JP2010091809A - Microscope apparatus - Google Patents

Abstract

A microscope apparatus capable of performing epifluorescence observation, confocal observation, and total reflection fluorescence observation, and suppressing an increase in size and cost.
A scanning means for scanning the specimen with illumination light from a first light source condensed on the specimen through the objective lens, and observation light from the specimen to the objective lens and scanning means. A confocal observation device 25 having a light receiving means 38 for receiving light through 32 and an introduction member U1 for introducing the light of the second light source into the optical path of the confocal observation device 25, and the light of the second light source to the objective lens 8 And an epi-illumination device 26 that irradiates the specimen 5 via the light, and fluorescence detection optical systems 10, 17, 18, and 27 that detect the fluorescence of the specimen 5 excited by the light of the confocal observation device 25 or the epi-illumination device 26. An imaging position conversion member U2 that forms a light source image of the first light source on the pupil plane P of the objective lens 8, and an introduction member U1 and an imaging position conversion member U2 in the optical path of the confocal observation device 25. And an insertion / removal mechanism for switching to the same position for insertion.
[Selection] Figure 2

Description

  The present invention relates to a microscope apparatus.

Conventionally, all of the confocal microscope, the total reflection fluorescence microscope, and the epifluorescence microscope have been widely used as devices for observing a specimen such as a living cell. In recent years, the confocal observation and the total reflection fluorescence observation have been switched. An operable microscope has been proposed (see, for example, Patent Document 1).
JP 2005-121796 A

  However, in the conventional microscope as described above, switching between the confocal observation and the total reflection fluorescence observation is performed by placing an optical member for condensing the illumination light on the total reflection condition region of the pupil plane of the objective lens in the illumination optical system. This is done by inserting and removing. Therefore, the conventional microscope requires a large-scale switching mechanism for realizing this, so that there is a problem in that the size and cost are increased.

  Therefore, the present invention has been made in view of the above problems, and provides a microscope apparatus capable of performing epi-illumination observation, confocal observation, and total reflection fluorescence observation, and suppressing an increase in size and cost. For the purpose.

In order to solve the above problems, the present invention
Scanning means for scanning the specimen with illumination light from a first light source condensed on the specimen via the objective lens, and light reception for receiving observation light from the specimen via the objective lens and the scanning means. A confocal observation device having means;
An epi-illumination device having an introduction member for introducing illumination light from a second light source into the optical path of the confocal observation device, and irradiating the sample with illumination light from the second light source via the objective lens;
A fluorescence detection optical system that detects fluorescence from the specimen excited by the illumination light of the confocal observation device or the epi-illumination device, and
An imaging position converting member for forming a light source image of the first light source on a pupil plane of the objective lens;
Provided is a microscope apparatus, further comprising: an insertion / removal mechanism for switching and inserting the introduction member and the imaging position conversion member to substantially the same position in the optical path of the confocal observation apparatus. .

  According to the present invention, it is possible to perform epi-illumination fluorescence observation, confocal observation, and total reflection fluorescence observation, and it is possible to provide a microscope apparatus that suppresses an increase in size and cost.

Hereinafter, a microscope apparatus according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.
FIG. 1 is a diagram showing an outline of a microscope apparatus according to an embodiment of the present invention, and FIG. 2 is a diagram showing an optical system of the microscope apparatus according to the embodiment of the present invention.
As shown in FIG. 1, a microscope apparatus 1 according to this embodiment includes an inverted microscope body 2 and a control device 3.

The microscope main body 2 can rotate and switch a transmission illumination optical system 6 that guides light from a transmission illumination light source 4 (described later) to a specimen 5, a stage 7 on which the specimen 5 is placed, and a plurality of objective lenses 8 having different magnifications in order from above. The revolver 9, the fluorescent cube holder 10, the imaging optical system 11, and the eyepiece tube 12 are provided.
The fluorescent cube holder 10 is a holding member that is capable of rotating and switching two fluorescent cubes 15 and 16 by a known rotating mechanism. The fluorescent cube 15 includes a dichroic mirror 15a and an emission filter disposed on the transmission optical path thereof. 15b. The fluorescent cube 16 includes a dichroic mirror 16a, an emission filter 16b disposed on the transmission optical path, and a wavelength selection filter 16c disposed on the incident optical path.
As shown in FIG. 2, the imaging optical system 11 includes, in order from the fluorescent cube holder 10, the imaging lens 17, a prism 18 that can be inserted into and removed from the optical path by a known slide mechanism, a mirror 19a, a relay lens 20a, and a mirror 19b. And a relay lens 20b.
The eyepiece tube 12 includes a lens 21, a mirror 22, and an eyepiece (not shown).

As shown in FIG. 1, a transmission illumination light source 4 for transmitting and illuminating the specimen 5 is provided on the outside of the microscope main body 2, and confocal observation for confocal observation of the specimen 5 is provided on the side. An apparatus 25, an epi-illumination device 26 for epi-illuminating the specimen 5, and an image sensor 27 are provided. In FIG. 2, the transmitted illumination light source 4 and the transmitted illumination optical system 6 are not shown.
As shown in FIG. 2, the confocal observation device 25 includes, in order from the fluorescent cube holder 10 side, an imaging lens 29, an exchange unit holder 30, a pupil relay lens 31, a two-dimensional scanner 32 that scans light two-dimensionally, a beam It includes a splitter 33, a collector lens 34, an optical fiber 35, a laser light source (not shown), an imaging lens 36 disposed on the reflection optical path of the beam splitter 33, a pinhole 37, and a light receiving element (PMT) 38.
The exchange unit holder 30 is a holding member that holds the two exchange units U1 and U2 by switching them to substantially the same position in the optical path by a known slide mechanism. The exchange unit U1 includes a dichroic mirror 39, and the exchange unit U2 includes a correction lens 40 described later.

The epi-illumination device 26 includes a field stop 42, a collector lens 43, an optical fiber 44, and an epi-illumination light source (not shown) in order from the replacement unit holder 30 side as shown in FIG. 29 is shared with the confocal observation device 25. In the present embodiment, a laser light source is used as the epi-illumination light source.
The control device 3 includes a personal computer 45 that controls various devices mounted on the microscope body 2, a monitor 46, and the like.

In the microscope apparatus 1 according to the present embodiment having such a configuration, as described below, transmitted illumination observation in which the specimen 5 is transmitted and illuminated to observe the transmitted light, epi-illuminated fluorescence observation in which the specimen 5 is reflected and illuminated to observe fluorescence, Scanning fluorescence observation in which illumination light is scanned two-dimensionally to irradiate the specimen 5 to observe fluorescence, confocal observation in which illumination light is scanned two-dimensionally to irradiate the specimen 5 and observe reflected light, and specimen The total reflection fluorescence observation for observing the fluorescence by totally reflecting illumination 5 can be switched appropriately.
FIGS. 3A, 3B, and 3C show a case in which the specimen 5 is subjected to epifluorescence observation, a scanning fluorescence observation and a confocal observation in the microscope apparatus 1 according to the present embodiment. FIG. 5 is a diagram showing the main part of an optical system and the behavior of illumination light when performing total reflection fluorescence observation.

When performing transmission illumination observation of the specimen 5 with the microscope apparatus 1 according to the present embodiment, the two fluorescent cubes 15 and 16 of the fluorescent cube holder 10 and the prism 18 are retracted out of the optical path in advance.
Under this premise, the illumination light from the transmitted illumination light source 4 is applied to the specimen 5 on the stage 7 through the transmitted illumination optical system 6, and the transmitted light (observation light) transmitted through the specimen 5 is mounted on the revolver 9. It is condensed by the objective lens 8 that is present. This observation light is imaged on the primary image plane Q via the imaging lens 17 of the imaging optical system 11 and the mirror 19a, and then sequentially passes through the relay lens 20a, the mirror 19b, and the relay lens 20b to the eyepiece tube 12. Incident. The observation light incident on the eyepiece tube 12 is guided to an eyepiece lens (not shown) via a lens 21 and a mirror 22. As a result, the observer can look through the eyepiece lens (not shown) and observe the transmitted image of the specimen 5, that is, can observe the transmitted illumination of the specimen 5.

When the epi-fluorescence observation of the specimen 5 is performed by the microscope apparatus 1 according to the present embodiment, the fluorescent cube 16 of the fluorescent cube holder 10, the prism 18, and the replacement unit U1 of the replacement unit holder 30 are arranged in advance in the optical path. .
Under this premise, laser light from an epi-illumination light source (not shown) is guided by an optical fiber 44 as shown in FIG. 3A and emitted from its end face, and then is substantially parallel through a collector lens 43. It becomes light and is reflected by the dichroic mirror 39 of the exchange unit U 1 through the field stop 42. The substantially parallel laser light is condensed by the imaging lens 29, transmitted through the wavelength selection filter 16c of the fluorescent cube 16 and reflected by the dichroic mirror 16a, so that light having a predetermined excitation wavelength is selected. The sample 5 on the stage 7 is irradiated through the objective lens 8.

  As a result, the fluorescence expressed in the specimen 5 is collected by the objective lens 8 and passes through the dichroic mirror 16a and the emission filter 16b of the fluorescent cube 16, so that light having an unnecessary wavelength is cut. The fluorescence is reflected by the prism 18 through the imaging lens 17 of the imaging optical system 11 and imaged on the imaging surface of the imaging device 27. As a result, the fluorescence image of the sample 5 is picked up by the image pickup device 27, and this is processed by the PC 45 and displayed on the monitor 46. In this way, the observer can perform epi-fluorescence observation of the specimen 5 using the microscope apparatus 1.

When the scanning fluorescence observation of the specimen 5 is performed by the microscope apparatus 1 according to the present embodiment, the fluorescent cube 15 and the prism 18 of the fluorescent cube holder 10 are arranged in the optical path in advance, and two replacements of the replacement unit holder 30 are performed. The units U1 and U2 are retracted out of the optical path.
Under this premise, laser light from a laser light source (not shown) is guided by an optical fiber 35 and emitted from its end face, and then passes through a beam splitter 33 as a substantially parallel laser light through a collector lens 34. . The laser beam that has passed through the beam splitter 33 is reflected by the two-dimensional scanner 32, and after being imaged on the image plane R by the pupil relay lens 31 as shown in FIG. 3B, passes through the exchange unit holder 30, It becomes substantially parallel laser light again through the imaging lens 29. The substantially parallel laser light is reflected by the dichroic mirror 15a of the fluorescent cube 15 so that light having a predetermined wavelength is selected, and then condensed by the objective lens 8 and applied to the sample 5 on the stage 7. . Since this laser beam is two-dimensionally scanned by the two-dimensional scanner 32, the entire observation area of the specimen 5 is irradiated. As a result, the specimen 5 is excited by the laser light to exhibit fluorescence and reflect the laser light.

  The fluorescence expressed in the sample 5 is collected by the objective lens 8 and passes through the dichroic mirror 15a and the emission filter 15b of the fluorescent cube 15, so that light having an unnecessary wavelength is cut. The fluorescence is reflected by the prism 18 through the imaging lens 17 of the imaging optical system 11 and imaged on the imaging surface of the imaging device 27. As a result, the fluorescence image of the sample 5 is picked up by the image pickup device 27, and this is processed by the PC 45 and displayed on the monitor 46. In this way, the observer can perform scanning fluorescence observation of the specimen 5 with the microscope apparatus 1.

On the other hand, the laser light reflected by the specimen 5 is collected by the objective lens 8 and reflected by the dichroic mirror 15 a of the fluorescent cube 15. The laser light passes through the imaging lens 29 and the pupil relay lens 31, is sequentially reflected by the two-dimensional scanner 32 and the beam splitter 33, passes through the pinhole 37, and reaches the light receiving element 38. Since the light reaching the light receiving element 38 is descanned by the two-dimensional scanner 32, the light receiving element 38 detects observation light over the entire observation region of the specimen 5. Then, the PC 45 generates a two-dimensional image of the sample 5 based on the intensity signal of the laser beam obtained by the light receiving element 38 and displays it on the monitor 46. In this way, the observer can perform confocal observation of the specimen 5 with the microscope apparatus 1.
In the microscope apparatus 1 according to the present embodiment, the fluorescence image obtained from the image sensor 27 and the confocal image obtained from the light receiving element 38 can be displayed on the monitor 46 so as to be observed.

When total reflection fluorescence observation of the specimen 5 is performed by the microscope apparatus 1 according to the present embodiment, the fluorescent cube 15 of the fluorescent cube holder 10, the prism 18, and the replacement unit U2 of the replacement unit holder 30 are arranged in the optical path in advance. To do.
The replacement unit U2 of the replacement unit holder 30 in the present embodiment includes a correction lens 40 including only a biconvex positive lens as shown in FIG. When the replacement unit U2 is disposed in the optical path in the replacement unit U2, the correction lens 40 is moved from the light source image position (image plane R) of the laser light source (not shown) of the confocal observation device 25 to the front side of the correction lens 40. The distance L to the point S is arranged at a position where it is substantially equal to the focal length f of the correction lens 40. With this arrangement, the correction lens 40 can convert light emitted from one point on the image plane R into a substantially parallel light beam.

  Under the above premise, laser light from a laser light source (not shown) is guided by an optical fiber 35 and emitted from its end face, and then converted into substantially parallel laser light by a collector lens 34 and then transmitted through a beam splitter 33. Then, it is reflected by the two-dimensional scanner 32. At this time, the reflection angle of the two-dimensional scanner 32 is controlled by the PC 45 so that the reflected light is incident on the total reflection condition region in the pupil plane P of the objective lens 8 described later. Then, the laser light reflected by the two-dimensional scanner 32 is imaged on the image plane R by the pupil relay lens 31 as shown in FIG. 3C, and then is substantially parallel by the correction lens 40 of the exchange unit U2. Converted to light. The substantially parallel laser light is focused by the imaging lens 29 and is reflected by the dichroic mirror 15a of the fluorescent cube 15 so that light having a predetermined wavelength is selected, and then the pupil plane P of the objective lens 8 is selected. The light is condensed at one point on the total reflection condition region. The total reflection condition region is an annular zone on the pupil plane P that allows the light to be incident on the sample 5 at a total reflection angle when the light is incident on the pupil plane P of the objective lens 8. A range of shapes.

  The laser beam focused on the total reflection condition region on the pupil plane P of the objective lens 8 is incident on the sample 5 through the objective lens 8 at a total reflection angle. Specifically, the medium of the sample 5 and the sample 5 are held. The sample 5 enters the sample 5 at an incident angle that is totally reflected at a boundary surface with a glass substrate (not shown). As described above, in the microscope apparatus 1, the image plane R and the sample plane are conjugate in the scanning fluorescence observation and the confocal observation described above, whereas the exchange unit U2 is arranged in the illumination optical path. The image plane R and the pupil plane P of the objective lens 8 are conjugate, and the total reflection illumination of the sample 5 is realized by guiding the laser beam to the total reflection condition region of the pupil plane P by the two-dimensional scanner 32 as described above. be able to.

  When the specimen 5 is totally reflected and illuminated as described above, evanescent light is generated at the boundary surface, and the portion near the boundary surface of the specimen 5 is excited by the evanescent light to express fluorescence. This fluorescence is collected by the objective lens 8 and passes through the dichroic mirror 15a and the emission filter 15b of the fluorescent cube 15, so that light having an unnecessary wavelength is cut. The fluorescence is reflected by the prism 18 through the imaging lens 17 of the imaging optical system 11 and imaged on the imaging surface of the imaging device 27. As a result, the fluorescence image of the sample 5 is picked up by the image pickup device 27, and this is processed by the PC 45 and displayed on the monitor 46. In this way, the observer can perform total reflection fluorescence observation of the specimen 5 with the microscope apparatus 1.

As described above, in this microscope apparatus 1, transmission illumination observation, epifluorescence observation, scanning fluorescence observation, confocal observation, and total reflection fluorescence observation of the specimen 5 can be switched as appropriate.
The microscope apparatus 1 can replace the exchange unit U1 for switching between epifluorescence observation and confocal observation and the exchange unit U2 for switching between confocal observation and total reflection fluorescence observation with an exchange unit holder 30. In other words, the two simple exchange units U1 and U2 are exchanged at the same position in the optical path (the position where the optical path of the epi-illumination device 26 joins the optical path of the confocal observation device 25). Thus, a large-scale switching mechanism like a conventional microscope is not required, and the increase in size and cost of the microscope main body 2 can be suppressed.

In the present embodiment, the two replacement units U1, U2 are held by the replacement unit holder 30 so as to be switchable as described above. However, the present invention is not limited to this configuration, and a slot may be formed in place of the replacement unit holder 30, and a configuration may be adopted in which the observer appropriately selects replacement units U1 and U2 separately prepared separately and is inserted into and removed from the slot. Thereby, it becomes possible to suppress the enlargement and cost increase of the microscope main body 2 more effectively.
Further, the correction lens 40 of the exchange unit U2 in the present embodiment is not limited to the above-described configuration. For example, in order from the two-dimensional scanner 32 side, a positive junction of a biconvex positive lens and a biconcave negative lens is performed. You may comprise by a lens, or you may comprise by the lens group which has the positive refractive power which consists of a biconvex positive lens and a biconcave negative lens.

Further, in the present embodiment, the installation location of the replacement unit holder 30 is high in the degree of freedom of space and is close to the two-dimensional scanner 32, which is suitable for the position adjustment of the correction lens 40 of the replacement unit U2. Therefore, the correction lens 40 of the exchange unit U2 in the present embodiment includes a known moving mechanism (not shown) for adjusting the position in the optical axis direction. As a result, when the objective lens 8 is switched to an objective lens having a different magnification by rotating the revolver 9 and total reflection fluorescence observation is performed, the position of the correction lens 40 is adjusted by the moving mechanism, thereby switching the objective lens. The laser beam is condensed on the pupil plane, and the reflection angle of the two-dimensional scanner 32 is changed to guide the laser beam to the total reflection condition area of the pupil plane. Thereby, even when the objective lens 8 is switched, total reflection illumination of the specimen 5 can be realized.
Further, when performing total reflection fluorescence observation with the microscope apparatus 1, a configuration in which the laser light condensed on the pupil plane P of the objective lens 8 is scanned in the annular total reflection condition region by the two-dimensional scanner 32. Thus, better total reflection illumination can be realized.

Further, when scanning microscope observation, confocal observation, or total reflection fluorescence observation is performed with the microscope apparatus 1, laser light is emitted by selecting a use wavelength in a laser light source (not shown) in the confocal observation apparatus 25. Therefore, the wavelength selection filter is unnecessary for the fluorescent cube 15 to be used. However, the present invention is not limited to this, and it is of course possible to use a fluorescent cube provided with a wavelength selection filter. In this case, since it is possible to remove autofluorescence generated in each optical element constituting the confocal observation device 25, it is possible to prevent occurrence of flare and ghost.
As described above, according to the present embodiment, it is possible to perform epi-illumination fluorescence observation, confocal observation, and total reflection fluorescence observation, and it is possible to realize a microscope apparatus that is suppressed in size and cost.

It is a figure showing the outline of the microscope device concerning the embodiment of the present invention. It is a figure which shows the optical system of the microscope apparatus which concerns on embodiment of this invention, and the transmission illumination light source and the transmission illumination optical system are abbreviate | omitted. (A), (b), and (c) are the case of performing epifluorescence observation of a specimen, the case of performing scanning and confocal observation, and the total reflection fluorescence observation, respectively, with the microscope apparatus according to the embodiment of the present invention. It is a figure which shows the principal part of the optical system in the case of performing, and the behavior of illumination light.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Microscope main body 3 Control apparatus 4 Transmitted illumination light source 5 Specimen 8 Objective lens 10 Fluorescent cube holder 12 Eyepiece tube 15, 16 Fluorescent cube 18 Prism 25 Confocal observation apparatus 26 Epi-illumination apparatus 30 Exchange unit holder 32 Two-dimensional scanner 40 Correction lens P Pupil plane R Image plane (light source image) of objective lens 8
U1, U2 exchange unit

Claims (4)

Scanning means for scanning the specimen with illumination light from a first light source condensed on the specimen via the objective lens, and light reception for receiving observation light from the specimen via the objective lens and the scanning means. A confocal observation device having means;
An epi-illumination device having an introduction member for introducing illumination light from a second light source into the optical path of the confocal observation device, and irradiating the sample with illumination light from the second light source via the objective lens;
A fluorescence detection optical system that detects fluorescence from the specimen excited by the illumination light of the confocal observation device or the epi-illumination device, and
An imaging position converting member for forming a light source image of the first light source on a pupil plane of the objective lens;
A microscope apparatus, further comprising: an insertion / removal mechanism for switching and inserting the introduction member and the imaging position conversion member to substantially the same position in the optical path of the confocal observation apparatus.   The microscope apparatus according to claim 1, wherein the imaging position conversion member includes at least one lens and has a positive refractive power as a whole.   The distance from the position of the light source image of the first light source to the position of the front principal point of the imaging position conversion member is substantially equal to the focal length of the imaging position conversion member. Microscope device.   2. The insertion / removal mechanism includes a holding member that holds the introduction member and the imaging position conversion member so that the introduction member and the imaging position conversion member can be inserted and switched to substantially the same position in the optical path of the confocal observation apparatus. The microscope apparatus according to claim 3.

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