 3 can be secured to improve the operability of the sample A, and the distance between the columns 10 and 11 can be made closer to achieve compactness in the width direction.

Furthermore, the height position of the 2nd base 7b with respect to the 1st base 7a can be arbitrarily set by enabling the space | interval member 8 to be exchangeable. Therefore, the interval can be set in accordance with the size of the sample A placed on the stage 3.
Further, since the sample A is not directly fixed on the stage 3 but is fixed to the tray member 9 fixed to the stage 3, the operability of the sample A is further improved. Furthermore, since the tray member 9 is made of a transparent or black material, it is possible to prevent the light that comes off the sample A and impinges on the tray member 9 from entering the objective lens units 4a to 4d as stray light.

  Further, according to the microscope observation apparatus 1 according to the present embodiment, since the camera 6 is installed on the upper plate 12 supported by the two columns 10 and 11, the camera 6 is unlikely to vibrate, and the observation image Blur can be prevented. Further, since the camera 6 is detachably provided, the camera 6 can be selected and used in accordance with the type of the sample A to be observed and the observation method. In addition, by making the camera 6 rotatable about the optical axis C, the angle of the camera 6 can be set in accordance with the direction of the sample A.

  If the entire microscope observation apparatus 1 according to the present embodiment is arranged in a dark screen or dark box for observation, it is possible to prevent external light or the like from entering the objective lens units 4a and 4b. . In particular, in the case of fluorescence observation, since the amount of fluorescence is weak, observation in a dark curtain or dark box is preferable. If the turret 13, the first arm 14, the second arm 17, the camera 6, etc. can be remotely operated by any driving means, observation in the dark screen or in the dark box is easy. In the case of manual operation, a part of the dark curtain may be raised, or an openable / closable window may be provided in a part of the dark box.

Next, a microscope observation apparatus 60 according to the second embodiment of the present invention will be described with reference to FIG. In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation apparatus 1 according to the first embodiment described above, and the description will be simplified.
As shown in FIG. 16, the microscope observation apparatus 60 according to the present embodiment is the first embodiment in that the turret 13, the arm 17, and the second turret 62 are rotatably attached to a single column 61. This is different from the microscope observation apparatus 1 according to the above.

  That is, as shown in FIG. 16, the microscope observation apparatus 60 according to the present embodiment includes a base 7 on which the stage 3 on which the sample A is mounted is fixed, a support column 61 extending vertically from the base 7, and the support column. From the bottom, a turret 13 equipped with a plurality of objective lens units 4a to 4d, an arm 17 equipped with a zoom mechanism 15 and a plurality of imaging lens units 5a to 5c are installed in the middle position in the length direction of 61. Turrets 62 are independently attached to the column 61 so as to be rotatable about the vertical axis. An upper plate 12 is fixed to the upper end of the column 61, and a camera 6 with the optical axis C directed downward is fixed to the upper plate 12. The light source is omitted.

  When observing the sample A placed on the stage 3, the objective lens units 4a to 4d and the imaging lens units 5a to 5c are selected in accordance with the magnification to be observed, and the positions coincide with the optical axis C of the camera 6. If the zoom mechanism 15 is arranged at a position that coincides with the optical axis C of the camera 6, the observation is performed. The objective lenses 4a to 4d having different magnifications are the same as in the first embodiment in that imaging lenses 5a to 5c suitable for the objective lenses 4a to 4d can be selected.

  According to the microscope observation apparatus 60 according to the present embodiment, the configuration is simple compared to the microscope observation apparatus 1 according to the first embodiment. In addition, since all the optical systems are collected in one support 61, there is an advantage that it can be manufactured compactly in the width direction. In addition, since the stage 3 attached to the base 7 can also be arranged in a relatively wide space without being surrounded, there is an advantage that the operability is good.

Next, a microscope observation apparatus 70 according to a third embodiment of the present invention will be described below with reference to FIG.
Also in the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation apparatuses 1 and 60 according to the above-described embodiments, and the description will be simplified.
The microscope observation apparatus 70 according to the present embodiment is the same as the microscope observation apparatus 60 according to the second embodiment in that it has a single column 61 fixed to the base 7.

  As shown in FIG. 17, the microscope observation apparatus 70 according to the present embodiment includes a first lens group 71 in which a low-magnification objective lens unit 4a and an imaging lens unit 5a are combined, and a high-magnification objective lens unit. 4d, the zoom mechanism 15, and the second lens group 72 that combines the imaging lens unit 5b. In the figure, only one each of the first lens group 71 and the second lens group 72 is shown, but a plurality of first lens groups 71 having different magnifications may be provided. These lens groups 71 and 72 are fixed to the same rotational radius position on the turret 13 rotatably supported by the support column 61 with an interval in the circumferential direction.

The microscope observation apparatus 70 according to the present embodiment is a fluorescence observation apparatus, and includes a light source 2, a shutter 73, a filter turret 74, and a switch 48 on the light source 2 side. The lighting devices 16 and 50 are connected to the switch 48. A dichroic mirror 47 for performing epi-illumination on the sample A for high magnification observation is disposed between the zoom mechanism 15 and the imaging lens unit 5b.
In the figure, reference numeral 75 denotes a computer provided with a control device for controlling the light source 2, shutter 73, filter turrets 25 and 74, switch 48, stage 3, turret 13 and camera 6, and reference numeral 76 denotes a monitor.

A case where the sample A is observed with fluorescence using the microscope observation apparatus 70 according to the present embodiment configured as described above will be described below.
First, a dye A is injected into a sample A such as an experimental small animal, or a sample A is injected or expressed with a fluorescent protein (step S1), and the prepared sample A is placed on the stage 3 (step S2).
Next, an arbitrary magnification is selected, and the lens groups 71 and 72 corresponding to the magnification are made to coincide with the optical axis C of the camera 6. Then, according to the magnification, when the magnification is low, the second illumination device 50 irradiates the entire sample A with light and acquires a bright field image (step S3). In this state, the stage 3 is actuated to move the sample A to the position to be imaged, and the lens groups 71 and 72 are adjusted to adjust the focus (step S4).

  Next, the dye for which fluorescence observation is desired is selected (step S5), and the imaging wavelength corresponding to the dye is determined by the filter turret 25 (step S6). Further, the illumination wavelength (excitation wavelength) corresponding to the selected dye is set by the filter turret 74 (step S7). Then, the exposure amount is determined and photographed (step S8), the image is stored (step S9), and when performing observation over time, the above photographing operation is repeated with time (step S10).

  According to the microscope observation apparatus 70 according to the present embodiment, a plurality of objective lens units 4a and 4d and imaging lens units 5a and 5b are used as lens groups 71 and 72, or a lens group to which a zoom mechanism 15 is further added. Since it is provided as 72, the magnification can be arbitrarily changed without performing the in-focus adjustment so that the numerical aperture does not become excessively small even at a low magnification. As a result, a bright image can be taken even at a low magnification.

  In addition to the fluorescence from the target fluorescent substance, a lot of autofluorescence is generated from the sample A such as an experimental small animal. Therefore, by analyzing the spectrum of fluorescence emitted from a substance such as a fluorescent dye and determining the ratio of the amount of fluorescence at two different wavelengths, fluorescence other than the fluorescence of the target fluorescent substance is separated from the obtained fluorescence image. Spectral deconvolution processing means for removal may be provided in the computer 24.

  That is, for example, as shown in US Pat. No. 6,403,332, when the fluorescence spectrum of a target fluorescent substance and the fluorescence spectrum of a substance that emits autofluorescence are known, among these fluorescence spectra, A ratio of fluorescence intensities corresponding to appropriate two wavelengths can be obtained in advance. Therefore, by obtaining these ratios in advance, the fluorescence spectrum of the target fluorescent substance can be extracted from the observed fluorescence.

  When the autofluorescent substance in the sample A is unknown or uncertain, as shown in Japanese Patent Laid-Open No. 7-50031, a fluorescence image from the sample A is taken and the fluorescence spectrum from the sample A is captured. It is preferable to perform spectral blind deconvolution that simultaneously calculates the spatial distribution of the fluorescent substance. According to this method, the fluorescence spectrum of each fluorescent substance and the existence ratio of the fluorescent substance in each pixel of the fluorescent image can be simultaneously obtained from the captured fluorescent image, and the distribution of the fluorescent substance in the sample A can be determined.

Next, a microscope observation apparatus 80 according to a fourth embodiment of the present invention will be described below with reference to FIGS.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation apparatus 1 according to the first embodiment described above, and the description will be simplified.

  The microscope observation device 80 according to the present embodiment replaces the switch 48, the optical fiber 49, and the illumination device 50 of the microscope observation device 1 according to the first embodiment with the holding member 81 in the low-magnification imaging lens unit 5a. And a relay optical system 84 fixed to the support 11 via a holding member 83. In the first embodiment, the zoom mechanism 15 is rotatably supported by the support column 11. In the present embodiment, the zoom mechanism 15 is fixed to the objective lens 4c for high magnification observation. Yes.

  The reflecting member 82 is arranged to face the front of the first illumination device 16 when the low-magnification imaging lens unit 5a is arranged on the optical axis C. As a result, the illumination light guided from the light source 2 via the fiber 46 and emitted from the first illumination device 16 is turned back toward the relay optical system 84 by the reflecting member 82.

  FIG. 20 shows a sectional view of the relay optical system 84. The relay optical system 84 includes a cylindrical outer cylinder 85 disposed outside the turret 13 in the circumferential direction, a plurality of lenses 87 a and 87 b held in the outer cylinder via a spacing tube 86, and an outer cylinder. And a reflecting member 88 that turns the illumination light relayed by the lenses 87a and 87b in the direction of the sample A.

  Here, the spacing tube 86 adjusts the luminous flux of the illumination light applied to the sample A. For example, when the sample A is a mouse, the length of the spacing tube 86 is adjusted so that the illumination light beam has a diameter of 100 mm on the stage 3 since the body length of the mouse is about 100 mm. Note that it is not always necessary to illuminate the entire sample A, and only the maximum observation range may be illuminated.

The operation of the microscope observation apparatus 80 according to this embodiment configured as described above will be described below.
19 and 20, in the low magnification observation system in which the imaging lens unit 5a for low magnification observation is arranged on the optical axis C, the illumination light emitted from the light source 2 passes through the optical fiber 46. To the lighting device 16. Since the reflecting member 82 is disposed in front of the lighting device 16, the illumination light emitted from the lighting device 16 is deflected by the reflecting member 82 and travels toward the relay optical system 84. Then, the luminous flux is adjusted in the relay optical system 84, and the illumination light deflected by the reflecting member 88 is irradiated onto the sample A.

  The return light emitted from the sample A is selectively collected by the turret 13 by any one of the objective lenses 4a, 4b, and 4d arranged coaxially with the imaging lens unit 5a. Is imaged.

  On the other hand, as shown in FIG. 21, in the high magnification observation system in which the zoom mechanism 15 and the imaging lens 5b for high magnification observation are arranged on the optical axis C, the illumination light emitted from the light source 2 passes through the fiber 46. And sent to the lighting device 16. By retracting the imaging lens 5a, the reflecting member 82 is removed from the front of the illumination device 16, and instead, the dichroic mirror 47 provided at the upper end of the zoom mechanism 15 is faced. Therefore, the illumination light emitted from the illumination device 16 is deflected vertically downward by the dichroic mirror 47, collected by the objective lens 4c, and then irradiated onto the sample A.

  Return light emitted from the sample A is collected by the objective lens 4c, magnified by the zoom mechanism 15, passes through the dichroic mirror 47, and forms an image on the camera 6 by the imaging lens 5b.

  According to the microscope apparatus 80 according to the present embodiment, since the illumination light is not passed through the objective lenses 4a, 4b, and 4d in the low magnification observation, the autofluorescence generated in the objective lenses 4a, 4b, and 4d can be reduced. As a result, an image with good contrast can be obtained. Further, in the high magnification observation system, since the light beam focused by the zoom mechanism 15 only needs to be applied to the dichroic mirror 47, there is an advantage that the size of the dichroic mirror 47 can be reduced.

Next, a microscope observation apparatus 90 according to a fifth embodiment of the present invention will be described below with reference to FIGS.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation apparatus 80 according to the fourth embodiment described above, and the description thereof is omitted.

  As shown in FIG. 22, the microscope observation device 90 according to the present embodiment replaces the reflecting member 82 fixed to the imaging lens unit 5 a of the microscope observation device 80 according to the fourth embodiment with the objective lenses 4 a to 4. Between 4d and the imaging lens units 5a and 5b, a turret 91 (rotary turret) supported so as to be rotatable around the axis of the support column 10 is provided. A plurality of dichroic mirrors 47 a, 47 b, 47 c having different characteristics and a reflecting member 82 are fixed to the turret 91.

  As shown in FIG. 23, each dichroic mirror 47a, 47b, 47c is attached to a through-hole 92 penetrating the turret 91 in the vertical direction via a holder 93. Each dichroic mirror 47a, 47b, 47c is set to be disposed at a position facing the illumination device 16 when the central axis of the through-hole 92 to which they are attached is aligned with the optical axis C. Yes.

  As shown in FIG. 23, the holder 93 is formed in a substantially cylindrical shape, is positioned by being fitted into a stepped through hole 92 provided in the turret 91, and is set from the outer peripheral surface of the turret 91. It is fixed by. The holder 93 is provided with through holes 93a and 93b penetrating in the axial direction and the radial direction. At the upper end of the holder 93, the dichroic mirrors 47a to 47c are disposed at positions where the through holes 93a penetrating in the axial direction are closed. Either is fixed.

  The inner diameters of the through holes 93a and 93b of the holder 93 and the sizes of the dichroic mirrors 47a to 47c are large enough to transmit the light beams collected by the objective lens units 4a to 4d. The dichroic mirrors 47a to 47c are arranged at an angle of 45 ° with respect to each of the illumination light and the optical axis C at the point where the illumination light irradiated from the illumination device 16 and the optical axis C intersect. It is fixed to the holder 93.

  Further, as shown in FIG. 24, the reflecting member 82 is attached to the vicinity of one through hole 92 a provided in the turret 91 with a screw 96 via a holder 95. The reflecting member 82 is fixed so as to be disposed at a position facing the illumination device 16 when the central axis of the through hole 92a is aligned with the optical axis C.

  As shown in FIG. 24, the reflecting member 82 is bonded to a holder 95 fixed to the end of the turret 91 and has an angle that deflects the illumination light emitted from the illumination device 16 in the direction of the relay optical system 84. Is set to Further, when the reflecting member 82 is disposed on the illumination optical path, the size of the through hole 92a disposed on the optical axis C is such that the light beams collected by the objective lenses 4a to 4d can pass therethrough. Is formed.

The operation of the microscope observation apparatus 90 according to the present embodiment configured as described above will be described below.
In order to perform high-magnification observation using the microscope observation apparatus 90 according to the present embodiment, the turret 13 is rotated to place the objective lens 4c and the zoom mechanism 15 on the optical axis C, and the first arm 14 is rotated. Thus, the imaging lens unit 5b is arranged on the optical axis C.

  Further, by rotating the turret 91 in accordance with the purpose of the observation, the dichroic mirrors 47a to 47c or the through holes 92a having different characteristics are selectively arranged on the optical axis C. The dichroic mirrors 47a to 47c are detachable by loosening the set screw 94 and removing the holder 93 from the turret 91, and those having characteristics necessary for observation can be appropriately replaced and arranged.

When the dichroic mirrors 47a to 47c are arranged on the optical axis C, the illumination light from the light source 2 is emitted from the illumination device 16 through the optical fiber 46, and is disposed in front of the illumination device so as to face the dichroic mirror 47a. ˜47c is deflected vertically downward, and the sample A is irradiated through the zoom mechanism 15 and the objective lens 4c.
In this case, the return light from the sample A is collected by the objective lens 4c, enlarged by the zoom mechanism 15, and then transmitted through the through-hole 93a of the holder 93 of the turret 91 and the dichroic mirrors 47a to 47c to form the imaging lens. The image is formed on the camera 6 by the unit 5b.

On the other hand, by arranging the through hole 92 a on the optical axis C, the illumination light from the light source 2 is deflected toward the relay optical system 84 by the reflecting member 82. For this reason, the sample A is not illuminated through the zoom mechanism 15 and the objective lens 4c, but is obliquely illuminated from an oblique side while bypassing the zoom mechanism 15 and the objective lens 4c.
In this case, the return light from the sample A is collected by the objective lens 4c, magnified by the zoom mechanism 15, and then passes through the through hole 92a of the turret 91 and is imaged on the camera 6 by the imaging lens unit 5b. .

  On the other hand, when performing low-magnification observation using the microscope observation apparatus 90 according to the present embodiment, the turret 13 is rotated to selectively select one of the low-magnification objective lenses 4a, 4b, and 4d as the optical axis. Place on C. Further, the first arm 14 is rotated to place the objective lens unit 5a on the optical axis C.

  Further, by rotating the turret 91, the dichroic mirrors 47a to 47c or the through holes 92a having different characteristics are selectively arranged on the optical axis C. When the dichroic mirrors 47a to 47c are arranged, the illumination light emitted from the light source 2 is sent to the illumination device 16 via the optical fiber 46, deflected by the dichroic mirrors 47a to 47c on the turret 91, and the objective lenses 4a, After being condensed by either 4b or 4d, the sample A is irradiated.

  When the through hole 92 a is arranged on the optical axis C, the illumination light from the light source 2 is deflected by the reflecting member 82 toward the relay optical system 84. For this reason, the sample A is not incident through the objective lenses 4a, 4b and 4d, but is deviated from the oblique side while bypassing the objective lenses 4a, 4b and 4d. The return light from the sample A is collected by the objective lenses 4a, 4b, and 4d, passes through the through-hole 92a of the turret 91, and is imaged on the camera 6 by the imaging lens 5a.

  According to the microscope observation apparatus 90 according to the present embodiment configured as described above, the turret 91 is rotated and the dichroic mirrors 47a to 47c or the through holes 92a are selectively arranged in both high magnification and low magnification observations. By doing so, it is possible to select the coaxial illumination that illuminates the sample A through the objective lenses 4a to 4d and the oblique illumination that illuminates the sample A from the oblique side while bypassing the objective lenses 4a to 4d. Further, since various dichroic mirrors 47a to 47c can be selected, the characteristics of the dichroic mirrors 47a to 47c can be selected according to the return light to be observed.

Next, a microscope observation apparatus 100 according to a sixth embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation apparatus 80 according to the fourth embodiment described above, and the description thereof is omitted.

  The microscope observation apparatus 100 according to the present embodiment is different from the fourth embodiment in that the relay optical system 101 is held on the turret 13 instead of the support 11. Furthermore, two sets of relay optical systems 101 are arranged for each of the objective lenses 4a to 4d at a position sandwiching the objective lenses 4a to 4d.

  The relay optical system 101 includes two cylindrical outer cylinders 102 and 103, condenser lenses 104 and 105 held by the outer cylinders 102 and 103, a half mirror 106, and mirrors 107, 108, and 109. Yes. The outer cylinders 102 and 103 are fixed to the turret 13 with a set screw (not shown) while penetrating the turret 13 in the thickness direction.

  In one outer cylinder 102 of the relay optical system 101, a half mirror 106, a condensing lens 104, and a mirror 108 are arranged in a substantially straight line from the top in this order. In the other outer cylinder 103, the mirror 107, the condensing lens 105, and the mirror 109 are arranged in a substantially straight line from above.

When the objective lenses 4a to 4d and the imaging lens unit 5a are arranged on the optical axis C, the outer cylinder 102 is vertically adjusted by a reflecting member 82 fixed to the imaging lens unit 5a via a holding member 81. The illumination light deflected downward is arranged at a position where it enters.
In this state, the half mirror 106 is disposed vertically below the reflecting member 82 so that the observation light deflected by the reflecting member 82 enters. Further, the mirror 107 is disposed at a position separated in the horizontal direction so that the illumination light deflected by the half mirror 106 enters.

  The positions of the outer cylinders 102 and 103 with respect to the turret 13, the positions and angles of the half mirrors 106, mirrors 107, 108 and 109, and the condenser lenses 104 and 105 in the outer cylinders 102 and 103 are within the observation range of the objective lenses 4 a to 4 d. Each relay optical system 101 is set at an optimum position so that illumination can be performed without waste.

The operation of the microscope observation apparatus 100 according to the present embodiment configured as described above will be described below.
When using the microscope observation apparatus 100 according to this embodiment to perform low-magnification observation in which the zoom mechanism 15 is not arranged on the optical axis C, the turret 13 is rotated to select one of the objective lenses 4a, 4b, and 4d. Are selectively arranged on the optical axis C. At this time, the relay optical system 101 provided for each objective lens 4a, 4b, 4d is also set.

  The illumination light emitted from the light source 2 is deflected by the reflecting member 82 and directed to the relay optical system 101. The illumination light transmitted through the half mirror 106 at the upper end of the outer cylinder 102 is adjusted by the condenser lens 104, deflected by the mirror 108, and applied to the sample A. The illumination light reflected by the half mirror 103 is deflected by the mirror 107 in the direction of the condensing lens 105, the light beam is adjusted by the condensing lens 105, deflected by the mirror 109, and irradiated on the sample A.

Thus, according to the microscope observation apparatus 100 according to the present embodiment, it is possible to irradiate the sample A with the observation light emitted from one light source 2 through two paths. In addition, since the two relay optical systems 101 arranged with the objective lenses 4a to 4d interposed therebetween adjust the positions of the optical elements in accordance with the objective lenses 4a to 4d, the objective lenses 4a to 4d. A range that matches the observation range of
In the present embodiment, the number of the light sources 2 is not limited to one but may be plural. In that case, at least the number of relay optical systems 101 equal to the number of the light sources 2 is required for each of the objective lenses 4a to 4d.

Next, a microscope observation apparatus 110 according to the seventh embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation apparatus 80 according to the fourth embodiment described above, and the description thereof is omitted.

  The microscope observation apparatus 110 according to the present embodiment is held by lenses 113 and 114 held by holders 111 and 112 fixed to the objective lenses 4a to 4d and a holder 115 fixed by the imaging lens units 5a and 5b. The relay optical system 120 includes a half mirror 116 and a mirror 117, and a mirror 119 held by a holder 118 fixed to the base 12. The half mirror 116 and the mirror 117 are arranged at positions separated in the vertical direction. Further, for example, as shown in FIG. 26, when the imaging lens unit 5a is disposed on the optical axis C, the half mirror 116 is disposed in front of the illumination device 16, and the illumination device 16, half mirror 116 and the mirror 118 are arranged in a substantially straight line in the horizontal direction.

As a result, the illumination light emitted from the illumination device 16 and transmitted through the half mirror 116 is reflected by the mirror 118 and directed to the sample A. The illumination light deflected vertically downward by the half mirror 116 is further deflected by the mirror 117 and directed to the sample A from another path. Further, the condensing lenses 113 and 114 are arranged so as to condense the illumination light deflected by the mirrors 117 and 118 onto the observation portion of the sample A.
In this case, the condensing lenses 113 and 114 are not necessarily fixed to all the objective lenses 4a to 4d.

  According to the microscope observation device 110 according to the present embodiment, in the low-magnification observation in which the joint lens unit 5a is disposed on the optical axis C, the illumination light emitted from the illumination device 16 is transmitted through the half mirror 116, Divided into the deflection side. The illumination light transmitted through the half mirror 116 is directly deflected in the direction of the sample A by the mirror 118. On the other hand, the illumination light deflected by the half mirror 116 is directed again toward the sample A by being deflected again by the mirror 117. The illumination light deflected by the mirrors 117 and 118 is irradiated so as to be condensed on the observation portion of the sample A by the relay optical system 401.

As described above, according to the microscope observation apparatus 110 according to the present embodiment, the same effect as in the sixth embodiment can be obtained, and the illumination light can be directly directed to the sample A by the mirror 118. The sample A can be efficiently illuminated by reducing the number of mirror reflections.
In addition, you may implement 5th Embodiment and 6th Embodiment, or combining 5th Embodiment and 7th Embodiment. In that case, both effects can be obtained simultaneously.

  Moreover, in the said embodiment, although the stage 3 installed on the 1st base 7a and moving the sample A mounted in the black tray 9 which absorbs a transparent material or light was adopted in the horizontal 2 direction and the perpendicular direction, it was employ | adopted. Instead, as shown in FIGS. A fixed stage 130 fixed to the first base 7a or provided integrally with the first base 7a may be employed.

  In the stage 130 of FIGS. 27 and 28, the mounting surface 131 on which the sample A is mounted is the bottom surface of the recess 133 that is recessed by one step from the peripheral edge 132. The volume of the recess 133 is preferably large enough to store a body fluid or a liquid W such as physiological saline that flows when the sample A is cut open. Thereby, it is possible to prevent the liquid W from leaking out of the stage 130 during observation and entering the portion around the stage 130 that is difficult to wipe or difficult to clean. In particular, when observing in a dark box, it is difficult to visually observe that the liquid W has leaked from the stage 130, so it is effective to use such a stage 130.

  The stage 140 of FIGS. 29 and 30 includes a circumferential groove 142 that is recessed by one step around the mounting surface 141 on which the sample A is mounted. The volume of the circumferential groove 142 is preferably large enough to store a body fluid or a liquid W such as physiological saline that flows when the sample A is cut open. Thereby, there is an effect similar to that of the above-described stage 130 in which the liquid W is prevented from leaking out of the stage 140 during observation and entering the portion around the stage 140 that is difficult to wipe or difficult to clean. Further, according to the stage 140, the liquid W that has flowed out accumulates in the circumferential groove 142 that is lower than the mounting surface 141, so that the sample A can be prevented from being immersed in the liquid W.

  Further, the stage 150 of FIGS. 31 and 32 is a recess 151 in which the mounting surface on which the sample A is mounted is recessed in a concave shape. Also in this case, the volume of the recess 151 is preferably large enough to store the body fluid flowing out when the sample A is incised and the liquid W such as physiological saline. According to this stage 150, there exists an effect similar to the said stage 150. FIG.

  Further, the stage 160 of FIG. 33 is provided with a notch 161 in a part of the peripheral edge 132 of the stage 130 of FIGS. 27 and 28, and can discharge the liquid W accumulated in the recess 133 to the outside. It is like that. A tray 162 is provided outside the notch 161 so that the liquid W that has flowed out can be stored. By doing so, it is possible to prevent the sample A from being immersed in the liquid W, similarly to the stage 140 of FIG. In addition, you may provide the notch 161 and the saucer 162 in the stage 150 of FIG. Further, through holes (not shown) for allowing the liquid W to flow downward may be provided on the mounting surfaces 131 and 151 of the stages 130 and 150, and a container for receiving the liquid W may be disposed at the tip of the through holes. .

1 is a perspective view showing a microscope observation apparatus according to a first embodiment of the present invention. It is the longitudinal cross-sectional view which fractured | ruptured a part which shows the tray member on the stage in the microscope observation apparatus of FIG. It is a longitudinal cross-sectional view which shows the low magnification objective lens unit of the microscope observation apparatus of FIG. It is a longitudinal cross-sectional view which shows the other low magnification objective lens unit similar to FIG. It is a longitudinal cross-sectional view which shows the other low magnification objective lens unit similar to FIG. It is a longitudinal cross-sectional view which shows the high magnification objective lens unit of the microscope observation apparatus of FIG. It is the longitudinal cross-sectional view which fractured | ruptured a part which shows the attachment structure of the 2nd arm of the microscope observation apparatus of FIG. It is the longitudinal cross-sectional view which fractured | ruptured a part which shows the attachment structure of the camera of the microscope observation apparatus of FIG. It is a top view which shows the attachment structure of FIG. It is a top view explaining arrangement | positioning of the support | pillar and space | interval member of the microscope observation apparatus of FIG. It is a top view which shows the arrangement | positioning area | region of the optical axis of the camera of the microscope observation apparatus of FIG. It is a schematic diagram explaining the optical path detour means of the microscope observation apparatus of FIG. It is a schematic diagram which shows the modification of the optical path detour means of FIG. It is a schematic diagram which shows the other modification of the optical path detour means of FIG. It is the longitudinal cross-sectional view which fractured | ruptured a part which shows the attachment structure of the imaging lens unit of the microscope observation apparatus of FIG. It is a perspective view which shows the microscope observation apparatus which concerns on the 2nd Embodiment of this invention. It is a whole block diagram which shows the microscope observation apparatus which concerns on the 3rd Embodiment of this invention. It is a flowchart which shows the observation procedure in the microscope observation apparatus of FIG. It is a perspective view which shows the microscope observation apparatus which concerns on the 4th Embodiment of this invention. FIG. 20 is a partial vertical cross-sectional view illustrating incident illumination by the microscope observation apparatus in FIG. 19. It is a perspective view which shows the state at the time of the high magnification observation by the microscope observation apparatus of FIG. It is a perspective view which shows the microscope observation apparatus which concerns on the 5th Embodiment of this invention. It is a partial longitudinal cross-sectional view explaining the coaxial illumination by the microscope observation apparatus of FIG. It is a partial longitudinal cross-sectional view explaining the incident illumination by the microscope observation apparatus of FIG. It is a partial longitudinal cross-sectional view which shows the microscope observation apparatus which concerns on the 6th Embodiment of this invention. It is a partial longitudinal cross-sectional view which shows the microscope observation apparatus which concerns on the 7th Embodiment of this invention. It is a perspective view which shows the 1st modification of the stage in the microscope observation apparatus of this invention. It is a longitudinal cross-sectional view of the stage of FIG. It is a perspective view which shows the 2nd modification of the stage in the microscope observation apparatus of this invention. FIG. 30 is a longitudinal sectional view of the stage of FIG. 29. It is a perspective view which shows the 3rd modification of the stage in the microscope observation apparatus of this invention. FIG. 32 is a longitudinal sectional view of the stage of FIG. 31. It is a perspective view which shows the 4th modification of the stage in the microscope observation apparatus of this invention.

Explanation of symbols

A Sample C Optical axis 1, 70, 80, 90, 100, 110 Microscope observation device 2 Light source (laser light source)
3, 130, 140, 150, 160 Stages 4a to 4d Objective lens 5a, 5b Imaging lens 6 Imaging means 7a First base 7b Second base (base)
8 Spacing member 9 Tray member 10, 11 Prop 12 Upper plate (beam member)
13 Turret (Objective lens switching mechanism: Movable bracket: Lens group switching mechanism)
14 First arm (imaging lens switching mechanism: movable bracket)
15 Zoom mechanism 17 Second arm (movable bracket)
18 Bracket (movable bracket)
19 Fixing bracket 20 Bearing 21 Assembly 30, 31, 32 Prism (optical path detour means)
35 Objective Confocal Adjustment Mechanism 36 Imaging Confocal Mechanism (Confocal Adjustment Mechanism)
47, 47a to 47c Dichroic mirror 71, 72 Lens group 70 Fluorescence microscope observation device 75 Computer (processing means)
82, 116, 117 Reflective member 84, 101 Relay optical system 91 Turret (rotary turret)

Claims (26)

A light source that irradiates the sample placed on the stage with excitation light or illumination light;
A plurality of objective lenses that are arranged opposite to the stage and collect fluorescence or reflected light from the sample and have different magnifications ;
A plurality of imaging lenses having different magnifications that are used in combination with each objective lens to form an image on the sample of each objective lens;
Imaging means for imaging an image on the sample that are imaged by the imaging lens,
An objective lens switching mechanism you arranged on the optical axis by switching the objective lens,
And Ruyuizo lens switching mechanism be disposed on the optical axis by switching each imaging lens,
When the high-magnification objective lens and the high-magnification imaging lens are combined and arranged on the optical axis, excitation light or illumination light from the light source is emitted from above the sample along the optical axis. Switching to epi-axial illumination to irradiate, and when the low-magnification objective lens and the low-magnification imaging lens are combined and placed on the optical axis, excitation light or illumination light from the light source is converted to the optical axis. And an illumination switching means for switching to oblique illumination that irradiates the sample from a direction inclined with respect to the specimen . The illumination switching unit includes a relay optical system that relays illumination light that illuminates a sample, and a reflection member that is held by the imaging lens and deflects illumination light from the light source toward the relay optical system. Item 2. The observation device according to Item 1. The illumination switching means selectively holds a relay optical system that relays illumination light that illuminates the sample, and a reflection member that deflects illumination light from a plurality of dichroic mirrors and a light source toward the relay optical system. The observation apparatus according to claim 1, further comprising: a rotating turret disposed to face the light source.   The observation apparatus according to claim 2, wherein the relay optical system is held by an objective lens or an objective lens switching mechanism.   The relay optical system divides illumination light from a light source into two or more and irradiates the sample with two or more divided illumination lights from different directions. Observation device. When the high magnification objective and a high magnification of the imaging lens is selected, including a zoom mechanism that is inserted and arranged on the optical axis between these high magnification objective and a high magnification of the imaging lens The observation apparatus according to claim 1.   The observation apparatus according to claim 6, wherein the zoom mechanism is detachable from the optical axis when a low-magnification objective lens and a low-magnification imaging lens are selected.   The observation apparatus according to claim 1, further comprising a focusing adjustment mechanism that adjusts an imaging position of the imaging lens.   The high-magnification imaging lens bypasses the optical path between the imaging lens and the imaging means, and the optical path bypass that matches the linear distance from the imaging lens to the image position of the imaging means with the low-magnification imaging lens. The observation apparatus according to any one of claims 1 to 8, wherein a means is provided.   The observation apparatus according to claim 9, wherein the optical path detour means is provided with an optical path length adjustment means capable of adjusting an optical path length thereof.   The observation device according to claim 9 or 10, wherein the optical path detouring means is provided with an angle adjusting means capable of adjusting an inclination angle of an optical axis thereof.   The observation apparatus according to claim 1, further comprising an objective confocal adjustment mechanism that adjusts a position conjugate with an imaging position of the imaging lens of the objective lens.   The observation apparatus according to claim 1, wherein the objective lens, the zoom mechanism, and the imaging lens are attached so as to be rotatable around the same axis line arranged along the vertical direction. The objective lens, the zoom mechanism, and the imaging lens are attached to be rotatable around two or more axes arranged along the vertical direction,
The observation apparatus according to claim 1, wherein the objective lens and the zoom mechanism are attached so as to be rotatable around different axes. A base installed horizontally,
Two or more struts extending vertically from the base along the axis;
A beam member spanning the upper ends of these columns,
The observation apparatus according to claim 14, wherein the imaging unit is fixed to the beam member.   The observation apparatus according to claim 15, wherein the optical axis is disposed at a position away from a plane including an axis of the two or more support columns.   The objective lens, the zoom mechanism, and the imaging lens are fitted to the support column from above and fixed to the support column, and a movable bracket that fixes the objective lens, the zoom lens, or the imaging lens, The observation device according to any one of claims 13 to 16, wherein the observation device is rotatably mounted around an axis of a support column by an assembly including a bearing that is assembled so that the movable bracket can be horizontally rotated with respect to the fixed bracket.   The base includes a first base that fixes the stage, and a second base that is spaced above the first base, and the first base and the second base are fixed by a spacing member. The observation apparatus according to claim 13, wherein the support column is fixed to the second base.   The observation device according to claim 18, wherein the spacing member is replaceable.   The observation apparatus according to claim 1, wherein a tray member on which a sample is fixed can be fixed to the stage in a positioning state.   21. The observation apparatus according to claim 20, wherein the tray member is made of a material that is transparent or absorbs light.   The observation apparatus according to any one of claims 1 to 21, wherein the imaging unit is provided in a replaceable manner.   The observation apparatus according to any one of claims 1 to 22, wherein the imaging unit is provided to be rotatable around the optical axis. A laser light source for irradiating the sample placed on the stage with excitation light;
A plurality of objective lenses with different magnifications that are arranged opposite to the stage and expand the fluorescence from the sample,
A plurality of lens groups including an imaging lens that is used in combination with each objective lens and forms an image of fluorescence from the sample magnified by each objective lens;
Imaging means for imaging fluorescence from the sample imaged by the imaging lens;
A lens group switching mechanism for switching the lens group;
When the high-magnification objective lens and the high-magnification imaging lens are combined and arranged on the optical axis, excitation light or illumination light from the light source is emitted from above the sample along the optical axis. Switching to epi-axial illumination to irradiate, and when the low-magnification objective lens and the low-magnification imaging lens are combined and placed on the optical axis, excitation light or illumination light from the light source is converted to the optical axis. A fluorescence switching device comprising illumination switching means for switching to oblique illumination for irradiating the sample from a direction inclined with respect to the sample .   The fluorescence observation apparatus according to claim 24, further comprising processing means for performing spectral deconvolution processing on the captured fluorescence. The fluorescence observation apparatus according to claim 25 , wherein the processing means performs a spectral blind deconvolution process.

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