JP2010164834A - Microscope apparatus and fluorescent cube - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope apparatus enabling confocal observation and total-reflection fluorescent observation, and suppressing increase in size and cost thereof. <P>SOLUTION: The microscope apparatus includes: an illumination optical system including a scanning means 32 for scanning the surface of a sample 5 with illumination luminous flux, and configured to guide the illumination luminous flux from a light source to the sample 5; a fluorescence detection optical system for detecting the fluorescence from the sample 5; and a plurality of fluorescent cubes 16 and 70 disposed in the illumination optical system to guide the illumination luminous flux to the sample 5. At least one of the fluorescent cubes 16 and 70 includes a light condensing position changing means including: a sheet-like optical member 51 having a cone-shaped recessed part; a polarizing element 71 for changing the polarized state of the illumination light; and an optical member 52 having a plurality of the same-shaped lens parts 52a adjacent to one another on almost the same circumference around the optical axis; configured to make the principal light beam of the illumination luminous flux nearly parallel to the optical axis of the illumination optical system, change the polarized state of the illumination light, and condense the illumination luminous flux at the proper position in a prescribed zonal area separated from the optical axis of the pupil position P of an objective lens 8. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a microscope apparatus and a fluorescent cube.

  Conventionally, confocal microscopes and total reflection fluorescence microscopes have been widely used for observing specimens such as living cells, and recently, microscopes capable of switching between confocal observation and total reflection fluorescence observation have been proposed. (For example, refer to 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, and there is a problem that the size and cost are increased.

  Therefore, the present invention has been made in view of the above problems, and is capable of performing confocal observation and total reflection fluorescence observation, and provides a microscope apparatus and a fluorescent cube that can be reduced in size and cost. Objective.

In order to solve the above problems, the present invention
An illumination optical system that includes a scanning unit that scans the sample surface with the illumination light beam, and that guides the illumination light beam from the light source to the sample;
A fluorescence detection optical system for detecting fluorescence from the specimen;
A plurality of fluorescent cubes arranged in the illumination optical system and guiding the illumination light flux to the specimen;
At least one of the fluorescent cubes includes a plate-shaped optical member having a mortar-shaped recess, a polarizing element that changes a polarization state of illumination light, and a plurality of adjacent elements on substantially the same circumference around the optical axis. An optical member having a lens portion of the same shape, making the principal ray of the illumination light beam substantially parallel to the optical axis of the illumination optical system, changing the polarization state of the illumination light, In addition, the present invention provides a microscope apparatus characterized by having a condensing position converting means for condensing the illumination light beam at an appropriate position in a predetermined annular zone area away from the optical axis of the pupil position of the objective lens. .
The present invention also provides
A fluorescent cube which is disposed in an optical path of the illumination optical system of the fluorescence microscope in an exchangeable manner,
It has a plate-shaped optical member having a mortar-shaped recess, a polarizing element that changes the polarization state of illumination light, and a plurality of lens portions of the same shape that are adjacent to each other on substantially the same circumference around the optical axis. An optical member, and when arranged in the optical path of the fluorescence microscope, the principal ray of the illumination light beam applied to the sample via the objective lens is made substantially parallel to the optical axis of the illumination optical system. A condensing position converting means for condensing the illuminating light beam at an appropriate position in a predetermined annular zone area away from the optical axis of the pupil position of the objective lens while changing the polarization state of the illuminating light. A fluorescent cube is provided.

  According to the present invention, it is possible to perform confocal observation and total reflection fluorescence observation, and it is possible to provide a microscope apparatus and a fluorescence cube that are suppressed in size and cost.

1 is a diagram showing an outline of a microscope apparatus 100 according to a first embodiment of the present invention. It is a figure which shows the optical system of the microscope apparatus 100 which concerns on 1st Embodiment of this invention. It is a figure which shows the optical system of the microscope apparatus 100 at the time of performing the total reflection fluorescence observation of the sample 5 in 1st Embodiment of this invention. (A) is a figure which shows the structure of the total reflection fluorescence cube 70 in 1st Embodiment of this invention, (b), (c), (d) is the all-around lens 52, all-around wedge 51, all-around, respectively. It is the figure which looked at the modification of the wedge 51 from the incident side of illumination light. (A) is a figure which shows the structure of the perimeter polarization element 71 of the total reflection fluorescence cube 70 in 1st Embodiment of this invention, (b) is a figure which shows the polarization direction of the illumination light irradiated to the sample 5. is there. FIG. 6 is a diagram showing the behavior of illumination light between the imaging lens 29 and the pupil plane P of the objective lens 8 in the microscope apparatus 100 according to the first embodiment of the present invention, and FIG. At the time of confocal observation, (b) shows the time of total reflection fluorescence observation, and (c) shows a partially enlarged view of (b). It is a figure which shows the optical system of the microscope apparatus 200 which concerns on 2nd Embodiment of this invention. (A) is a figure which shows the structure of the total reflection fluorescence cube 80 in 2nd Embodiment of this invention, (b) is a figure which shows the modification.

Hereinafter, a microscope apparatus according to each embodiment of the present invention will be described in detail with reference to the accompanying drawings.
(First embodiment)
FIG. 1 is a diagram showing an outline of a microscope apparatus according to the first embodiment of the present invention, and FIG. 2 is a diagram showing an optical system of the microscope apparatus according to the first embodiment of the present invention.
As shown in FIG. 1, the microscope apparatus 100 according to the present embodiment includes an inverted microscope main body 2 and a control device 3.

The microscope main body 2 includes a transmission illumination optical system 6 that guides light from a transmission illumination light source 4 (to be described later) to the specimen 5, a stage 7 on which the specimen 5 is placed, and a plurality of objective lenses having different magnifications in order from the top along the optical axis. 8 is provided with a revolver 9, a fluorescent cube holder 10, an imaging optical system 11, and an eyepiece tube 12.
The fluorescent cube holder 10 is a holding member that is capable of selectively rotating the fluorescent cube 16 and the total reflection fluorescent cube 70 by a known rotating means. 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. The total reflection fluorescent cube 70 will be described later.
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, the transmitted illumination optical system 6, and the control device 3 are not shown.
As shown in FIG. 2, the confocal observation device 25 sequentially includes an imaging lens 29, a dichroic mirror 55 that can be inserted into and removed from the optical path by a known slide mechanism, a pupil relay lens 31, and two light beams. A two-dimensional scanner (galvanomirror) 32 that scans in dimension, a beam splitter 33, a collector lens 34, an optical fiber 35, a laser light source (not shown), and an imaging lens 36 disposed on the reflection optical path of the beam splitter 33; It consists of a pinhole 37 and a light receiving element (PMT) 38. In the present embodiment, a light source that outputs linearly polarized laser light is used as the laser light source (not shown).

As shown in FIG. 2, the epi-illumination device 26 is composed of a field stop 42, a collector lens 43, an optical fiber 44, and an epi-illumination light source (not shown) in this order from the dichroic mirror 55 side. 25 and shared. In the present embodiment, a laser light source is used as the epi-illumination light source.
The control device 3 includes a personal computer (PC) 45 that controls various devices mounted on the microscope body 2, a monitor 46, and the like.

  In the microscope apparatus 100 according to the present embodiment having such a configuration, as described below with reference to FIGS. 2 and 3, transmitted illumination observation in which the specimen 5 is transmitted and illuminated to observe the transmitted light, and the specimen 5 is incident illumination. Epi-illumination observation for observing fluorescence, scanning-type fluorescence observation for irradiating the specimen 5 by scanning the illumination light two-dimensionally and observing the fluorescence, and illuminating the specimen 5 for two-dimensional scanning with illumination light and reflecting Confocal observation for observing light and total reflection fluorescence observation for observing fluorescence by illuminating the sample 5 with total reflection can be appropriately switched. FIG. 3 is a diagram showing an optical system of the microscope apparatus 100 when performing total reflection fluorescence observation of the specimen 5 in the first embodiment of the present invention.

When performing transmission illumination observation of the specimen 5 with the microscope apparatus 100 according to the present embodiment, the fluorescent cube 16, the total reflection fluorescent cube 70, and the prism 18 of the fluorescent cube holder 10 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 100 according to the present embodiment, the fluorescent cube 16, the prism 18, and the dichroic mirror 55 of the fluorescent cube holder 10 are arranged in advance in the optical path.
Under this assumption, laser light from an epi-illumination light source (not shown) is guided by an optical fiber 44 and emitted from its end face, and then becomes a substantially parallel light beam through a collector lens 43, passes through a field stop 42, and reaches a dichroic mirror 55. Reflected by. The substantially parallel light beam is collected 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, and then the objective lens. The sample 5 on the stage 7 is irradiated through 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 100.

When scanning microscope observation and confocal observation of the specimen 5 are performed by the microscope apparatus 100 according to the present embodiment, the fluorescence cube 16 and the prism 18 of the fluorescence cube holder 10 are arranged in the optical path in advance, and the dichroic mirror 55 is arranged. Is withdrawn from the optical path.
Under this premise, laser light from a laser light source (not shown) is guided by the optical fiber 35 and emitted from the end face, and then passes through the beam splitter 33 through the collector lens 34 as a substantially parallel light beam. Then, the substantially parallel light beam is reflected by the two-dimensional scanner 32, is imaged on the image plane R by the pupil relay lens 31, and then becomes a substantially parallel light beam again through the imaging lens 29. The laser light thus converted into a substantially parallel light beam passes through the wavelength selection filter 16c of the fluorescent cube 16 and is reflected by the dichroic mirror 16a, so that light of a predetermined wavelength is selected and then collected by the objective lens 8. The sample 5 on the stage 7 is irradiated with light. 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 illuminated with the laser light and exhibits fluorescence and reflects the laser light.

  The fluorescence expressed in the sample 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, an observer can perform scanning fluorescence observation of the specimen 5 using the microscope apparatus 100.

On the other hand, the laser beam reflected by the specimen 5 is collected by the objective lens 8, reflected by the dichroic mirror 16a of the fluorescent cube 16, and then transmitted through the wavelength selection filter 16c. 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. As a result, the PC 45 generates a two-dimensional image of the specimen 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 by using the microscope apparatus 100.
In the microscope apparatus 100 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 and observed.

When total reflection fluorescence observation of the specimen 5 is performed by the microscope apparatus 100 according to the present embodiment, the total reflection fluorescence cube 70 of the fluorescence cube holder 10 and the prism 18 are arranged in the optical path in advance as shown in FIG. The dichroic mirror 55 is retracted out of the optical path.
Here, the total reflection fluorescent cube 70 of the fluorescent cube holder 10 according to the present embodiment emits illumination light inclined by a predetermined angle with respect to the optical axis in order from the illumination light incident side, as shown in FIG. An all-around wedge 51 that is substantially parallel to the optical axis, an all-round polarizing element 71 that changes the polarization state of illumination light from the all-around wedge 51, and illumination light that has passed through the all-round polarizing element 71 is the pupil plane P of the objective lens 8. It has an omnidirectional lens 52 having a plurality of lens portions 52a for condensing on it, a dichroic mirror 50a, and an emission filter 50b disposed on the transmission light path of the observation light. Detailed configurations of the all-around wedge 51, the all-round polarizing element 71, and the all-around lens 52 will be described later.

Under the above premise, laser light from a laser light source (not shown in FIG. 3) is guided by an optical fiber 35, emitted from its end face, converted into a substantially parallel light beam by a collector lens 34, and then transmitted through a beam splitter 33. Reflected by the two-dimensional scanner 32. At this time, the reflection angle of the two-dimensional scanner 32 is such that the substantially parallel light beam reflected by the two-dimensional scanner 32 is within the total reflection condition region on the pupil plane P of the objective lens 8 as shown in FIG. It is controlled by the PC 45 so as to be incident on a position (position D) that is decentered by a distance d from the positioned optical axis.
Here, the total reflection condition region is a ring 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. The range of the band shape. The distance d is set according to the total reflection condition region that varies depending on the type of the objective lens.

  The substantially parallel light beam reflected by the two-dimensional scanner 32 is imaged on the image plane R by the pupil relay lens 31, and then becomes a substantially parallel light beam again through the image forming lens 29, and is inclined by an angle α with respect to the optical axis. It is inclined and enters the entire circumferential wedge 51 in the total reflection fluorescent cube 70. The substantially parallel light beam incident on the all-around wedge 51 is refracted by the all-around wedge 51 so that its optical axis I1 is substantially parallel to the optical axis of the imaging lens 29 and decentered by a distance d. (See also FIG. 6B.) The substantially parallel light flux passes through the omnidirectional polarization element 71 and becomes light having a predetermined polarization direction, and enters the lens portion 52 a of the omnidirectional lens 52. Then, this light is reflected by the dichroic mirror 50a through the lens portion 52a, so that light having a predetermined wavelength is selected, and then at a position D in the total reflection condition region on the pupil plane P of the objective lens 8. Focused. In the case of performing scanning fluorescence observation and confocal observation with the microscope apparatus 100, as shown in FIG. 6A, the (+) maximum field angle light beam indicated by the dotted line from the imaging lens 29, indicated by the solid line. Neither the image center beam nor the (−) maximum field angle beam indicated by a broken line is collected at the pupil position P of the objective lens 8.

  The laser beam having a predetermined polarization direction collected in the total reflection condition region on the pupil plane P of the objective lens 8 in this way enters the sample 5 through the objective lens 8 at a total reflection angle. The sample 5 is incident on the sample 5 at an incident angle at which it is totally reflected at the boundary surface between the medium 5 and the glass substrate (not shown) that holds the sample 5, whereby the total reflection illumination of the sample 5 can be realized.

  When the specimen 5 is totally reflected and illuminated, evanescent light is generated at the boundary surface, and the evanescent light illuminates the vicinity of the boundary surface of the specimen 5 to express fluorescence. This fluorescence is collected by the objective lens 8 and passes through the dichroic mirror 50a and the emission filter 50b of the total reflection fluorescent cube 70, 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 using the microscope apparatus 100.

  Here, the configuration of the all-around wedge 51, the all-round polarizing element 71, and the all-around lens 52 in the above-described total reflection fluorescent cube 70 will be described in detail. FIGS. 4B and 4C are views of the omnidirectional lens 52 and the omnidirectional wedge 51 of the total reflection fluorescent cube 70 according to the first embodiment of the present invention as viewed from the illumination light incident side. FIG. 5 is a diagram showing the configuration of the all-round polarizing element 71 of the total reflection fluorescent cube 70 in the first embodiment of the present invention.

As shown in FIGS. 4A and 4C, the all-around wedge 51 is formed by forming a mortar-shaped dent in a prism member having a plane-parallel plate shape, and the dent is directed toward the illumination light incident side. Yes. Then, as shown in FIGS. 6B and 6C, the all-around wedge 51 emits illumination light (substantially parallel light beam) incident on the all-around wedge 51 at an inclination angle α with respect to the optical axis of the imaging lens 29. In order to refract the optical axis I1 so as to be substantially parallel to the optical axis of the imaging lens 29, the following relational expression (1) is satisfied.
(1) α = (n−1) × δ
However,
α: The inclination angle of the illumination light incident on the entire wedge 51 with respect to the optical axis of the imaging lens 29 δ: The apex angle of the entire wedge 51 (the angle between the incident surface and the exit surface of the illumination light in the entire wedge 51)
n: Refractive index with respect to d-line (wavelength 587.6 nm) of the medium constituting the entire wedge 51

As shown in FIGS. 4A and 4B, the omnidirectional lens 52 includes a plurality of lens portions 52a having the same shape adjacent to each other on substantially the same circumference centered on the optical axis, that is, each lens portion 52a. Is made of a glass member arranged adjacent to the optical axis by being decentered by a distance d. In the present embodiment, the lens portion 52a is composed of 12 plano-convex lens portions, and is arranged with the convex surface facing the illumination light incident side.
Note that the all-around wedge 51 and the all-around lens 52 having the above-described configuration do not need to have illumination light incident on the respective center portions, and therefore may have a shape that is hollowed out at the center portion. The material of the all-around wedge 51 and the all-around lens 52 is not limited to glass or plastic. The all-around lens 52 can also be constituted by a Fresnel lens, and the all-around wedge 51 can be constituted by bonding a plurality of wedge prisms having the same shape as shown in FIG.

As shown in FIG. 5A, the omnidirectional polarizing element 71 includes a plurality of wave plates 71a processed in the same fan shape provided corresponding to the plurality of lens portions 52a of the omnidirectional lens 52 described above. It is arranged adjacent to the axis and forms a circular appearance. In the present embodiment, the plurality of wave plates 71a are composed of twelve half-wave plates, which are shown in detail in FIG. 5A so that the directions of the optical axes of the adjacent half-wave plates are different. As shown by thick arrows, the optical axes perpendicular to the bisector of the central angle of the sector shape are respectively provided.
The location of the all-round polarizing element 71 in the total reflection fluorescent cube 70 is not limited to the position between the all-around wedge 51 and the all-around lens 52, but the incident side of the all-around wedge 51 and the exit of the all-around lens 52. It may be on the side. In particular, as in this embodiment, by arranging the all-round polarizing element 71 between the all-around wedge 51 and the all-around lens 52, the illumination light (substantially parallel light beam) from the all-around wedge 51 is sent to the all-round polarizing element 71. It becomes possible to make it enter perpendicularly with respect to the wavelength plate 71a, and to maximize the characteristics of the wavelength plate 71a.
In addition, the laser light source (not shown) used for the total reflection fluorescence observation of the specimen 5 in the present embodiment outputs linearly polarized laser light as described above, and such laser light is used as illumination light all around. The polarization direction when entering the polarizing element 71 is indicated by a thin arrow in FIG.

Under such a configuration, when linearly polarized illumination light enters the wave plate 71a, which is a half-wave plate, the polarization direction is rotated by 2θ and is emitted from the wave plate 71a (wave plate). The angle between the optical axis 71a and the polarization direction of the illumination light is denoted by θ. Specifically, for example, when the illumination light indicating the polarization direction in FIG. 5A is incident on A of the wave plate 71a, the polarization direction is 2θ as shown in A in FIG. 5B. The sample 5 is emitted after being rotated only by maintaining the polarization direction. The same applies to B and C in FIG. 5, and the polarization direction of the illumination light irradiated onto the specimen 5 is different as shown in FIG. 5B. Therefore, it is possible to change the polarization direction of the illumination light irradiated on the specimen 5 depending on which waveplate 71a the illumination light is incident on, that is, by making the illumination light incident on an arbitrary waveplate 71a. It is possible to specify the polarization direction of the illumination light irradiated on the specimen 5.
Note that the all-round polarizing element 71 having the above-described configuration does not require illumination light to be incident on the central portion, similarly to the above-described all-around wedge 51 and all-around lens 52, and thus may have a shape that has a hollow center portion. .

  As described above, the illumination light is incident on one lens portion 52a of the all-round lens 52 in the total reflection fluorescent cube 70, and the illumination light is applied to one point (position D) in the total reflection condition region on the pupil plane P of the objective lens 8. The case where the sample 5 is totally reflected only from one direction side by focusing is described. The microscope apparatus 100 according to the present embodiment is not limited to this, and by controlling the reflection angle of illumination light by the two-dimensional scanner 32 by the PC 45, the arbitrary wavelength plate 71 a in the all-round polarizing element 71 and the all-around lens 52 are included. It is also possible to cause the illumination light to enter the arbitrary lens portion 52a and to collect the illumination light at an arbitrary point in the total reflection condition region. Thereby, the sample 5 can be totally reflected and illuminated from an arbitrary direction by linearly polarized light having an arbitrary polarization direction. In other words, it is possible to perform total reflection illumination of the specimen 5 by selectively switching the combination of the direction in which the specimen 5 is irradiated with the illumination light and the polarization direction of the illumination light.

  Here, in general, when performing total reflection fluorescence observation, when observing fluorescence expressed from a specimen by irradiating the specimen with linearly polarized light in a predetermined polarization direction as illumination light, the degree of fluorescence expression depends on the orientation of the fluorescent molecules in the specimen. Different. For this reason, fluorescence can be observed when illumination light having a certain polarization direction is used, but a phenomenon may occur in which fluorescence cannot be observed when illumination light having a polarization direction different from the illumination light is used. In this way, in total reflection fluorescence observation, the information and characteristics of the specimen obtained differ depending on the polarization direction of the illumination light, so it is desirable that the polarization direction of the illumination light can be changed according to the required information and characteristics. On the other hand, the microscope apparatus 100 according to the present embodiment can illuminate the sample 5 with the linearly polarized light having an arbitrary polarization direction as described above, and therefore the fluorescence of the sample 5 in consideration of the polarization direction of the illumination light. It is possible to perform total reflection illumination of the specimen 5 with illumination light having a polarization direction optimum for the direction of molecules.

  Further, in the microscope apparatus 100 according to the present embodiment, the illumination light is scanned by controlling the two-dimensional scanner 32 by the PC 45, whereby a plurality of lens portions 52a (more preferably, all the lens portions 52a are included in the all-around lens 52. It is also possible to cause the illumination light to sequentially enter the light and to collect the illumination light at a plurality of points in the total reflection condition region. As a result, the specimen 5 can be sequentially totally reflected from a plurality of directions, and speckles caused by the surface reflection of each optical element arranged in the optical path from the optical fiber 35 to the objective lens 8 can be obtained. It is possible to eliminate unevenness of the pattern, that is, the intensity of illumination light, and realize more uniform total reflection illumination. Further, the illumination light is sequentially incident on the plurality of wavelength plates 71 a in the all-round polarizing element 71 by scanning the illumination light and sequentially entering the plurality of lens portions 52 a in the all-around lens 52. Therefore, total reflection illumination is performed while sequentially changing the polarization direction of the illumination light, and thus fluorescent molecules having different directions in the specimen 5 can be appropriately expressed and observed. In such a case, an image of the sample 5 is acquired each time the illumination light is scanned, and a plurality of acquired images are integrated and averaged by the PC 45, thereby acquiring a good image of the sample 5. It becomes possible.

  In the microscope apparatus 100 according to the present embodiment, when the total reflection fluorescence observation is performed by switching a plurality of objective lenses provided in the revolver 9, a total reflection fluorescence cube having the above configuration is prepared in advance for each objective lens. The total reflection fluorescence observation can be easily realized by mounting on the fluorescence cube holder 10 and switching the total reflection fluorescence cube together with the objective lens. Even in such a case, the PC 45 controls the reflection angle of the illumination light by the two-dimensional scanner 32 in accordance with the switching between the objective lens and the total reflection fluorescent cube, so that the position within the total reflection condition region that differs for each objective lens. Illumination light can be appropriately incident on D.

As described above, in the microscope apparatus 100 according to the present embodiment, the transmission illumination observation, the epifluorescence observation, the scanning fluorescence observation, the confocal observation, and the total reflection fluorescence observation of the sample 5 can be switched as appropriate.
In the microscope apparatus 100 according to this embodiment, the total reflection fluorescent cube 70 having the above-described configuration is mounted on the fluorescent cube holder 10 that is usually provided in a general fluorescent microscope, and this is used to perform confocal observation and total reflection fluorescence. Since switching to observation is realized, a large-scale switching mechanism such as a conventional microscope is not required, and it is possible to effectively suppress an increase in size and cost of the microscope main body 2 and confocal observation. Switching to total reflection fluorescence observation can be performed simply and quickly.

  Further, when performing total reflection fluorescence observation with the microscope apparatus 100 according to the present embodiment, not only the sample 5 is totally reflected from only one direction as described above, but also from an arbitrary direction or a plurality of different directions. Since total reflection illumination is possible, a better image of the specimen 5 from which the speckle pattern is eliminated can be acquired. Further, as described above, since the total reflection illumination can be performed by changing the polarization direction of the illumination light, the image of the specimen 5 in which the fluorescent molecules are appropriately expressed regardless of the orientation of the fluorescent molecules in the specimen 5. Can be obtained.

In addition, the all-round polarizing element 71 in the total reflection fluorescent cube 70 in the microscope apparatus 100 according to the present embodiment is not limited to the above configuration, and may be configured as follows (this is described in the second embodiment described later). The same applies to the form.)
For example, when linearly polarized laser light output from a light source used for total reflection fluorescence observation is converted into circularly polarized light in the optical path and guided to the total reflection fluorescent cube 70, or a light source that outputs circularly polarized laser light as the light source , Each fan-shaped wave plate 71a of the all-round polarizing element 71 is composed of twelve quarter-wave plates each having an optical axis perpendicular to the bisector of the fan-shaped central angle. Also good. With this configuration, it is possible to achieve the same effect as when each wave plate 71a is formed of a half-wave plate as in the present embodiment.

  Also, for example, when linearly polarized laser light output from a light source used for total reflection fluorescence observation is converted into circularly polarized light or random polarized light in the optical path and guided to the total reflection fluorescent cube 70, or circularly polarized light or random polarized light is used as the light source. In the case of using a light source that outputs the laser beam, each fan-shaped wave plate 71a of the all-round polarizing element 71 is provided with an optical axis perpendicular to the bisector of the central angle of the fan-shaped, and circularly polarized light and random You may comprise with the polarizer which converts polarized light into linearly polarized light. With this configuration, it is possible to achieve the same effect as when each wave plate 71a is formed of a half-wave plate as in the present embodiment.

  Further, for example, each of the fan-shaped wave plates 71a of the all-round polarizing element 71 is constituted by twelve half-wave plates configured so that the directions of the respective optical axes coincide with each other. It is good also as a structure further provided with the well-known rotation means for rotating the omnidirectional polarizing element 71 centering on an optical axis further, comprised by the sheet | seat of circular 1/2 wavelength plate. With this configuration, even when the illumination light is incident on any lens portion 52a in the all-around lens 52 by controlling the reflection angle of the two-dimensional scanner 32, the sample 5 is irradiated by rotating the all-round polarizing element 71. It is possible to arbitrarily change the polarization direction of the illumination light. That is, the sample 5 can be totally reflected and illuminated from an arbitrary direction by linearly polarized light having an arbitrary polarization direction. This also applies to the case where the omnidirectional polarizing element 71 is composed of a quarter-wave plate or a polarizer as described above.

For example, the all-round polarizing element 71 may be configured by a polarizer that converts illumination light into only s-polarized light, or a polarizer that converts illumination light into only p-polarized light. Thereby, the total reflection illumination of the specimen 5 can be realized with illumination light of only s-polarized light or only p-polarized light.
Here, the direction of the fluorescent molecule for the fluorescent molecule to express the fluorescence is determined by the direction of the dipole, and the fluorescent molecule is biased in the direction perpendicular to the polar axis of the dipole to express the fluorescence (see “Seventh Light No pencil "21 Dark Field Observation Method 4 page 424).
Therefore, when the sample is illuminated with s-polarized illumination light, fluorescent molecules oriented in parallel with the sample boundary surface are mainly excited, and the fluorescence intensity distribution becomes maximum in the optical axis direction (z direction). For this reason, as described above, by realizing total reflection illumination of the specimen 5 with illumination light of only s-polarized light, the light collection efficiency of the objective lens 8 is increased, and a bright fluorescent image can be obtained.
On the other hand, when the specimen is illuminated with p-polarized illumination light, not only fluorescent molecules oriented in parallel to the specimen boundary surface but also fluorescent molecules oriented in the vertical direction are excited, and the latter fluorescence intensity distribution is in a direction perpendicular to the optical axis. Maximum. For this reason, when the total reflection illumination of the specimen 5 by the illumination light of only p-polarized light is realized as described above, it is intended to collect the fluorescence expressed by the fluorescent molecules in the direction perpendicular to the specimen boundary surface with the objective lens. Since the fluorescence intensity distribution is extremely biased toward the high NA side, it is difficult to obtain a bright fluorescent image, and in particular, it is necessary to use an objective lens with a high NA, but the optical axis direction cannot be obtained with total reflection illumination using s-polarized light. It is possible to obtain information on fluorescent molecules in

  Further, for example, the omnidirectional polarizing element 71 may be constituted by a quarter wavelength plate that converts linearly polarized light into circularly polarized light. Thereby, the total reflection illumination of the specimen 5 can be realized with illumination light in which s-polarized light and p-polarized light are mixed, and the effects of both the total reflection illumination by s-polarization and the total reflection illumination by p-polarization described above can be achieved. It becomes possible.

(Second Embodiment)
In the present embodiment, portions having the same configuration as in the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions will be described in detail. FIG. 7 is a diagram showing an optical system of a microscope apparatus according to the second embodiment of the present invention. In FIG. 7, the transmitted illumination light source 4, the transmitted illumination optical system 6, and the control device 3 are not shown.

As shown in FIG. 7, the microscope apparatus 200 according to the present embodiment includes a first dichroic mirror 61 that can be inserted into and removed from the optical path by a known slide mechanism between the fluorescent cube holder 10 and the objective lens 8 in the microscope body 2. It has. On the reflected light path of the first dichroic mirror 61, an imaging unit 64 including an emission filter 62, an imaging lens 63, and an imaging element 27 is provided.
In addition, a second slide which can be inserted into and removed from the optical path by a known slide mechanism between the imaging lens 17 of the imaging optical system 11 and the mirror 19a in the microscope body 2 in place of the prism 18 of the first embodiment. A dichroic mirror 65 is provided. A confocal observation device 25 similar to that in the first embodiment is disposed on the reflected light path of the second dichroic mirror 65.
In the microscope body 2, only the epi-illumination device 26 similar to that in the first embodiment is disposed on the incident light path of the illumination light of the fluorescent cube holder 10.

  The fluorescent cube holder 10 includes a total reflection fluorescent cube 80 shown in an enlarged view in FIG. 8A in place of the total reflection fluorescent cube 70 of the first embodiment. In the present embodiment, the total reflection fluorescent cube 80 includes, in order from the illumination light incident side (imaging lens 17 side), an all-around wedge 51, an all-round polarizing element 71, and an all-around lens having the same configuration as that of the first embodiment. 52.

In the microscope apparatus 200 according to the present embodiment having such a configuration, the transmission illumination observation, the epifluorescence observation, the scanning fluorescence observation, the confocal observation, and the total reflection fluorescence observation of the sample 5 are appropriately switched as described below. be able to.
When performing transmission illumination observation of the specimen 5 with the microscope apparatus 200 according to the present embodiment, the fluorescent cube 16 and the total reflection fluorescent cube 80 of the fluorescent cube holder 10, the first dichroic mirror 61, and the second dichroic mirror 65 are preliminarily provided. Is withdrawn from the optical path. Under this premise, the observer can perform transmitted illumination observation of the specimen 5 as in the first embodiment.

When the epi-fluorescence observation of the specimen 5 is performed by the microscope apparatus 200 according to the present embodiment, the fluorescent cube 16 of the fluorescent cube holder 10 and the first dichroic mirror 61 are arranged in advance in the optical path.
Under this assumption, the laser light emitted from the optical fiber 44 passes through the field stop 42 as a substantially parallel light beam via the collector lens 43. The substantially parallel light beam is collected by the imaging lens 29, passes through the wavelength selection filter 16c of the fluorescent cube 16 and is reflected by the dichroic mirror 16a, so that light having a predetermined excitation wavelength is selected, and then the first dichroic light. The light passes through the mirror 61 and is further irradiated onto the specimen 5 through the objective lens 8.

  As a result, the fluorescence expressed in the sample 5 is collected by the objective lens 8, reflected by the first dichroic mirror 61, and transmitted through the emission filter 62, so that light having an unnecessary wavelength is cut. This fluorescence is imaged on the imaging surface of the imaging device 27 by the imaging lens 63. As a result, the imaging device 27 captures a fluorescent image of the specimen 5 and displays it on the monitor 46, so that the observer can perform epifluorescence observation of the specimen 5.

When performing the scanning fluorescence observation and the confocal observation of the specimen 5 by the microscope apparatus 200 according to the present embodiment, the first dichroic mirror 61 and the second dichroic mirror 65 are arranged in the optical path in advance, and the fluorescence cube holder 10 The fluorescent cube 16 and the total reflection fluorescent cube 80 are retracted out of the optical path.
Under this premise, the laser light emitted from the optical fiber 35 is imaged on the image plane R in the same manner as in the first embodiment, and then is reflected by the second dichroic mirror 65, whereby light having a predetermined wavelength is obtained. Is selected. This laser light again becomes a substantially parallel light beam through the imaging lens 17, is condensed by the objective lens 8, and is irradiated on the sample 5. The laser light is two-dimensionally scanned by the two-dimensional scanner 32 as in the first embodiment. Therefore, the specimen 5 is excited by the laser light to express fluorescence and reflects the laser light.

Fluorescence expressed in the sample 5 is collected by the objective lens 8, reflected by the first dichroic mirror 61, and transmitted through the emission filter 62, so that light having an unnecessary wavelength is cut. This fluorescence is imaged on the imaging surface of the imaging device 27 via the imaging lens 63. As a result, the imaging device 27 captures a fluorescent image of the specimen 5 and displays it on the monitor 46, so that the observer can perform scanning fluorescence observation of the specimen 5.
On the other hand, the laser beam reflected by the specimen 5 is condensed by the objective lens 8, passes through the first dichroic mirror 61 and the imaging lens 17, and then is reflected by the second dichroic mirror 65, which is the same as in the first embodiment. Similarly, it reaches the light receiving element 38. The light reaching the light receiving element 38 is descanned by the two-dimensional scanner 32. Then, a two-dimensional image of the sample 5 based on the light intensity signal obtained by the light receiving element 38 is displayed on the monitor 46, so that the observer can perform confocal observation of the sample 5.

When total reflection fluorescence observation of the specimen 5 is performed by the microscope apparatus 200 according to this embodiment, the total reflection fluorescence cube 80, the first dichroic mirror 61, and the second of the fluorescence cube holder 10 are preliminarily shown in FIG. A dichroic mirror 65 is disposed in the optical path.
Under this premise, the laser light emitted from the optical fiber 35 is imaged on the image plane R in the same manner as in the first embodiment, and then is reflected by the second dichroic mirror 65, whereby light having a predetermined wavelength is obtained. Is selected. 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 position D in the total reflection condition region on the pupil plane P of the objective lens 8 as in the first embodiment. Yes. Such laser light again becomes a substantially parallel light beam through the imaging lens 17 and is incident on the total reflection fluorescent cube 80 with an inclination angle α with respect to the optical axis.

  Similar to the first embodiment, the substantially parallel light beam incident on the total reflection fluorescent cube 80 is refracted by the entire circumferential wedge 51 to become a substantially parallel light beam that is substantially parallel to the optical axis and decentered by the distance d. The light passes through the circumferential polarization element 71 and becomes light having a predetermined polarization direction. Then, the substantially parallel light beam passes through the entire circumference lens 52, passes through the first dichroic mirror 61, and then is condensed at a position D in the total reflection condition region on the pupil plane P of the objective lens 8. This laser light is incident on the sample 5 through the objective lens 8 at a total reflection angle, and thereby, it is possible to realize total reflection illumination of the sample 5 by laser light having a predetermined polarization direction.

  When the sample 5 is illuminated by total reflection, fluorescence is generated from the sample 5 as in the first embodiment. The fluorescence is condensed by the objective lens 8, then reflected by the first dichroic mirror 61 and transmitted through the emission filter 62, so that light having an unnecessary wavelength is cut. The image is formed on the surface. As a result, the imaging device 27 captures a fluorescent image of the sample 5 and displays it on the monitor 46, whereby the observer can perform total reflection fluorescence observation of the sample 5.

  As in the first embodiment, the microscope apparatus 200 is not limited to the total reflection illumination of the specimen 5 only from one direction, and the reflection angle of the illumination light of the two-dimensional scanner 32 is controlled by the PC 45. Thus, since the sample 5 can be totally reflected from an arbitrary direction or a plurality of different directions, a better image of the sample 5 in which the speckle pattern is eliminated can be acquired. Further, similarly to the first embodiment, since the total reflection illumination can be performed by changing the polarization direction of the illumination light, the fluorescent molecules are appropriately expressed regardless of the orientation of the fluorescent molecules in the sample 5. An image of the specimen 5 can be acquired.

  As described above, in the microscope apparatus 200 according to the present embodiment, the transmission illumination observation, the epifluorescence observation, the scanning fluorescence observation, the confocal observation, and the total reflection fluorescence observation of the sample 5 can be switched as appropriate. The same effects as those of the first embodiment can be obtained. Further, the total reflection fluorescent cube 80 having the above-described configuration does not need to include the dichroic mirror 50a in the total reflection fluorescent cube 70 of the first embodiment, so that the all-round wedge 51 and the all-round lens 52 can be more easily accommodated and stored. It is possible to arrange.

In the microscope apparatus 200 according to the present embodiment, the configuration of the total reflection fluorescent cube 80 used in the total reflection fluorescence observation of the specimen 5 is not limited to the above-described configuration. For example, the total reflection described below with reference to FIG. A fluorescent cube 81 can also be used.
As shown in FIG. 8B, the total reflection fluorescent cube 81 includes a cone-shaped mirror 68, a mortar-shaped mirror 69, an all-round polarizing element 71 having the same configuration as that of the first embodiment, in order from the imaging lens 17 side. And an all-round lens 52.

  The cone-shaped mirror 68 is a reflecting member formed by forming a mirror surface on the side surface of the conical member, and is disposed on the optical axis with the mirror surface facing the imaging lens 17 side. The mortar-shaped mirror 69 is a reflecting member in which a mortar-shaped dent is provided on a parallel flat plate member to form a mirror surface, and a through-hole is provided at the center of the mirror surface. It is arranged on the optical axis toward the mold mirror 68 side. The distance between the cone-shaped mirror 68 and the mortar-shaped mirror 69 and the angle of each mirror surface are set so that a substantially parallel light beam traveling on the optical axis is decentered by a distance d substantially parallel to the optical axis. Has been. The cone-shaped mirror 68 and the mortar-shaped mirror 69 can also be configured by bonding a plurality of mirrors having the same shape.

  When total reflection fluorescence observation of the specimen 5 is performed using the total reflection fluorescent cube 81 having the above-described configuration, the illumination light (substantially parallel light beam) traveling on the optical axis and entering the total reflection fluorescent cube 81 is a mortar-shaped mirror 69. , And is sequentially reflected by the cone-shaped mirror 68 and the mortar-shaped mirror 69 to form ring-shaped illumination light substantially parallel to the optical axis of the imaging lens 17. The light enters the wave plate 71a. The illumination light incident on each wavelength plate 71 a passes through each wavelength plate 71 a and becomes light having a different polarization direction, and is incident on each lens portion 52 a of the all-around lens 52. The illumination light that has passed through the lens unit 52a is collected on the circumference D of the distance d in the total reflection condition area D on the pupil plane P of the objective lens 8, that is, the radius around the optical axis in the total reflection condition area. Lighted. Therefore, the ring-shaped illumination light is applied to the specimen 5 at the total reflection angle via the objective lens 8, and it is possible to realize a good total reflection illumination that effectively eliminates the speckle pattern. Become. At this time, since the ring-shaped illumination light is composed of a plurality of linearly polarized light beams having different polarization directions, the fluorescent molecules in the specimen 5 can be appropriately expressed regardless of the direction. In such a case, it is not necessary to scan the illumination light by the two-dimensional scanner 32, and it is not necessary to take and accumulate a plurality of images of the specimen 5, so that the total reflection fluorescence observation can be performed more quickly. Is possible.

  As described above, according to each of the above-described embodiments, it is possible to perform confocal observation and total reflection fluorescence observation, and it is possible to realize a microscope apparatus that suppresses an increase in size and cost, and a fluorescent cube used in the microscope apparatus. .

DESCRIPTION OF SYMBOLS 100 Microscope apparatus 2 Microscope main body 3 Control apparatus 4 Transmission illumination light source 5 Specimen 8 Objective lens 10 Fluorescent cube holder 12 Eyepiece tube 16 Fluorescence cube 18 Prism 25 Confocal observation apparatus 26 Epi-illumination apparatus 32 Two-dimensional scanner 70 Total reflection fluorescence cube 71 All-round polarizing element 51 All-round wedge 52 All-round lens 80, 81 Total reflection fluorescent cube 68 Cone type mirror 69 Mortar-shaped mirror 200 Microscope device P Pupil plane R of objective lens 8 Image plane (light source image)

Claims (15)

An illumination optical system that includes a scanning unit that scans the sample surface with the illumination light beam, and that guides the illumination light beam from the light source to the sample;
A fluorescence detection optical system for detecting fluorescence from the specimen;
A plurality of fluorescent cubes arranged in the illumination optical system and guiding the illumination light flux to the specimen;
At least one of the fluorescent cubes includes a plate-shaped optical member having a mortar-shaped recess, a polarizing element that changes a polarization state of illumination light, and a plurality of adjacent elements on substantially the same circumference around the optical axis. An optical member having a lens portion of the same shape, making the principal ray of the illumination light beam substantially parallel to the optical axis of the illumination optical system, changing the polarization state of the illumination light, The microscope apparatus further comprises a condensing position converting means for condensing the illumination light beam at an appropriate position in a predetermined annular zone area away from the optical axis of the pupil position of the objective lens.   The fluorescent cube having the condensing position converting means further includes a dichroic mirror that guides the illumination light beam to the objective lens and separates reflected light and fluorescence from the specimen. The microscope apparatus according to 1.   Control means for controlling the scanning means to guide the illumination light to a predetermined position on the pupil plane of the objective lens when the fluorescent cube having the condensing position converting means is arranged in an optical path. The microscope apparatus according to claim 1 or 2.   The fluorescent cube having the condensing position converting means includes, instead of the plate-like optical member, a conical side-surface-shaped reflecting member and a mortar-shaped reflecting member having a through-hole at the center. The microscope apparatus according to any one of claims 1 to 3.   The microscope apparatus according to any one of claims 1 to 4, further comprising an epi-illumination optical system that guides an epi-illumination light beam from an epi-illumination light source to the specimen through the objective lens.   The polarizing element in the condensing position converting means includes a plurality of 1 / different directions of optical axes provided on substantially the same circumference around the optical axis corresponding to the lens portions in the optical member. The microscope apparatus according to claim 1, comprising a two-wavelength plate.   The polarizing element in the condensing position converting means includes a plurality of 1 / different directions of optical axes provided on substantially the same circumference around the optical axis corresponding to the lens portions in the optical member. The microscope apparatus according to claim 1, comprising a four-wave plate.   The polarizing element in the condensing position converting means has substantially the same circumference centered on the optical axis corresponding to the lens portions in the optical member in order to convert circularly polarized light and random polarized light into linearly polarized light. The microscope apparatus according to claim 1, wherein the microscope apparatus includes a plurality of polarizers having different transmission axis directions.   6. The polarizing element in the condensing position converting means is composed of a half-wave plate, and has a rotating means for rotating the half-wave plate around an optical axis. The microscope apparatus according to any one of the above.   The microscope apparatus according to any one of claims 1 to 5, wherein the polarizing element in the condensing position converting means is a quarter wavelength plate.   The microscope apparatus according to claim 10, further comprising a rotating unit that rotates the quarter-wave plate about an optical axis.   The polarizing element in the condensing position converting means is composed of a polarizer for converting circularly polarized light and random polarized light into linearly polarized light, and has rotating means for rotating the polarizer around an optical axis. The microscope apparatus according to any one of claims 1 to 5.   The microscope apparatus according to any one of claims 1 to 5, wherein the polarizing element in the condensing position converting unit includes a polarizer that converts the illumination light into only s-polarized light.   The microscope apparatus according to any one of claims 1 to 5, wherein the polarizing element in the condensing position converting unit includes a polarizer that converts the illumination light into only p-polarized light. A fluorescent cube which is disposed in an optical path of the illumination optical system of the fluorescence microscope in an exchangeable manner,
It has a plate-shaped optical member having a mortar-shaped recess, a polarizing element that changes the polarization state of illumination light, and a plurality of lens portions of the same shape that are adjacent to each other on substantially the same circumference around the optical axis. An optical member, and when arranged in the optical path of the fluorescence microscope, the principal ray of the illumination light beam applied to the sample via the objective lens is made substantially parallel to the optical axis of the illumination optical system. A condensing position converting means for condensing the illuminating light beam at an appropriate position in a predetermined annular zone area away from the optical axis of the pupil position of the objective lens while changing the polarization state of the illuminating light. A fluorescent cube characterized by containing.

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