JP4128387B2 - Microscope equipment - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microscope apparatus capable of selectively acquiring a confocal image (LSM image) by a laser microscope and an evanescent fluorescence image by a fluorescence microscope.
[0002]
[Prior art]
Recently, functional analysis of living cells has been actively performed. Among these functional analyzes of cells, in order to observe the function of the cell membrane, in particular, a fluorescence microscope that acquires an evanescent fluorescence image from the cell membrane and the vicinity thereof has attracted attention.
[0003]
This type of fluorescence microscope often uses laser light as a light source, and recently, a microscope capable of selectively acquiring a confocal image (LSM image) by a laser microscope and an evanescent fluorescence image by a fluorescence microscope. A device is considered.
[0004]
FIG. 3 shows a schematic configuration of a scanning laser microscope. Laser light emitted from a laser light source 1 is condensed on an incident end of an optical fiber 3 by a condensing lens 2 and passes through the optical fiber 3 from an emission end. The emitted light is converted into parallel light by the collimator lens 4, passes through the dichroic mirror 5, and enters the scanner 6. The scanner 6 is projected onto the exit pupil plane 9 a of the objective lens 9 by the pupil projection lens 7, and the laser beam deflected by the scanner 6 is placed on the stage 10 through the imaging lens 8 and the objective lens 9. The light is condensed and scanned in the X and Y directions, and the light from the specimen 11 is incident on the dichroic mirror 5 through the optical path opposite to that described above, and is reflected and reflected by the absorption filter 12, the pinhole imaging lens 13, and the pinhole 14. The LSM image is acquired by receiving light with the photomultiplier 16 via the condenser lens 15.
[0005]
On the other hand, FIG. 4 shows a schematic configuration of the fluorescence microscope, in which the laser light emitted from the laser light source 1 is condensed on the incident end of the optical fiber 3 by the condenser lens 2 and passes through the optical fiber 3 from the emission end. The emitted light beam is guided to the evanescent light projecting tube 17, converted into parallel light by the collimating lens 18 inside the evanescent light projecting tube 17, and then the object image projecting lens 19 passes through the dichroic mirror 20 to the objective lens 9. The light is condensed at the pupil position 9a. Then, by moving the exit end of the optical fiber 3 in the direction of the arrow shown in the figure, the condensing position of the laser light on the exit pupil plane 9a is moved in the range from the pupil center to the periphery of the objective lens 9, and the laser light When the condensing position is in the vicinity of the center of the pupil of the objective lens 9, the laser light illuminates the specimen 11 almost in parallel with the optical axis to enable normal fluorescence observation, and the condensing position of the laser light is the objective. When it is in the vicinity of the pupil of the lens 9, the evanescent fluorescence observation is made possible by illuminating the specimen 11 obliquely with respect to the optical axis. In this case, the specimen 11 is in a state where a cover glass is pasted on a cell or the like, and when the condensing position of the laser light is condensed around the pupil of the objective lens 9, the boundary between the cell and the cover glass is obtained. Illumination is performed at an angle equal to or greater than the critical angle at which total reflection occurs on the surface, and evanescent fluorescence observation of the cells is performed using illumination light that oozes about 100 nm to 200 nm on the cell side at the boundary surface when this total reflection occurs. The fluorescence observation image illuminated by the evanescent light is observed by the eyepiece lens 22 and the CCD 23 through the imaging lens 21.
[0006]
[Problems to be solved by the invention]
However, in order to selectively acquire the LSM image and the evanescent fluorescence image, if the respective configurations of the scanning laser microscope and the fluorescence microscope are prepared separately, the configuration becomes complicated and the operation becomes troublesome. In addition, there is a disadvantage that the whole apparatus becomes large and requires a large installation space, which is economically disadvantageous.
[0007]
On the other hand, Japanese Patent Application Laid-Open No. 6-27385 discloses a first optical path for condensing light from a light source through an objective lens on the specimen and spot illuminating the specimen, and light from the light source on the pupil plane of the objective lens. There has been disclosed a microscope in which the second optical path for condensing near and illuminating the field of view of the sample with a large beam diameter can be selectively switched.
[0008]
However, when the operation for acquiring the LSM image by the laser microscope and the evanescent fluorescence image by the fluorescence microscope is performed, the first and second optical paths are switched inside the scan unit. As a result, a large number of optical elements are required, and the configuration becomes complicated accordingly. Also, it is necessary to secure an internal space for this purpose, which causes problems such as an increase in size of the microscope itself.
[0009]
Therefore, as disclosed in the specification of Japanese Patent Application No. 2001-214373, a condensing lens that condenses the laser light at the pupil position of the objective lens and a predetermined distance offset in parallel to the laser light, the center of the objective lens A transparent parallel plane plate for allowing the laser beam to enter the position offset from the optical path of the laser beam is provided so as to be insertable / removable. LSM images can be acquired as described above. On the other hand, in the state where the condenser lens and the plane parallel plate are inserted in the optical path, the plane parallel plate is rotated to change the laser beam condensing position on the pupil plane of the objective lens. Therefore, it is possible to acquire an evanescent fluorescent image by evanescent illumination and to selectively acquire an LSM image and an evanescent fluorescent image. There.
[0010]
However, in this configuration, in order to perform evanescent fluorescence observation, a dedicated parallel plane plate must be prepared as a scanner that scans the laser beam on the pupil plane of the objective lens. Since driving means for rotationally driving with high precision is also required, there is a problem that the configuration becomes complicated and the cost becomes expensive.
[0011]
Therefore, it is conceivable that a scanner used for acquiring an LSM image cannot be used. However, in Japanese Patent Application No. 2001-214373, the evanescent fluorescence is observed in the optical path on the laser light source side from the scanner. Only the condensing lens for fluorescence observation is inserted, and the scanner cannot be projected onto the object image plane, so the laser light is moved slightly to scan the laser light to shift the condensing point of the laser light. Then, so-called vignetting occurs around the field of view, and appropriate evanescent illumination cannot be performed.
[0012]
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microscope apparatus that can selectively acquire an LSM image and an evanescent fluorescence image with a simple configuration.
[0013]
[Means for Solving the Problems]
According to a first aspect of the present invention, the light source for illuminating the specimen, the scanner for deflecting the illumination light from the light source and the deflection angle of which can be changed, and the optical path of the illumination light deflected by the scanner are arranged. , An objective lens that projects the image of the specimen, and a first lens that is disposed between the scanner and the objective lens, and that condenses the illumination light deflected by the scanner onto the specimen via the objective lens. A projection lens; and a second projection lens that is disposed between the scanner and the objective lens and collects illumination light deflected by the scanner on a pupil plane of the objective lens, The first and second projection lenses can be selectively inserted into the optical path.
[0014]
According to a second aspect of the present invention, in the first aspect of the present invention, the first and second projection lenses are stored in a unit containing lens storage means, and the lens storage means includes the scanner and the objective lens. It is characterized in that it can be inserted into and removed from the optical path between.
[0015]
According to a third aspect of the present invention, in the first or second aspect of the invention, the scanner may change the deflection angle of the illumination light at a predetermined angle in a state where the second projection lens is inserted in the optical path. The feature is that it can be stopped.
[0016]
As a result, according to the present invention, when the LSM image is observed by the lens switching unit, the illumination light deflected by the scanner is condensed on the specimen by switching the first projection lens on the optical path, and evanescent fluorescence is collected. In the case of observation, since the illumination light deflected by the scanner is condensed on the pupil plane of the objective lens by switching the second projection lens on the optical path, the LSM image can be surely obtained with a simple configuration. An evanescent fluorescence image can be selectively acquired.
[0017]
Further, according to the present invention, since the lens storage means has a unit configuration that can be inserted into and removed from the optical path, the lens switching means can be mounted by slightly modifying a conventionally used scanning laser microscope. It can be applied simply by configuring.
[0018]
  Furthermore, according to the present invention, it is possible to adjust the evanescent illumination during the evanescent fluorescence observation only by determining the stop position of the deflection angle of the scanner.
  According to a fourth aspect of the present invention, there is provided a light source for illuminating a specimen, a scanner for deflecting illumination light from the light source, the deflection angle being changeable, and an optical path of illumination light deflected by the scanner And an objective lens that projects an image of the specimen, and is arranged between the scanner and the objective lens, and the illumination light deflected by the scanner is condensed on the specimen via the objective lens. And a lens means capable of switching between a second state for condensing the illumination light on the pupil plane of the objective lens, and the scanner is conjugate with the objective image plane in the second state. It is characterized by being.
  According to a fifth aspect of the present invention, in the fourth aspect of the present invention, the first light receiving element that receives light from the sample when the lens means is in the first state, and the lens means is the second A first light receiving element that receives light from the specimen when in the state; and a dichroic mirror that guides light from the specimen to the second light receiving element when in the second state; In this state, light from the sample is received by the first light receiving element to acquire an LSM image, and in the second state, light from the sample is received by the second light receiving element. Thus, an evanescent fluorescence image is acquired.
  The invention of claim 6 is characterized in that, in the invention of claim 5, the depth of evanescent light oozing is varied by changing the stop position of the deflection angle of the scanner in the second state.
  According to a seventh aspect of the present invention, in the fourth to sixth aspects of the invention, the condensing position on the pupil plane of the objective lens is varied by changing a deflection angle of the scanner in the second state. It is said.
  The invention of claim 8 is characterized in that, in the invention of claim 5, the stop position of the deflection angle of the scanner is varied according to the type of the objective lens in the second state.
  A ninth aspect of the invention is characterized in that, in the first to third aspects of the invention, when the second projection lens is inserted, the scanner is conjugate with the object image plane.
  The invention of claim 10 is arranged in a light source for illuminating a specimen, a scanner for deflecting illumination light from the light source and changing the deflection angle, and an optical path of illumination light deflected by the scanner, An objective lens that projects an image of the specimen; and a microscope apparatus that forms a scanned image of the specimen by scanning the illumination light condensed by the objective lens with the scanner. The scanner and the objective lens And a projection lens that condenses the illumination light on the pupil plane of the objective lens and conjugates the scanner with the objective image plane, and the illumination light deflected by the scanner is Evanescent illumination of the specimen is made possible by focusing light on the periphery of the pupil plane of the objective lens through the projection lens. That.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
[0020]
FIG. 1 shows a schematic configuration of an upright microscope apparatus to which the present invention is applied.
[0021]
In the figure, reference numeral 31 denotes a laser light source. On the optical path of the laser light emitted from the laser light source 1, an incident end of an optical fiber 33 is disposed via a condensing lens 32 and is condensed by the condensing lens 32. Laser light is incident.
[0022]
A scan unit 34 is disposed at the emission end of the optical fiber 33. The scan unit 34 includes a collimating lens 35, a dichroic mirror 36, and a scanner 37 disposed along the optical path of a light beam led out into the optical fiber 33 and emitted from the exit end, and collimates the laser light incident from the optical fiber 33. The lens 35 converts the light into parallel light, passes through the dichroic mirror 36, and enters the scanner 37. The scanner 37 deflects the laser light from the laser light source 1 and changes the deflection angle. In this case, the scanner 37 is of a type that rotates a mirror, is supported rotatably about an axis 37a, and deflects (scans) the laser beam only in a linear direction (for example, the X direction). It has become.
[0023]
The dichroic mirror 36 of the scan unit 34 reflects fluorescence from the specimen 47 side, and as an absorption filter 38, a pinhole imaging lens 39, a pinhole 40, a condensing lens 41, and a light receiving element on the reflected light path. The photomultiple 42 is arranged.
[0024]
On the optical path of the laser light deflected by the scanner 37, an imaging lens 43, an objective lens 44, and a specimen 47 on the stage 46 of the microscope main body 45 are arranged.
[0025]
The microscope main body 45 is provided with a lens barrel 49 for deflecting the objective image and an eyepiece lens 48 for visually observing the objective image deflected by the lens barrel 49.
[0026]
A lens storage unit 51 is disposed in the optical path between the scanner 37 and the objective lens 44 as lens storage means. This lens storage unit 51 has a unit configuration in which a pupil projection lens 52 as a first projection lens, objective image projection lenses 53a and 53b as a second projection lens, and a dichroic mirror 54 are stored. The lens 52, the objective image projection lenses 53a and 53b, and the dichroic mirror 54 can be selectively switched on the optical path by lens switching means (not shown).
[0027]
In this case, the pupil projection lens 52 is inserted on the optical path when acquiring an LSM image, and the laser light deflected by the scanner 37 is collected on the specimen 47 via the imaging lens 43 and the objective lens 44. In addition, the scanner 37 is projected onto the exit pupil plane 44 a of the objective lens 44. Here, the reason why the scanner 37 is set at a conjugate position with the exit pupil plane 44a of the objective lens 44 is to prevent so-called vignetting around the visual field when the laser beam is scanned. .
[0028]
The objective image projection lenses 53a and 53b are inserted on the optical path when an evanescent fluorescent image is acquired, and the laser light deflected by the scanner 37 and emitted in parallel is supplied to the objective lens 44 via the imaging lens 43. In addition, the light is condensed on the exit pupil plane 44a and the scanner 37 is projected onto the object image plane 50. Here, the scanner 37 is set at a position conjugate with the objective image plane 50. When the LSM image is observed, the scanner 37 is projected onto the exit pupil plane 44a of the objective lens 44 by the pupil projection lens 52. This prevents the so-called vignetting around the field of view when the laser light is deflected. The objective image projection lenses 53a and 53b are close to a Kepler type afocal converter, and are an internal focus type that forms an intermediate image 44b of the pupil of the objective lens 44 between the objective image projection lenses 53a and 53b. In this case, the projection pupil plane 44a of the objective lens 44 is projected to infinity, and the scanner 37 is projected onto the objective image plane 50 in a small space.
[0029]
The dichroic mirror 54 has characteristics such that it transmits laser light that is wavelength-selective excitation light and reflects only the fluorescence emitted from the specimen 47. The reflected fluorescence is incident on the CCD 55 through the absorption filter 56. I try to let them. The dichroic mirror 54 is formed integrally with the objective image projection lenses 53a and 53b, and is inserted into and removed from the optical path together with the objective image projection lenses 53a and 53b.
[0030]
Although specific switching means for the optical paths of the pupil projection lens 52, the objective image projection lenses 53a and 53b, and the dichroic mirror 54 are not shown in the drawings, for example, a slide switching mechanism using a slider and a rotation switching means using a turret are known. The lens switching means can be used.
[0031]
Further, the lens storage unit 51 configured as described above is configured to be detachable with respect to the optical path between the scanner 37 and the objective lens 44.
[0032]
In such a configuration, first, a case where an LSM image is observed will be described.
[0033]
In this case, the lens storage unit 51 is inserted into the optical path between the scanner 37 and the objective lens 44, and the pupil projection lens 52 is inserted into the optical path by switching means (not shown). In the drawing, the pupil projection lens 52 inserted on the optical path, the objective image projection lenses 53a and 53b and the dichroic mirror 54 removed from the optical path are indicated by broken lines.
[0034]
Parallel laser light emitted from the laser light source 31 is condensed on the incident end face of the optical fiber 33 by the condenser lens 32, guided in the optical fiber 33, and reaches the emission end. The laser light emitted from the emission end of the optical fiber 33 is returned to parallel light by the collimating lens 35, passes through the dichroic mirror 36, and is guided to the scanner 37.
[0035]
In this state, the scanner 37 is rotated around an axis 37 a that is a point at which the optical axis is folded, and the laser light from the collimating lens 35 is deflected toward the specimen 47. Here, the laser light deflected by the scanner 37 is a state of a light beam when the solid line in the figure is deflected parallel to the optical axis, and a light beam when the broken line in the figure is deflected at a certain angle with respect to the optical axis. Represents a state.
[0036]
The laser light deflected by the scanner 37 is condensed on the specimen 47 through the pupil projection lens 52, the imaging lens 43, and the objective lens 44.
[0037]
In this case, the scanner 37 is projected onto the exit pupil plane 44a of the objective lens 44 by the pupil projection lens 52 via the imaging lens 43, and is set at a position conjugate with the exit pupil plane 44a of the objective lens 44.
[0038]
In this way, the laser light deflected by the scanner 37 is condensed on the sample 47 and scanned according to the deflection angle of the scanner 37.
[0039]
On the other hand, the fluorescence emitted from the sample 47 by being excited by the laser beam condensed on the sample 47 passes through the objective lens 44, the imaging lens 43, the pupil projection lens 52, and the scanner 37 in the reverse optical path as described above. Then, the light enters the dichroic mirror 36. Then, the light is reflected by the dichroic mirror 36, passes through the absorption filter 38, and an image of the sample 47 is formed on the pinhole 40 by the pinhole imaging lens 39.
[0040]
In this case, as the absorption filter 38, a filter that cuts off the laser light serving as excitation light and transmits only the fluorescence wavelength necessary for observation emitted from the specimen 47 is used. The pinhole 40 cuts image components other than the focus surface on the specimen 47.
[0041]
As a result, the fluorescence that has passed through the pinhole 40 is received by the photomultiplier 42 via the condenser lens 41 and converted into an electrical signal. Although not shown in the figure, an image is generated from a photomal electrical signal by processing such as an electric circuit or software for constructing an image, is displayed on a monitor, and can be observed as an LSM image.
[0042]
Next, a case where evanescent fluorescence observation is performed will be described.
[0043]
In this case, the pupil projection lens 52 is removed from the optical path using switching means (not shown), and the objective image projection lenses 53a and 53b and the dichroic mirror 54 are inserted into the optical path. In the drawing, the objective image projection lenses 53a and 53b and the dichroic mirror 54 inserted in the optical path and the pupil projection lens 52 removed from the optical path are indicated by solid lines.
[0044]
Also in this case, the laser light emitted from the laser light source 31 enters the collimating lens 35 via the optical fiber 33. Next, the laser light converted into a parallel light beam by the collimating lens 35 passes through the dichroic mirror 36.
[0045]
In the case of evanescent fluorescence observation, the dichroic mirror 36 is unnecessary, and may be removed from the optical path to reduce the loss of brightness.
[0046]
The laser light transmitted through the dichroic mirror 36 is guided to the scanner 37 and deflected toward the specimen 47 by the scanner 37.
[0047]
In the scanner 37, the deflection angle is variable as in the case of observing the LSM image. Here, the state of the light beam when the illustrated solid line is deflected parallel to the optical axis, and the broken line illustrated in FIG. It represents the state of the light beam when deflected at an angle.
[0048]
However, in the case of the evanescent fluorescence observation, unlike the case of the observation of the LSM image, it is not necessary to perform image construction by scanning the image plane, so the scanner 37 stops changing the deflection angle of the laser light at a predetermined angle. Is done.
[0049]
The laser light deflected by the scanner 37 is condensed on the exit pupil plane 44 a of the objective lens 44 through the objective image projection lenses 53 a and 53 b, the dichroic mirror 54, and the imaging lens 43.
[0050]
In this case, the scanner 37 is projected onto the objective image plane 50 by the objective image projection lenses 53 a and 53 b and is set at a position conjugate with the objective image plane 50.
[0051]
As a result, the laser light condensed on the exit pupil plane 44a of the objective lens 44 can move in the range from the pupil center to the periphery of the objective lens 44 only by rotating the scanner 37 and changing the deflection angle. it can.
[0052]
In this case, as shown in FIG. 2, when the laser beam condensed on the exit pupil plane 44 a of the objective lens 44 is in the range of a to b near the pupil center of the objective lens 44, the laser beam is Ordinary fluorescence observation can be performed by illuminating the specimen 47 in parallel with the optical axis via the lens 44. When the laser beam is in the range of b to c around the pupil of the objective lens 44, the laser beam illuminates the specimen 47 obliquely with respect to the optical axis via the objective lens 44, and the angle is as described above. If the critical angle is exceeded, evanescent illumination can be obtained and evanescent fluorescence observation can be performed.
[0053]
In this case, the specimen 47 is in a state in which a cover glass is pasted on a cell or the like. Therefore, the critical angle at which evanescent illumination becomes different depending on the refractive index of the cover glass, and even at an angle greater than the critical angle. Depending on the angle, the penetration depth of the evanescent light that oozes from the boundary surface between the cell and the cover glass to the cell side also varies. This oozing depth is the depth of the specimen 47 that the specimen 47 wants to observe, and varies depending on the purpose of the spectrographer.
[0054]
In this way, the angle of the laser light applied to the specimen 47 varies depending on the conditions of the specimen 47 and the depth to be observed. In this case, the laser beam condensed on the exit pupil plane 44a of the objective lens 44 has a larger irradiation angle to the specimen 47 with respect to the optical axis as it moves to the periphery of the pupil. The condensing position on the exit pupil plane 44a of the objective lens 44 is different. Even if the angle of irradiation of the specimen 47 with respect to the optical axis is the same, the condensing position of the laser light on the exit pupil plane 44a also differs depending on the type of the objective lens 44. Accordingly, the spectrographer performs evanescent fluorescence observation by setting the deflection angle of the scanner 37 to an optimum angle according to the application while taking these into consideration.
[0055]
On the other hand, the fluorescence emitted from the specimen 47 by the evanescent illumination enters the dichroic mirror 54 through the objective lens 44 and the imaging lens 43, is reflected from the dichroic mirror 54 toward the CCD 55, and passes through the absorption filter 56 to the CCD 55. Is projected onto the objective image plane 50 which is also the light receiving surface.
[0056]
In this case, as the absorption filter 56, a filter that cuts off laser light serving as excitation light and transmits only the fluorescence wavelength necessary for observation emitted from the specimen 47 is used.
[0057]
As a result, the objective image picked up by the CCD 55 is displayed on a monitor (not shown) and can be observed as an evanescent fluorescence image.
[0058]
Accordingly, in this case, the lens housing unit 51 that houses the pupil projection lens 52 and the objective image projection lenses 53a and 53b is arranged in the optical path between the scanner 37 and the objective lens 44, and in the case of observing an LSM image. The pupil projection lens 52 is switched to the optical path, the laser light deflected by the scanner 37 is condensed on the specimen 47, and the scanner 37 is projected onto the exit pupil plane 44a of the objective lens 44, while evanescent fluorescence observation is performed. In this case, the objective image projection lenses 53a and 53b are switched to the optical path, the laser beam deflected by the scanner 37 is condensed on the exit pupil plane 44a of the objective lens 44, and the scanner 37 is projected onto the objective image plane 50. So, in order to acquire the LSM image and the evanescent fluorescence image by the fluorescence microscope inside the conventional scan unit, Compared with those to switch the road, the optical element for the optical path switching is not required, it is possible to configure easily, can eliminate wasted interior space can be realized compact microscope itself. In addition, since the LSM image and the evanescent fluorescence image can be selectively acquired by the common scanner 37, a dedicated parallel plane plate for performing conventional evanescent fluorescence observation, and a driving means for accurately rotating and driving the parallel plane plate Compared to the one that needs to be prepared, the configuration is simple and the price can be reduced.
[0059]
Further, since the lens storage unit 51 arranged in the optical path between the scanner 37 and the objective lens 44 has a unit configuration that can be inserted into and removed from the optical path, a conventional scanning laser microscope is slightly used. This can be realized simply by modifying the lens storage unit 51 so that the lens storage unit 51 can be mounted.
[0060]
Further, in the case of LSM image observation, the scanner 37 is projected onto the exit pupil plane 44a of the objective lens 44 by the pupil projection lens 52, and in the case of evanescent fluorescence observation, the scanner 37 is objectively imaged by the objective image projection lenses 53a and 53b. Since the projection can be performed on the surface 50, it is possible to prevent the occurrence of vignetting around the visual field when the laser beam is scanned, and to perform appropriate illumination.
[0061]
Furthermore, the scanner 37 is used to repeatedly deflect the laser beam within a predetermined angle range in the observation of the LSM image, and is stopped and used in the state of the predetermined deflection angle in the evanescent fluorescence observation. Since the switching of these operations can be easily obtained by simply controlling the driving means (not shown) of the scanner 37, the switching between the LSM image observation and the evanescent fluorescence observation can be easily performed, and the evanescent fluorescence observation is possible. The evanescent illumination can be easily adjusted by simply adjusting the stop position of the deflection angle of the scanner 37.
[0062]
Furthermore, since the objective image projection lenses 53a and 53b and the dichroic mirror 54 are integrally formed and can be selectively switched on the optical path with the pupil projection lens 52, observation of LSM images and observation of evanescent fluorescence can be performed. It can be switched by one-touch operation.
[0063]
Furthermore, the objective image projection lenses 53a and 53b are close to a Kepler type afocal converter, and an internal focus type that forms an intermediate image 44b of the pupil of the objective lens 44 between the objective image projection lenses 53a and 53b. Therefore, the projection pupil plane 44a of the objective lens 44 can be projected to infinity, and operations such as projecting the scanner 37 onto the objective image plane 50 can be realized in a small space.
[0064]
Furthermore, by using a scanner that rotates the mirror as the scanner 37, the condensing position of the laser light on the exit pupil plane 44a of the objective lens 44 can be moved greatly, so that the normal fluorescence observation is changed to the evanescent observation. Can be easily adjusted.
[0065]
In the above-described embodiment, only one scanner 37 is provided and deflected (scanned) in the linear direction. However, two scanners 37 are provided to perform two-dimensional (XY direction) scanning. Also good. In the above-described embodiment, the switching is performed by alternately inserting and removing the pupil projection lens 52 and the objective image projection lenses 53a and 53b with respect to the optical path. The condensing position of the laser beam may be changed using other means such as inserting / removing only a part of the lens. Further, in the above-described embodiment, the objective image projection lenses 53a and 53b and the dichroic mirror 54 are integrated and inserted / removed from the optical path at the same time. May be disposed between the imaging lens 43 and the objective lens 44. In this case, in order to form an evanescent fluorescent image, an image forming lens may be provided separately after being reflected by the dichroic mirror 54. Furthermore, the dichroic mirrors 36 and 54 are arranged with the observation optical path side as a reflection surface. In order to reduce image deterioration due to the surface accuracy of the reflection surfaces of the dichroic mirrors 36 and 54, respectively, The round 42 side and the laser light source 31 side, and the CCD 55 side and the laser light source 31 side may be reversed. Further, the objective lens 44 may be a finite objective lens that does not use the imaging lens 43. Furthermore, the above-described embodiment can be applied to, for example, a caged reagent releasing device or FRAP for forcibly fading a certain point of fluorescence. Furthermore, in the above-described embodiments, the upright microscope has been described as an example, but it can also be applied to an inverted microscope with the same configuration.
[0066]
In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary.
[0067]
Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0068]
【The invention's effect】
As described above, according to the present invention, it is possible to provide a microscope apparatus capable of selectively acquiring an LSM image and an evanescent fluorescence image with a simple configuration.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of an embodiment of the present invention.
FIG. 2 is a diagram for explaining evanescent fluorescence observation according to one embodiment.
FIG. 3 is a diagram showing a schematic configuration of a conventional scanning laser microscope.
FIG. 4 is a diagram showing a schematic configuration of a conventional fluorescence microscope
[Explanation of symbols]
31 ... Laser light source
32 ... Condensing lens
33 ... Optical fiber
34 ... Scanning unit
35 ... Collimating lens
36 ... Dichroic mirror
37 ... Scanner
37a ... axis
38 ... Absorption filter
39 ... Pinhole imaging lens
40 ... pinhole
41 ... Condensing lens
42 ... Photomaru
43 ... Imaging lens
44 ... Objective lens
44a ... exit pupil plane
44b ... Intermediate image
45 ... Microscope body
46 ... Stage
47 ... Sample
48 ... Eyepiece
49 ... Tube
50: Objective image plane
51 ... Lens storage unit
52 ... Pupil projection lens
53a. 53b ... Objective image projection lens
54 ... Dichroic mirror
55 ... CCD
56 ... Absorption filter

Claims (10)

A light source for illuminating the specimen;
A scanner that deflects illumination light from the light source and that can change the deflection angle;
An objective lens that is disposed in an optical path of illumination light deflected by the scanner and projects an image of the specimen;
A first projection lens disposed between the scanner and the objective lens, and condensing the illumination light deflected by the scanner onto the specimen via the objective lens;
A second projection lens disposed between the scanner and the objective lens and condensing the illumination light deflected by the scanner on the pupil plane of the objective lens;
And a microscope apparatus characterized in that the first and second projection lenses can be selectively inserted into the optical path. A lens housing means having a unit configuration housing the first and second projection lenses
2. The microscope apparatus according to claim 1, wherein the lens storage means can be inserted into and removed from an optical path between the scanner and the objective lens.   3. The microscope apparatus according to claim 1, wherein the scanner is configured to be able to stop changing the deflection angle of the illumination light at a predetermined angle in a state where the second projection lens is inserted in the optical path. . A light source for illuminating the specimen;
A scanner that deflects illumination light from the light source and that can change the deflection angle;
An objective lens that is arranged in an optical path of illumination light deflected by the scanner and projects an image of the specimen;
A first state in which illumination light, which is disposed between the scanner and the objective lens and is deflected by the scanner, is condensed on the sample via the objective lens, and the pupil plane of the objective lens Lens means capable of switching the second state of focusing on, and
The microscope apparatus according to claim 1, wherein the scanner is conjugate with an object image plane in the second state. A first light receiving element for receiving light from the specimen when the lens means is in the first state;
A second light receiving element for receiving light from the specimen when the lens means is in the second state;
A dichroic mirror for guiding light from the specimen to the second light receiving element in the second state;
Light from the specimen is received by the first light receiving element in the first state to acquire an LSM image, and light from the specimen is received in the second light receiving element in the second state. 5. The microscope apparatus according to claim 4, wherein an evanescent fluorescence image is acquired by receiving the light. 6. The microscope apparatus according to claim 5, wherein in the second state, the penetration depth of the evanescent light is varied by changing a stop position of the deflection angle of the scanner. 7. The microscope apparatus according to claim 4, wherein, in the second state, a condensing position on the pupil plane of the objective lens is varied by changing a deflection angle of the scanner. 6. The microscope apparatus according to claim 5, wherein in the second state, the stop position of the deflection angle of the scanner is varied according to the type of the objective lens. 4. The microscope apparatus according to claim 1, wherein when the second projection lens is inserted, the scanner is conjugate with an objective image plane. A light source for illuminating the specimen;
A scanner that deflects illumination light from the light source and that can change the deflection angle;
An objective lens that is disposed in an optical path of illumination light deflected by the scanner and projects an image of the specimen, and scans the specimen by scanning the illumination light condensed by the objective lens with the scanner. In a microscope apparatus for forming an image,
A projection lens that is disposed between the scanner and the objective lens, condenses the illumination light on the pupil plane of the objective lens and conjugates the scanner and the objective image plane;
The microscope apparatus, wherein the illumination light deflected by the scanner is condensed on the periphery of the pupil plane of the objective lens through the projection lens, thereby enabling evanescent illumination of the specimen.

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