JP2007072391A - Laser microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a laser microscope capable of accurately acquiring an LSM image at a different deep part on a sample with high NA, while keeping the total reflection illuminating range fixed. <P>SOLUTION: The laser microscope which has a laser scanning illumination optical system 10 and a total reflection illumination optical system 20, and where the two illumination optical systems hold an objective 12 in common has a plane parallel plate 23, arranged on a total reflection illuminating optical path nearer to the light source side than to the imaging lens 13 and constituted so that the tilt angle varies as a means of adjusting the angle of total reflected illuminating principal rays, capable of changing the angle formed by the principal rays of the total reflection illuminating luminous flux with the pupil surface of the objective 12 to an angle, at which the total reflection illumination range on the sample 50 is kept the same, according to the change of a distance between the objective 12 and the sample 50. Thus, the total reflected illumination range on the sample 50 is kept fixed, regardless of the variations in the distance between the objective 12 and the sample 50. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microscope capable of acquiring, for example, a confocal image (LSM image) by scanning laser illumination and an evanescent fluorescence image (TIRFM image) by total reflection illumination.

In recent years, for example, in functional analysis of living cells, in order to observe the function of a cell membrane, a confocal image (LSM image) by scanning laser illumination and an evanescent fluorescence image (TIRFM image) by total reflection illumination can be acquired. It has been needed.
For this reason, conventionally, for example, a microscope for observing a confocal image by scanning laser illumination and an evanescent fluorescence image by total reflection illumination has been proposed, as in the microscope described in Patent Document 1 below.
JP 2003-307682 A

  In the microscope described in Patent Document 1, a lens for condensing illumination light on a specimen via an objective lens and a lens for condensing illumination light on the exit pupil of the objective lens are selectively inserted into the optical path. Thus, the LSM image and the TIRFM image can be selectively acquired.

In addition, for example, a microscope has been proposed in which an objective lens is arranged above and below a specimen and a laser can be incident from above and below, like a microscope described in Patent Document 2 below.
JP 2002-55282 A

  In the microscope described in Patent Document 2, total reflection illumination is performed from the objective lens on the lower side of the specimen, and LSM observation can be performed from the objective lens on the upper side of the specimen.

In recent years, in order to observe the function of cell membranes in detail in functional analysis of living cells, a confocal image (LSM image) obtained by scanning laser illumination is acquired while giving a stimulus by totally reflecting the surface of the sample. Has been needed.
However, the microscope described in Patent Document 1 cannot acquire an LSM image while illuminating the surface of the sample with TIRFM. Further, in a microscope capable of acquiring a conventional LSM image and a TIRFM image, such as the microscope described in Patent Document 1, the focus position in the specimen of the objective lens for scanning in the optical axis direction in LSM observation Is changed accordingly, the illumination range (TIRFM illumination range) of the specimen surface irradiated with the total reflection illumination light changes, and there is a problem that evanescent fluorescence cannot be obtained in a desired observation target region.

Moreover, in order to observe a living cell with a microscope as described in Patent Document 2, a water immersion objective lens must be used as the upper LSM objective lens.
That is, when a living cell is observed from above with an upright microscope, a glass bottom dish is usually used. This is because if a live cell is sandwiched between cover glasses, the movement of the live cell is inhibited and the live cell is easily damaged. For this reason, when observing a living cell from the upper side via the LSM objective lens, a liquid intervenes between the living cell on the glass bottom dish and the LSM objective lens. Here, when an oil immersion objective lens is used as the LSM objective lens, the oil comes into contact with the living cells, and the living cells are easily damaged. Therefore, when observing live cells upright, it is most efficient to use a water immersion objective lens.
However, the water immersion objective lens has a smaller NA than the oil immersion objective lens.
For this reason, the microscope as described in Patent Document 2 cannot accurately observe a high cell with high NA when observing a living cell.

  The present invention has been made in view of the above problems, and while maintaining the total reflection illumination range constant, it is possible to accurately acquire LSM images at different depths in a specimen with high NA, and preferably LSM images and TIRFM images. An object of the present invention is to provide a laser microscope capable of simultaneously obtaining the above.

  In order to achieve the above object, a laser microscope according to the present invention includes a laser scanning illumination optical system and a total reflection illumination optical system, and the two illumination optical systems share an objective lens. The total reflection illumination can change the angle between the principal ray of the total reflection illumination light beam and the pupil plane of the objective lens to an angle that keeps the total reflection illumination range in the sample in the same range according to the change in the distance to A chief ray angle adjusting means is provided, and the total reflection illumination range on the specimen can be kept constant regardless of the variation in the distance between the objective lens and the specimen.

  The laser microscope according to the present invention includes a laser scanning illumination optical system and a total reflection illumination optical system, and the two illumination optical systems share an imaging lens and an objective lens. The angle of the principal ray of the total reflection illumination light beam and the pupil plane of the objective lens can be changed to an angle at which the total reflection illumination range of the sample is kept in the same range according to the change in the distance of A light beam angle adjusting means is provided, and the total reflection illumination range to the specimen can be kept constant regardless of the variation in the distance between the objective lens and the specimen.

  Further, in the laser microscope of the present invention, the total reflection illumination principal ray angle adjusting means is disposed on the total reflection illumination optical path on the light source side with respect to the imaging lens, and has a variable inclination angle. A face plate is preferred.

  Further, in the laser microscope of the present invention, the total reflection illumination principal ray angle adjusting means is disposed on the total reflection illumination optical path on the light source side with respect to the imaging lens, and at least one of the mutual positional relationship is vertical or horizontal. A pair of wedge-shaped prisms configured to be variable in direction is preferable.

  In the laser microscope of the present invention, the total reflection illumination principal ray angle adjusting means is disposed on the total reflection illumination optical path closer to the light source than the imaging lens, and is parallel to each other and inclined with respect to the optical axis. However, it is preferable that at least one of the reflecting surfaces is configured to be movable in a direction in which the mutual distance changes.

  Further, in the laser microscope of the present invention, the total reflection illumination optical system has a fiber on the total reflection illumination optical path on the light source side than the imaging lens, and the total reflection illumination principal ray angle adjusting means includes: The exit end face of the fiber is preferably configured to be adjustable in angle.

  The laser microscope according to the present invention further includes interlock control means for linking the adjustment of the total reflection illumination principal ray angle by the total reflection illumination principal ray angle adjustment means with a change in the distance between the objective lens and the specimen. Is preferred.

  In the laser microscope of the present invention, a total reflection fluorescent image pickup device that picks up an evanescent fluorescent image by the total reflection illumination of the total reflection illumination optical system, and a total reflection that forms the evanescent fluorescent image on the image pickup surface of the image pickup device. A fluorescence image imaging optical system, and in conjunction with a change in the distance between the objective lens and the specimen, the relative distance between the total reflection fluorescent image imaging element and the total reflection fluorescent image imaging optical system is determined by the evanescent It is preferable to have a second interlock control unit that changes the fluorescent image so that it is focused on the imaging surface of the image sensor.

  According to the present invention, it is possible to obtain a laser microscope capable of accurately acquiring LSM images at different depths in a specimen with high NA, and preferably simultaneously acquiring LSM images and TIRFM images while keeping the total reflection illumination range constant. It is done.

First Embodiment FIG. 1 is a schematic configuration diagram of a laser microscope according to a first embodiment of the present invention, and FIG. 2 is a principle explanatory view showing an operation of a main part in the laser microscope of FIG.
The microscope according to the first embodiment includes a laser scanning illumination optical system 10, a total reflection illumination optical system 20, a personal computer 30, and a monitor 40.

  The laser scanning illumination optical system 10 includes a laser light source unit 11, an objective lens 12, an imaging lens 13, a scanning unit 14, a pupil projection lens 15, and a detection optical system 16. In the figure, reference numeral 17 denotes, for example, a dichroic mirror that transmits the laser light from the laser light source unit 11 toward the sample 50 and reflects the light from the sample 50 that has passed through the pupil projection lens 15 toward the detection optical system 16. An optical branching member 18 such as a half mirror reflects the laser light from the laser light source 11 that has passed through the imaging lens 13 toward the specimen 50, and the light from the specimen 50 is reflected on the imaging lens 13 on the detection optical system 16 side. For example, a dichroic mirror or a half mirror.

The laser light source unit 11 includes a laser light source 11a, a coupling lens 11b, an optical fiber 11c, and a collimating lens 11d. The laser light emitted from the laser light source 11a is transmitted through the coupling lens 11b. The laser light introduced into the optical fiber 11c and emitted from the optical fiber 11c is converted into parallel light through the collimating lens 11d.
The scanning unit 14 is configured by a galvanometer mirror, and can scan the laser beam from the laser light source unit 11 in a two-dimensional direction on the sample 50.
The pupil projection lens 15 is disposed between the scanning unit 14 and the imaging lens 13, and laser light deflected by the scanning unit 14 is passed through the imaging lens 13 on the pupil plane of the objective lens 12. The light is projected onto the position and condensed onto the sample 50 via the imaging lens 13 and the objective lens 2.
The detection optical system 16 includes a pinhole imaging lens 16a, a pinhole 16b, a condenser lens 16c, and a photomultiplier 16d. The detection optical system 16 emits from the sample 50, and includes the objective lens 12 and the imaging lens 13. The light passing through the pupil projection lens 14 and reflected by the light branching member 17 is detected.

The total reflection illumination optical system 20 includes a laser light source unit 21, a lens 22, a light branching member 24, an objective lens 12, an imaging lens 13, a light branching member 18, and a detection optical system 25. Has been.
That is, in the laser microscope of the first embodiment, the objective lens 12, the light branching member 18, and the imaging lens 13 are used as common optical members for the laser scanning illumination optical system 10 and the total reflection illumination optical system 20.

The laser light source unit 21 includes a laser light source 21a, a coupling lens 21b, and an optical fiber 21c. The laser light source 21 introduces laser light emitted from the laser light source 21a into the optical fiber 21c through the coupling lens 21b. The light is emitted from the optical fiber 21c.
The lens 22 condenses the laser light emitted from the optical fiber 21 c at a predetermined position on the pupil plane of the objective lens 12 via the imaging lens 13, and the sample via the imaging lens 13 and the objective lens 12. 50 is configured to be projected on the diagonal.

The light branching member 24 is composed of a dichroic mirror having a characteristic of reflecting the laser light from the laser light source unit 21 and transmitting other wavelengths (laser light from the laser light source unit 11 and light from the sample 50). ing. The optical branching member 24 can be inserted into and removed from the optical path by using a mirror, and a TIRFM image can be obtained via the total reflection illumination optical system when inserted into the optical path, and the laser can be removed when removed from the optical path. An LSM image may be obtained via the scanning illumination optical system.
The light branching member 18 reflects the laser light from the laser light source unit 11 that has passed through the imaging lens 13 described above toward the sample 50 and directs the light from the sample 50 toward the imaging lens 13 on the detection optical system 16 side. In addition to the characteristic of reflecting, the laser light from the laser light source unit 21 that has passed through the imaging lens 13 is reflected toward the sample 50 and also has the characteristic of transmitting the evanescent fluorescence from the sample 50 toward the detection optical system 25. ing.

The detection optical system 25 includes an imaging lens 25a and a CCD 25b, and captures an evanescent fluorescence image emitted from the sample 50 and passed through the objective lens 12 and the light branching member 18.
The relative distance between the sample 50 and the objective lens 12 can be adjusted by moving a stage (not shown), for example.
The personal computer 30 is connected to the laser scanning illumination optical system 10 and the total reflection illumination optical system 20, and performs predetermined conversion processing on the signal information obtained via the respective detection optical systems 16 and 25. And a function capable of controlling the optical members constituting the laser scanning illumination optical system 10 and the total reflection illumination optical system 20 and the like.

Here, as a characteristic configuration of the present invention, in the laser microscope of the first embodiment, a transparent parallel plate 23 as a total reflection illumination principal ray angle adjusting means is provided between the lens 22 and the light branching member 24. Yes.
The transparent parallel flat plate 23 is configured to be rotatable about an axis 23a, and according to a change in the distance between the objective lens 12 and the sample 50, the principal ray of the total reflection illumination light beam and the pupil plane of the objective lens 12 The angle formed is rotated by a rotation amount that can be changed to an angle at which the total reflection illumination range in the sample 50 is maintained within the same range.

The relationship between the rotation of the parallel plate 23 accompanying the change in the distance between the objective lens 12 and the sample 50 and the total reflection illumination range will be described with reference to FIG. In FIG. 2, for convenience of explanation, the optical branching member 24 is omitted, and the configuration of FIG. 1 and the optical path length are not matched. Here, the focal position of the objective lens 12 is P.
When the surface 50a of the sample 50 is located at the position A1 (the position indicated by the broken line), the laser scanning illumination light is focused on a predetermined portion of the surface 50a of the sample 50. At this time, the transparent parallel plate 23 intersects perpendicularly to the optical axis, as indicated by a broken line. The total reflection illumination light M1 transmitted through the transparent parallel plate 23 follows a path indicated by a broken line, and is condensed at a predetermined position on the pupil plane of the objective lens 12, shifted to the objective lens 12, and then incident on the objective lens 12. A peripheral region 50b including the condensing position P of the laser scanning illumination light on the surface 50a of the sample 50 is projected through the lens 12 from an oblique direction.

On the other hand, when the surface 50a of the sample 50 is positioned at the position A2 (the position indicated by the solid line), the laser scanning illumination light is focused on a predetermined portion inside the sample 50.
Here, in the conventional laser microscope, the sample 50 is projected through the objective lens 12 along the same path as the total reflection illumination light M1. In this case, the projection region 50b ′ on the surface 50a of the sample 50 is displaced from the projection region 50b when the surface 50a of the sample 50 is positioned at the position A1.
On the other hand, in the laser microscope of the first embodiment, the transparent parallel plate 23 can be tilted by a predetermined angle with respect to the incident optical axis as shown by a solid line. The total reflection illumination light M2 incident on the transparent parallel plate 23 is refracted at the incident surface and the exit surface of the transparent parallel plate 23, and is emitted with the output optical axis shifted parallel to the incident optical axis, as indicated by the solid line. In addition, as compared with the total reflection illumination light M1, the principal ray travels a path having an angle different from the pupil plane of the objective lens 12, and is condensed at a predetermined position on the pupil plane of the objective lens 12, and then the objective. The light is incident on the lens 12, and the peripheral region 50 b on the extension of the condensing position P of the laser scanning illumination light on the surface 50 a of the sample 50 is projected through the objective lens 12 from an oblique direction.
Thus, in the laser microscope of the first embodiment, even if the distance between the objective lens 12 and the sample 50 varies by rotating the transparent parallel plate 23 as the total reflection illumination principal ray angle adjusting means by a predetermined amount, The same range (peripheral region 50b) in the sample 50 can be totally reflected.

  The rotational amount and rotational driving of the transparent parallel plate 23 can be automatically controlled via the personal computer 30 shown in FIG. In that case, a means for detecting the distance between the objective lens 12 and the surface 50a of the sample 50 (for example, a means for detecting the amount of movement of the stage on which the sample 50 is placed, the amount of rotation of the gear, and between the stage and the objective lens barrel) The personal computer 30 calculates the amount of rotation based on the distance detected by the distance detecting means, and drives the transparent parallel plate 23 to rotate with the calculated amount of rotation. do it.

  According to the laser microscope of the first embodiment configured as described above, the laser light emitted from the laser light source 11 of the laser scanning illumination optical system 10 passes through the light branching member 17 and is scanned in a predetermined direction by the scanning unit 14. It is scanned and transmitted through the light branching member 24, passes through the pupil projection lens 15 and the imaging lens 13, is reflected by the light branching member 18, passes through the objective lens 12, and is condensed on a predetermined portion of the sample 50. The light from the sample 50 follows the reverse path such as the objective lens 12, the light branching member 18, the imaging lens 13, the light branching member 24, the pupil projection lens 24, and the scanning unit 14, and enters the light branching member 17. To do. The light reflected by the light branching member 17 is received by the photomultiplier 16d of the detection optical system 16. The light received by the photomultiplier 16d is subjected to signal conversion processing by the personal computer 30 and displayed on the monitor 40. Thereby, an LSM image is obtained.

The laser light emitted from the laser light source unit 21 of the total reflection illumination optical system 20 passes through the lens 22, the transparent parallel plate 23, the light branching member 24, the imaging lens 13, and the light branching member 18, and the pupil of the objective lens 12. After being condensed at a predetermined position on the surface, it is projected obliquely onto the sample 50. Thereby, the surface of the sample 50 can be stimulated by total reflection illumination. The evanescent fluorescence generated by totally reflecting the sample 50 is incident on the light branching member 18 through the objective lens 12 together with the totally reflected illumination light. The light branching member 18 transmits only evanescent fluorescence and reflects other wavelengths. The evanescent fluorescence transmitted through the light branching member 18 is imaged by the CCD 25b of the detection optical system 25.
The captured image is displayed on the monitor 40 via the personal computer 30. As a result, when the CCD 25b is focused and imaged, a TIRFM image is obtained.

Here, when acquiring LSM images of the sample 50 at different depths, a stage (not shown) is driven to vary the distance between the objective lens 12 and the sample.
At this time, in the laser microscope of the first embodiment, the transparent parallel flat plate 23 is rotated by a predetermined amount in conjunction with the change in the distance between the objective lens 12 and the sample via the personal computer 30, so that the same range of the sample 50 ( The peripheral area 50b) can be totally reflected illuminated. Therefore, as described with reference to FIG. 2, LSM images at different depths in the sample 50 can be acquired while giving a stimulus by totally reflecting the same range on the surface of the sample 50.

The laser microscope according to the first embodiment includes a laser scanning illumination optical system 10 and a total reflection illumination optical system 20, and is a laser microscope in which two illumination optical systems share the objective lens 12. Since the illumination range can be kept constant, a microscope is used to obtain an LSM image of the deep part of the sample 50 while totally reflecting the vicinity of the cover glass supporting the bottom surface of the sample 50 with one objective lens from the lower side. Can be configured.
If the microscope is configured in this way, when the sample 50 to be observed is a living cell in the glass bottom dish, a liquid is interposed between the objective lens 12 and the cover glass at the bottom of the glass bottom dish. Thus, the LSM image can be obtained, and the sample 50 in the glass bottom dish does not need to be pressed with a cover glass or contacted with the liquid used for the immersion objective lens from above. Therefore, an LSM image can be obtained by using an oil immersion objective lens for the objective lens 12. The immersion type objective lens has a larger NA than the water immersion type objective lens.
For this reason, according to the laser microscope of the first embodiment, an LSM image can be obtained by using an oil immersion objective lens for the objective lens 12, and the objective on the lower side of the specimen as described in Patent Document 2 is used. Compared with a microscope that performs total reflection illumination from a lens and performs LSM observation from an objective lens on the upper side of the sample, the sample 50 can be observed with high NA and high accuracy.

In the laser microscope of the first embodiment, the imaging position of the CCD 25b in the detection unit 25 is conjugate with the focal position P of the objective lens 12 in the laser scanning illumination optical system 10. For this reason, if the distance between the objective lens 12 and the sample 50 is varied in order to acquire LSM images at different depths other than the surface of the sample 50, the evanescent fluorescence is incident on the CCD 25b in a defocused state. A TIRFM image cannot be acquired.
Therefore, at least one of the imaging lens 25a and the CCD 25b is moved by a predetermined amount along the optical axis in conjunction with the change in the relative distance between the sample 50 and the objective lens 12, and the evanescent fluorescent image is placed on the imaging surface of the CCD 25b. It is preferable to control via the personal computer 30 so that the relative distance between the imaging lens 25a and the CCD 25b is changed so that an image is formed in focus. In this way, it is possible to acquire a TIRFM image always in focus simultaneously with acquisition of LSM images at different depths.

Second Embodiment FIG. 3 is a schematic configuration diagram of a laser microscope according to a second embodiment of the present invention, and FIG. 4 is a principle explanatory view showing the operation of the main part of the laser microscope of FIG.
In the laser microscope of the second embodiment, as a total reflection illumination principal ray angle adjusting means, a pair of wedge-shaped prisms 23 a ′ and 23 b ′ are provided between the lens 22 and the light branching member 24 instead of the transparent parallel plate 23. Is provided.
As shown in FIG. 4, the pair of wedge-shaped prisms 23a ′ and 23b ′ are arranged with point symmetry and parallel intervals. One wedge-shaped prism 23b ′ is configured such that the positional relationship with the other wedge-shaped prism 23a ′ can be varied in the vertical or horizontal direction (vertical direction in FIG. 4). The amount of movement that can change the angle between the principal ray of the total reflected illumination light beam and the pupil plane of the objective lens 12 to an angle that maintains the total reflected illumination range in the sample 50 in accordance with the change in the distance of So you can move around.
Other configurations are substantially the same as those of the laser microscope of the first embodiment.

The relationship between the movement of the wedge-shaped prism 23b ′ accompanying the change in the distance between the objective lens 12 and the sample 50 and the total reflection illumination range will be described with reference to FIG. In FIG. 4, for convenience of explanation, the light branching member 24 is omitted, and the configuration of FIG. 3 and the optical path length are not matched. Here, the focal position of the objective lens 12 is P.
When the surface 50a of the sample 50 is located at the position A1 (the position indicated by the broken line), the laser scanning illumination light is focused on a predetermined portion of the surface 50a of the sample 50. At this time, the wedge-shaped prism 23b ′ is close to the wedge-shaped prism 23a ′ as indicated by a broken line. The total reflection illumination light M1 refracted at the exit surface of the wedge prism 23a ′ immediately enters the wedge prism 23b ′. The total reflection illumination light M1 refracted at the entrance surface of the wedge-shaped prism 23b ′ follows a path indicated by a broken line, and is condensed at a predetermined position on the pupil plane of the objective lens 12 which is shifted from the optical axis. The incident light is incident on the surface 50a of the sample 50 through the objective lens 12, and the peripheral region 50b including the condensing position P of the laser scanning illumination light is projected obliquely.

On the other hand, when the surface 50a of the sample 50 is positioned at the position A2 (the position indicated by the solid line), the laser scanning illumination light is focused on a predetermined portion inside the sample 50.
Here, in the conventional laser microscope, the sample 50 is projected through the objective lens 12 along the same path as the total reflection illumination light M1. In this case, the projection region 50b ′ on the surface 50a of the sample 50 is displaced from the projection region 50b when the surface 50a of the sample 50 is positioned at the position A1.
On the other hand, in the laser microscope of the second embodiment, the wedge-shaped prism 23b ′ can be positioned farther from the wedge-shaped prism 23a ′ than the broken-line position, as indicated by the solid line. The total reflection illumination light M2 refracted on the exit surface of the wedge prism 23a ′ extends longer than the position of the wedge prism 23b ′ indicated by the broken line and enters the wedge prism 23b ′. The total reflection illumination light M2 refracted at the entrance surface of the wedge-shaped prism 23b ′ has an angle that is different from the total reflection illumination light M1 with respect to the pupil plane of the objective lens 12 as compared with the total reflection illumination light M1. The laser scanning illumination light on the surface 50a of the sample 50 is incident on the objective lens 12 through the objective lens 12 after being condensed at a predetermined position shifted from the optical axis on the pupil plane of the objective lens 12. The peripheral region 50b on the extension of the condensing position P is projected obliquely.
As described above, in the laser microscope according to the second embodiment, the wedge prism 23b ′ as the total reflection illumination principal ray angle adjusting means is changed, and the positional relationship between the wedge prisms 23a ′ and 23b ′ is changed in the vertical or horizontal direction. Thus, even if the distance between the objective lens 12 and the sample 50 varies, the same range (peripheral region 50b) in the sample 50 can be totally reflected and illuminated.

  The moving amount and driving of the wedge prism 23b 'can be automatically controlled via the personal computer 30 shown in FIG. In that case, a means for detecting the distance between the objective lens 12 and the surface 50a of the sample 50 (for example, a means for detecting the amount of movement of the stage on which the sample 50 is placed, the amount of rotation of the gear, and between the stage and the objective lens barrel) The personal computer 30 calculates the amount of movement based on the distance detected by the distance detecting means, and drives the wedge-shaped prism 23b ′ with the calculated amount of movement. do it.

According to the laser microscope of the second embodiment configured as described above, the wedge-shaped prism 23b ′ is moved by a predetermined amount in conjunction with the change in the distance between the objective lens 12 and the sample via the personal computer 30. By changing the relative distance between the wedge-shaped prisms 23a ′ and 23b ′, the same range (peripheral region 50b) of the sample 50 can be totally reflected. Therefore, as described with reference to FIG. 4, LSM images at different depths in the sample 50 can be acquired while giving a stimulus by totally reflecting the same range of the surface of the sample 50.
In the laser microscope according to the second embodiment, at least one of the imaging lens 25a and the CCD 25b is moved by a predetermined amount along the optical axis in conjunction with the change in the relative distance between the sample 50 and the objective lens 12. The evanescent fluorescent image is controlled via the personal computer 30 so as to change the relative distance between the imaging lens 25a and the CCD 25b so that the imaging surface of the CCD 25b is focused on. It is preferable to do this. In this way, it is possible to acquire a TIRFM image always in focus simultaneously with acquisition of LSM images at different depths.
Other functions and effects are substantially the same as those of the laser microscope of the first embodiment.

Third Embodiment FIG. 5 is a schematic block diagram of a laser microscope according to a third embodiment of the present invention, and FIG. 6 is a principle explanatory view showing the operation of the main part of the laser microscope of FIG.
In the laser microscope of the third embodiment, a pair of mirrors 23a ″ and 23b ″ are provided between the lens 22 and the light branching member 24 as total reflection illumination principal ray angle adjusting means instead of the transparent parallel plate 23. ing.
The pair of mirrors 23a "and 23b" are arranged parallel to each other and inclined with respect to the optical axis. The one mirror 23b ″ is configured to be movable in a direction in which the distance between the reflecting surfaces of the pair of mirrors 23a ″ and 23b ″ changes, and according to a change in the distance between the objective lens 12 and the sample 50. Then, the angle formed by the principal ray of the total reflection illumination light beam and the pupil plane of the objective lens 12 can be moved by an amount of movement that can be changed to an angle at which the total reflection illumination range in the sample 50 is kept in the same range. ing.
Other configurations are substantially the same as those of the laser microscope of the first embodiment.

The relationship between the movement of the mirror 23b ″ accompanying the change in the distance between the objective lens 12 and the sample 50 and the total reflection illumination range will be described with reference to FIG. 6. In FIG. 5 is omitted and the optical path length is not matched, and here, the focal position of the objective lens 12 is P.
When the surface 50a of the sample 50 is located at the position A1 (the position indicated by the broken line), the laser scanning illumination light is focused on a predetermined portion of the surface 50a of the sample 50. At this time, as shown by the broken line, the mirror 23b ″ is close to the mirror 23a ″ in the distance between the reflecting surfaces. The total reflection illumination light M1 reflected by the reflecting surface of the mirror 23a "immediately enters the mirror 23b". The total reflection illumination light M1 reflected by the reflecting surface of the mirror 23b ″ follows a path indicated by a broken line, and is condensed at a predetermined position on the pupil plane of the objective lens 12 and then enters the objective lens 12. The peripheral region 50b including the condensing position P of the laser scanning illumination light on the surface 50a of the sample 50 is projected obliquely through the objective lens 12.

On the other hand, when the surface 50a of the sample 50 is positioned at the position A2 (the position indicated by the solid line), the laser scanning illumination light is focused on a predetermined portion inside the sample 50.
Here, in the conventional laser microscope, the sample 50 is projected through the objective lens 12 along the same path as the total reflection illumination light M1. In this case, the projection region 50b ′ on the surface 50a of the sample 50 is displaced from the projection region 50b when the surface 50a of the sample 50 is positioned at the position A1.
On the other hand, in the laser microscope of the third embodiment, the mirror 23b ″ can be positioned farther from the mirror 23a ″ than the broken line, as indicated by the solid line. The total reflection illumination light M2 reflected by the reflection surface of the mirror 23a ″ extends longer than the position of the mirror 23b ″ indicated by the broken line and enters the mirror 23b ″. The total reflection illumination light reflected by the reflection surface of the mirror 23b ″. M2 follows a path having an angle different from that of the principal ray of the objective lens 12 as compared with the total reflection illumination light M1, as indicated by a solid line, and the optical axis on the pupil plane of the objective lens 12 After being condensed at a predetermined position shifted from each other, the light is incident on the objective lens 12, and the peripheral region 50 b on the extension of the condensing position P of the laser scanning illumination light on the surface 50 a of the sample 50 through the objective lens 12 is obliquely viewed. Project.
Thus, in the laser microscope of the third embodiment, the mirror 23b ″ as the total reflection illumination principal ray angle adjusting means is moved so that the distance between the reflection surfaces of the mirrors 23a ″ and 23b ″ changes. Even if the distance between the objective lens 12 and the sample 50 varies, the same range (the peripheral region 50b) in the sample 50 can be totally reflected.

  The moving amount and driving of the mirror 23b ″ can be automatically controlled via the personal computer 30 shown in FIG. 5. In that case, the distance between the objective lens 12 and the surface 50a of the sample 50 is detected. Means (for example, means for detecting the amount of rotation of the stage on which the sample 50 is mounted, means for detecting the amount of rotation of the gear, and sensor for detecting the distance between the stage and the objective lens barrel) are provided. The movement amount may be calculated based on the distance detected in (5), and the mirror 23b ″ may be driven with the calculated movement amount.

According to the laser microscope of the third embodiment configured as described above, the mirror 23b ″ is moved by a predetermined amount in conjunction with the change in the distance between the objective lens 12 and the sample via the personal computer 30, and the mirror 23a. The same range (peripheral region 50b) of the sample 50 can be totally reflected by changing the relative distance of the reflection surface of “, 23b” .For this reason, as described with reference to FIG. LSM images at different depths in the sample 50 can be acquired while stimulating the same area of the 50 surface with total reflection illumination.
In the laser microscope according to the third embodiment, at least one of the imaging lens 25a and the CCD 25b is moved by a predetermined amount along the optical axis in conjunction with the change in the relative distance between the sample 50 and the objective lens 12. The evanescent fluorescent image is controlled via the personal computer 30 so as to change the relative distance between the imaging lens 25a and the CCD 25b so that the imaging surface of the CCD 25b is focused on. It is preferable to do this. In this way, it is possible to acquire a TIRFM image always in focus simultaneously with acquisition of LSM images at different depths.
Other functions and effects are substantially the same as those of the laser microscope of the first embodiment.

Fourth Embodiment FIG. 7 is a schematic configuration diagram of a laser microscope according to a fourth embodiment of the present invention, and FIG. 8 is a principle explanatory view showing an operation of a main part in the laser microscope of FIG.
In the laser microscope of the fourth embodiment, instead of providing an optical member such as the transparent parallel plate 23 as a total reflection illumination principal ray angle adjusting means, a total reflection illumination is performed according to a change in the distance between the objective lens 12 and the sample 50. The angle of the exit end face of the optical fiber 21c is adjusted by an angle that can change the angle between the principal ray of the luminous flux and the pupil plane of the objective lens 12 so that the total reflection illumination range in the sample 50 is kept in the same range. It is configured to be possible.
Other configurations are substantially the same as those of the laser microscope of the first embodiment.

The relationship between the direction of the emission end face of the optical fiber 21c and the total reflection illumination range according to the change in the distance between the objective lens 12 and the sample 50 will be described with reference to FIG. In FIG. 8, for convenience of explanation, the light branching member 24 is omitted, and the configuration of FIG. 7 and the optical path length are not matched. Here, the focal position of the objective lens 12 is P.
When the surface 50a of the sample 50 is located at the position A1 (the position indicated by the broken line), the laser scanning illumination light is focused on a predetermined portion of the surface 50a of the sample 50. At this time, the emission end face of the optical fiber 21c is perpendicular to the axis passing through the center of the lens 22, as indicated by a broken line. The total reflection illumination light M1 emitted from the emission end face of the optical fiber 21c and incident on the lens 22 follows a path indicated by a broken line, and is condensed at a predetermined position on the pupil plane of the objective lens 12, and then the objective lens. 12, and the peripheral region 50 b including the condensing position P of the laser scanning illumination light on the surface 50 a of the sample 50 is projected from the oblique direction through the objective lens 12.

On the other hand, when the surface 50a of the sample 50 is positioned at the position A2 (the position indicated by the solid line), the laser scanning illumination light is focused on a predetermined portion inside the sample 50.
Here, in the conventional laser microscope, the sample 50 is projected through the objective lens 12 along the same path as the total reflection illumination light M1. In this case, the projection region 50b ′ on the surface 50a of the sample 50 is displaced from the projection region 50b when the surface 50a of the sample 50 is positioned at the position A1.
On the other hand, in the laser microscope of the fourth embodiment, the emission end face of the optical fiber 21c can be tilted with respect to the axis passing through the center of the lens 22, as indicated by the solid line. The total reflection illumination light M2 emitted from the emission end face of the optical fiber 21c and incident on the lens 22 has an angle different from that of the total reflection illumination light M1 and the pupil surface of the objective lens 12 of the principal ray, as shown by a solid line. The laser scanning illumination light on the surface 50a of the sample 50 is incident on the objective lens 12 through the objective lens 12 after being condensed at a predetermined position shifted from the optical axis on the pupil plane of the objective lens 12. The peripheral region 50b on the extension of the condensing position P is projected obliquely.
As described above, in the laser microscope of the fourth embodiment, the distance between the objective lens 12 and the sample 50 is changed by making the angle of the exit end face of the optical fiber 21c adjustable as a total reflection illumination principal ray angle adjusting means. In addition, the same range (peripheral region 50b) in the sample 50 can be totally reflected.

  The direction of the emission end face of the optical fiber 21c and the driving in a predetermined direction can be automatically controlled via the personal computer 30 shown in FIG. In that case, a means for detecting the distance between the objective lens 12 and the surface 50a of the sample 50 (for example, a means for detecting the amount of movement of the stage on which the sample 50 is placed, the amount of rotation of the gear, and between the stage and the objective lens barrel) The personal computer 30 calculates the direction and the driving amount based on the distance detected by the distance detecting means, and uses the calculated direction and driving amount to determine the exit end face of the optical fiber 21c. The direction may be driven in a predetermined direction.

According to the laser microscope of the fourth embodiment configured as described above, by adjusting the direction of the emission end face of the optical fiber 21c in conjunction with the change in the distance between the objective lens 12 and the sample via the personal computer 30. The same range (peripheral region 50b) of the sample 50 can be totally reflected. Therefore, as described with reference to FIG. 8, LSM images at different depths in the sample 50 can be acquired while giving the same range of the surface of the sample 50 by performing a totally reflected illumination.
Also in the laser microscope of the fourth embodiment, at least one of the imaging lens 25a and the CCD 25b is moved by a predetermined amount along the optical axis in conjunction with the change in the relative distance between the sample 50 and the objective lens 12. The evanescent fluorescent image is controlled via the personal computer 30 so as to change the relative distance between the imaging lens 25a and the CCD 25b so that the imaging surface of the CCD 25b is focused on. It is preferable to do this. In this way, it is possible to acquire a TIRFM image always in focus simultaneously with acquisition of LSM images at different depths.
Other functions and effects are substantially the same as those of the laser microscope of the first embodiment.

Fifth Embodiment FIG. 9 is a schematic configuration diagram of a laser microscope according to a fifth embodiment of the present invention.
The laser microscope of the fifth embodiment is provided with the light branching member 26a, the lens 26b, the mirrors 26c and 26d, and the lens instead of including the laser light source unit 21 to the light branching member 24 in the laser microscopes of the first to fourth embodiments. 26e and a light branching member 26f are provided, and a part of the laser light from the pupil projection lens 15 in the laser scanning illumination optical system 10 is used as total reflection illumination light.
And the transparent parallel plate 23 as a total reflection illumination principal ray angle adjustment means is provided between the lens 26e and the light branching member 26f.

  The light branching members 26a and 26f reflect only the wavelength for total reflection illumination of the laser light from the laser light source unit 11 that has passed through the pupil projection lens 15, and transmit the other wavelengths. It consists of an ic mirror. When the light branching members 26a and 26f are configured to be inserted into and removed from the optical path using a mirror, and when inserted into the optical path, a TIRFM image is obtained via the total reflection illumination optical system and removed from the optical path. Alternatively, an LSM image may be obtained via a laser scanning illumination optical system. In this case, the optical members from the light branching member 26a to the light branching member 26f may be configured as a unit so as to be inserted into and removed from the optical path of the laser scanning illumination optical system.

The lenses 26 b and 26 e condense the laser light that has passed through the pupil projection lens 15 to a predetermined position on the pupil plane of the objective lens 12 through the imaging lens 13 and through the imaging lens 13 and the objective lens 12. It is configured to project on the sample 50 from an oblique direction.
The transparent parallel flat plate 23 as the total reflection illumination principal ray angle adjusting means is configured to be rotatable like the transparent parallel flat plate 23 in the laser microscope of the first embodiment. The total reflection illumination principal ray angle adjusting means may be constituted by a pair of wedge prisms in the laser microscope of the second embodiment, instead of the transparent parallel plate 23. Or you may comprise with a pair of mirror in the laser microscope of 3rd embodiment.
Other configurations and operational effects are almost the same as those of the laser microscope of the first embodiment.

Sixth Embodiment FIG. 10 is a schematic configuration diagram of a laser microscope according to a sixth embodiment of the present invention.
In the laser microscope of the sixth embodiment, the laser scanning illumination optical system 10 and the total reflection illumination optical system 20 have imaging lenses 13 and 27, respectively, and a light branching member 28 on a common optical path. The objective lens 12 is configured to be shared. The light branching member 18 is configured by a dichroic mirror, a half mirror, and the like, reflects the laser light from the laser light source unit 11 that has passed through the imaging lens 13 toward the sample 50, and reflects the light from the sample 50 as an imaging lens. 13 has a characteristic of transmitting evanescent fluorescence from the specimen 50 generated by irradiating light from the laser light source unit 21 toward the detection optical system 25 in addition to the characteristic of reflecting toward the light source 13. The light branching member 28 is composed of a dichroic mirror, a half mirror, and the like, and transmits the laser light from the laser light source unit 11 that has passed through the imaging lens 13 and the light branching member 18 toward the sample 50, and is transmitted from the sample 50. In addition to the characteristic of transmitting light toward the light branching member 18, the laser light from the laser light source unit 21 that has passed through the imaging lens 27 is reflected toward the sample 50, and evanescent fluorescence from the sample 50 is detected by the detection optical system 25. It has the property of transmitting toward
As in the laser microscope of the first embodiment, a transparent parallel plate 23 as a total reflection illumination principal ray angle adjusting means is provided between the lens 22 and the imaging lens 27.
The total reflection illumination principal ray angle adjusting means may be constituted by a pair of wedge prisms in the laser microscope of the second embodiment, instead of the transparent parallel plate 23. Or you may comprise with a pair of mirror in the laser microscope of 3rd embodiment.
Other configurations and operational effects are almost the same as those of the laser microscope of the first embodiment.

  The laser microscope of the present invention is useful in fields where it is necessary to observe an LSM image with a microscope while stimulating a sample surface with TIRFM illumination in medicine, biology, pharmacy, agriculture, or the like.

It is a schematic block diagram of the laser microscope concerning 1st embodiment of this invention. FIG. 2 is a principle explanatory view showing an operation of a main part in the laser microscope of FIG. 1. It is a schematic block diagram of the laser microscope concerning 2nd embodiment of this invention. FIG. 4 is a principle explanatory view showing an operation of a main part in the laser microscope of FIG. 3. It is a schematic block diagram of the laser microscope concerning 3rd embodiment of this invention. FIG. 6 is a principle explanatory view showing an operation of a main part in the laser microscope of FIG. 5. It is a schematic block diagram of the laser microscope concerning 4th embodiment of this invention. FIG. 8 is a principle explanatory view showing an operation of a main part in the laser microscope of FIG. 7. It is a schematic block diagram of the laser microscope concerning 5th embodiment of this invention. It is a schematic block diagram of the laser microscope concerning 6th embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Laser scanning illumination optical system 11 Laser light source part 11a Laser light source 11b Coupling lens 11c Optical fiber 11d Collimate lens 12 Objective lens 13, 25, 27 Imaging lens 14 Scanning means 15 Pupil projection lens 16, 25 Detection optical system 16a Pinhole connection Image lens 16b Pinhole 16c Condensing lens 16d Photomultiplier 17, 18, 24, 26a, 26f, 28 Optical branching member 20 Total reflection illumination optical system 21 Laser light source 21a Laser light source 21b Coupling lens 21c Optical fiber 22, 26b, 26e Lens 23 Transparent parallel plate 23 'A pair of wedge-shaped prisms 23a', 23b 'Wedge-shaped prism 23 "A pair of mirrors 23a", 23b ", 26c, 26d Mirror 25b CCD
30 Personal computer 40 Monitor 50 Sample 50a Surface 50 of sample 50b Peripheral region including focusing position P of laser scanning illumination light on sample 50

Claims (8)

In a laser microscope having a laser scanning illumination optical system and a total reflection illumination optical system, and the two illumination optical systems share an objective lens,
In accordance with a change in the distance between the objective lens and the specimen, the angle formed by the principal ray of the total reflected illumination light beam and the pupil plane of the objective lens is changed to an angle at which the total reflected illumination range in the specimen is maintained in the same range. A laser microscope characterized by having a total reflection illumination principal ray angle adjusting means, and capable of keeping the total reflection illumination range on the specimen constant regardless of variations in the distance between the objective lens and the specimen. . In a laser microscope having a laser scanning illumination optical system and a total reflection illumination optical system, and the two illumination optical systems share an imaging lens and an objective lens,
In accordance with a change in the distance between the objective lens and the specimen, the angle formed by the principal ray of the total reflected illumination light beam and the pupil plane of the objective lens is changed to an angle at which the total reflected illumination range in the specimen is maintained in the same range. A laser microscope characterized by having a total reflection illumination principal ray angle adjusting means, and capable of keeping the total reflection illumination range on the specimen constant regardless of variations in the distance between the objective lens and the specimen. . The total reflection illumination principal ray angle adjusting means,
3. The laser microscope according to claim 1, wherein the laser microscope is a parallel plane plate that is disposed on a total reflection illumination optical path closer to the light source than the imaging lens and has a variable inclination angle. The total reflection illumination principal ray angle adjusting means,
A pair of wedge-shaped prisms arranged on the total reflection illumination optical path on the light source side with respect to the imaging lens, and at least one of which is configured to be capable of changing the mutual positional relationship in the vertical or horizontal direction. The laser microscope according to claim 1 or 2. The total reflection illumination principal ray angle adjusting means,
A pair disposed on the total reflection illumination optical path on the light source side than the imaging lens, configured to be parallel to each other and inclined with respect to the optical axis, and at least one of which can move in a direction in which the mutual distance changes. The laser microscope according to claim 1, wherein the laser microscope is a reflective surface. The total reflection illumination optical system has a fiber on the total reflection illumination optical path on the light source side of the imaging lens,
3. The laser microscope according to claim 1, wherein the total reflection illumination chief ray angle adjusting means is an emission end face of the fiber configured to be adjustable in angle.   The interlock control means for linking the adjustment of the total reflection illumination chief ray angle by the total reflection illumination chief ray angle adjustment means with a change in the distance between the objective lens and the specimen. The laser microscope according to claim 3, which is dependent on 2. A total reflection fluorescent image pickup element that picks up an evanescent fluorescent image by the total reflection illumination of the total reflection illumination optical system; and a total reflection fluorescent image pickup optical system that forms the evanescent fluorescent image on an image pickup surface of the image pickup element. ,
In conjunction with the change in the distance between the objective lens and the specimen, the relative distance between the total reflection fluorescent image imaging device and the total reflection fluorescent image imaging optical system is indicated, and the evanescent fluorescent image is the imaging surface of the imaging device. The laser microscope according to claim 1, further comprising a second interlocking control unit that changes the image so that the image is focused in a focused state.

Images