US20070183030A1 - Total reflection fluorescent microscope - Google Patents
Total reflection fluorescent microscope Download PDFInfo
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- US20070183030A1 US20070183030A1 US11/734,208 US73420807A US2007183030A1 US 20070183030 A1 US20070183030 A1 US 20070183030A1 US 73420807 A US73420807 A US 73420807A US 2007183030 A1 US2007183030 A1 US 2007183030A1
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- 238000005286 illumination Methods 0.000 claims abstract description 111
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- 230000007246 mechanism Effects 0.000 description 5
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/0088—Inverse microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
- G02B21/10—Condensers affording dark-field illumination
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
Abstract
A fluorescent microscope comprises a light source, an optical illumination system which forms an optical path to irradiate a specimen with a light beam from the light source, an objective lens which condenses the light beam of the optical illumination system onto the specimen, an optical device which is disposed on the optical path of the optical illumination system and which decenters the light beam by decentering an optical axis of the optical path, and a slit which passes the light beam decentered by the optical device through a total reflection illumination region on an emission pupil surface of the objective lens.
Description
- The present application is a Divisional Application of U.S. application Ser. No. 10/848,626 filed May 18, 2004, which is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2003-143382, filed May 21, 2003, the entire contents of which are incorporated herein by reference.
- 1. Field of the Invention
- The present invention relates to a total reflection fluorescent microscope, which can perform fluorescent observation by total reflection illumination.
- 2. Description of the Related Art
- In recent years, functions of biological cells have been vigorously analyzed. In the function analysis of the cells, attentions have been paid especially to a total reflection fluorescent microscope which acquires a total reflection fluorescent image from a cell membrane and its vicinity as a device for observing the function of the cell membrane.
- Total reflection illumination which locally illuminates only a sample in the vicinity of a glass surface is used in the total reflection fluorescent microscope. In the total reflection illumination, an evanescent light is used which oozes to a sample side by about several hundreds of nanometers in a boundary surface between glass and sample, and a background noise (scattered light and the like) is remarkably low. Therefore, fluorescent observation of even a molecular of fluorescent dyestuff is possible by the total reflection fluorescent microscope.
- Additionally, in general, in the total reflection fluorescent microscope, a laser light beam is used as a light source. A total reflection fluorescent microscope in which the laser light beam is introduced into an optical illumination system of the microscope via a glass fiber is described, for example, in Jpn. Pat. Appln. KOKAI Publication No. 2002-169097.
- However, the laser light source which produces the laser light beam is expensive, and additionally a monochromatic light is produced. Therefore, for example, in order to cope with fluorescent reagents having various excitation wavelength characteristics, a plurality of laser light sources have to be prepared. Therefore, the total reflection fluorescent microscope becomes further expensive, and additionally a large occupying space is also required for installing a plurality of laser light sources.
- To solve the problem, as described in Jpn. Pat. Appln. KOKAI Publication No. 2002-236258, a total reflection fluorescent microscope has been proposed in which inexpensive white light sources such as a mercury lamp and a xenon lamp are used instead of the laser light source. The total reflection fluorescent microscope according to the Jpn. Pat. Appln. KOKAI Publication No. 2002-236258 is configured as follows. A ring slit for transmitting a light beam in an annular form is disposed in the optical illumination system disposed on an optical path of the light emitted from the white laser light beam. Moreover, when an image of the ring slit is projected onto an emission pupil surface of an objective lens, an illuminative light is guided only to an orbicular total reflection region around an emission pupil of the objective lens. Moreover, total reflection is performed in a boundary surface between a specimen and cover glass to produce is the evanescent light, and a fluorescent dyestuff is excited.
- A fluorescence microscope according to a first aspect of the present invention is characterized by comprising a light source; an optical illumination system which forms an optical path to irradiate a specimen with a light beam from the light source; an objective lens which condenses the light beam of the optical illumination system onto the specimen; an optical device which is disposed on the optical path of the optical illumination system and which decenters the light beam by decentering an optical axis of the optical path; and a slit which passes the light beam decentered by the optical device through a total reflection illumination region on an emission pupil surface of the objective lens.
- A fluorescence microscope according to a second aspect of the present invention is characterized by comprising: a light source; an optical illumination system which forms an optical path to irradiate a specimen with a light beam from the light source; an objective lens which condenses the light beam of the optical illumination system onto the specimen; and a slit which passes the light beam from the light source through a total reflection illumination region on an emission pupil surface of the objective lens, in which an emission position of the light beam emitted from the light source is movable between an optical axis of the optical illumination system and a position shifting from the optical axis by a predetermined distance.
- Advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
- The accompanying drawings, which are incorporated in and configure a part of the specification, illustrate embodiments of the invention, and together with the general description given above and the detailed description of the embodiments given below, serve to explain the principles of the invention.
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FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention; -
FIG. 2 is a diagram showing a schematic configuration of a main part of the first embodiment; -
FIGS. 3A to 3C are explanatory views of a slit for use in the first embodiment; -
FIG. 4 is an explanatory view of a state in which a wedge prism and slit are removed from an optical path is of an optical illumination system in the first embodiment; -
FIG. 5 is a diagram showing a schematic configuration of a main part of a second embodiment; -
FIG. 6 is a diagram showing a light flux refracted by the wedge prism for use in the second embodiment: -
FIGS. 7A to 7D are explanatory views of a slit having a crescent opening for use in the second embodiment; -
FIGS. 8A to 8E are explanatory views of a slit having a small-diameter opening for use in the second embodiment; -
FIGS. 9A to 9D are explanatory views of a slit having an annular opening for use in the second embodiment; -
FIGS. 10A and 10B are diagrams showing a schematic configuration of a main part of a modification of the second embodiment; -
FIG. 11 is a diagram showing a schematic configuration of a third embodiment of the present invention; -
FIG. 12 is a diagram showing a light flux refracted by a conical prism for use in the third embodiment; -
FIG. 13 is an explanatory view of a state of a light source image projected on the annular opening of the third embodiment; -
FIGS. 14A and 14B are diagrams showing a schematic configuration of a main part of a modification of the third embodiment; -
FIG. 15 is a diagram showing a schematic configuration of a fourth embodiment of the present invention; -
FIG. 16 is an explanatory view of an LED image projected on the crescent opening of the fourth embodiment; -
FIG. 17 is an explanatory view of a state of an LED image projected on the annular opening of the fourth embodiment; -
FIG. 18 is a diagram showing a schematic configuration of a fifth embodiment of the present invention; -
FIGS. 19A and 19B are explanatory views of a slit having a crescent opening for use in the fifth embodiment; -
FIGS. 20A and 20B are explanatory views of a slit having an annular opening for use in the fifth embodiment; -
FIG. 21 is an explanatory view of a slit showing a small-diameter opening for use in the fifth embodiment; -
FIG. 22 is an explanatory view of a state in which an afocal converter, wedge prism, and slit are detached from an optical path of an optical illumination system in the fifth embodiment; -
FIG. 23 is a diagram showing a schematic configuration of a sixth embodiment of the present invention; -
FIG. 24 is a diagram showing a schematic configuration of a seventh embodiment of the present invention; and -
FIG. 25 is a diagram showing a schematic configuration of an eighth embodiment of the present invention. - Embodiments of the present invention will be described hereinafter with reference to the drawings.
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FIG. 1 is a diagram showing a schematic configuration of a total reflection fluorescent microscope to which the present invention is applied. In this case,FIG. 1 shows an example of an inverted microscope for performing observation by an objective lens disposed below a specimen. - In
FIG. 1 , astage 2 is disposed in an upper part of a microscopemain body 1. Aspecimen 3 is laid on thestage 2. In this case, as shown inFIG. 2 , a cover glass 7 is disposed under thespecimen 3. Anobjective lens 4 is disposed under the cover glass 7 via oil (not shown). - A
revolver 5 is disposed under thespecimen 3. Therevolver 5 is held in the microscopemain body 1. Therevolver 5 holds a plurality ofobjective lenses 4. When therevolver 5 rotates, it is possible to selectively dispose theobjective lens 4 having a magnification or a type required for the observation on an observationoptical axis 6. When therevolver 5 vertically moves along the observationoptical axis 6 by an operation of a focusing handle (not shown) to change a relative distance between thespecimen 3 and theobjective lens 4 on thestage 2, thespecimen 3 can be focused. - A
light source 11 for illuminating thespecimen 3 is used in total reflection fluorescent illumination or usual fluorescent illumination in which total reflection is not performed. As thelight source 11, high-luminance arc light sources such as a mercury lamp and xenon lamp are used. It is to be noted that these arc light sources preferably have micro luminescent spots, and the light source is selected having a luminescent spot in which a projected image on an emission pupil surface of the objective lens is smaller than an emission pupil diameter of the objective lens. - A
collector lens 12 is provided on an illuminativelight axis 18 of the optical path from thelight source 11. Thecollector lens 12 condenses the light beams from thelight source 11 and emits a parallel light beam. - A wedge-shaped
plane plate 41 for decentering the optical path is disposed in the optical path of the parallel light beam from thecollector lens 12. The wedge-shapedplane plate 41 refracts the parallel light beam emitted from thecollector lens 12 at a predetermined angle with respect to the illuminativelight axis 18. - A
condenser 14 and aslit 15 are disposed in the optical path of a light flux refracted by the wedge-shapedplane plate 41. Thecondenser 14 condenses the light flux refracted by the wedge-shapedplane plate 41 on a surface of aslit 15, and alight source image 11 a of thelight source 11 is projected. - In the
slit 15,slits having openings FIGS. 3A to 3C are used. Theseopenings light source images 11 a) condensed by thecondenser 14. The light beams transmitted through theopenings specimen 3 and the cover glass 7. - It is to be noted that in any of three types of
slits 15, two slits are disposed closely along an illuminativelight axis 18. This is because the light beam that is not totally reflected, generated by frame reflection inside the illuminative light axis or by a diffracted light in theslit 15, is cut. The wedge-shapedplane plate 41 and slit 15 move along a plane vertical to the illuminativelight axis 18 by known switching mechanism such as a slider, and are detachably inserted with respect to the optical path. When the wedge-shapedplane plate 41 and slit 15 are inserted into the optical path, the total reflection fluorescent illumination can be selected. When the wedge-shapedplane plate 41 and slit 15 are removed from the optical path, the usual fluorescent illumination that does not perform the total reflection can be selected. Theslit 15 is movable along a plane crossing the illuminativelight axis 18 at right angles (in a vertical direction of an arrow shown inFIG. 1 in this case) in a state in which the slit is inserted in the optical path. - A field stop (FS) 16 and an
FS projection lens 17 are disposed in the optical path of the light transmitted through theslit 15. The field stop (FS) 16 is used to restrict an illumination field, and a slit diameter can be varied. TheFS projection lens 17 projects the field stop (FS) 16 on three surfaces of thespecimen 3, and projects the image of theslit 15 on an emission pupil surface of theobjective lens 4. - A
rotatable cassette 19 which holds two ormore mirror units FS projection lens 17. Themirror units cassette 19 by known means such as a dovetail. - The
cassette 19 is rotated around arotation axis 20. By this rotation, themirror units optical axis 6 in accordance with wavelength characteristics of a fluorescent dyestuff with which thespecimen 3 is dyed. InFIG. 1 , themirror unit 18 a is switched (disposed) on the observationoptical axis 6. - In the
mirror unit 18 a, anexcitation filter 211,dichroic mirror 212, andabsorption filter 213 are integrally disposed as a set. Theexcitation filter 211 selectively transmits a wavelength required for exciting thespecimen 3 among the light beams emitted from theFS projection lens 17. Thedichroic mirror 212 reflects an excitation wavelength from theexcitation filter 211, and transmits a fluorescent wavelength emitted from thespecimen 3. Thedichroic mirror 212 is inclined by 45° with respect to both the illuminativelight axis 18 and the observationoptical axis 6 in such a manner that an excited light along the illuminativelight axis 18 from theexcitation filter 211 is guided in a direction matching the optical axis (observation optical axis 6). Theabsorption filter 213 selectively transmits only the wavelength required for the observation in fluorescence emitted from thespecimen 3. - An
optical relay system 231 is disposed in a transmission optical path of theabsorption filter 213. An image of thespecimen 3 formed by theobjective lens 4 is relayed to the vicinity of aneyepiece lens 232. Theeyepiece lens 232 is used in such a manner that the image of thespecimen 3 relayed through theoptical relay system 231 is visually observable. - Since the
mirror unit 18 b is configured in the same manner as in themirror unit 18 a, the description is omitted. - A
transmission illumination section 26 includes an optical transmission illumination system in a case where transmission illumination observation is performed. - A case where the total reflection fluorescent illumination is performed in the above-described configuration will be described.
- When an illuminative light beam is emitted from the
light source 11, the light beam is formed into the parallel light beam by thecollector lens 12 and is incident upon the wedge-shapedplane plate 41. - The wedge-shaped
plane plate 41 refracts the parallel light beam from thecollector lens 12 at a predetermined angle to emit a light flux (light source image) refracted with respect to the illuminativelight axis 18. The light flux is condensed onto theslit 15 by thecondenser 14. - In this case, in the
slit 15, the crescent opening 43 a (or theopening slit 15 on aslit 15 surface as shown inFIG. 3A . A circular portion of the opening 43 a is formed substantially in parallel with a circumferential direction (i.e., on a substantially concentric circle). - Moreover, the
light source image 11 a is projected as the image of thelight source 11 in the crescent opening 43 a of theslit 15. - In the
light source image 11 a projected on theslit 15, the light beam transmitted through the opening 43 a is incident upon theexcitation filter 211 via theFS projection lens 17. Theexcitation filter 211 selects the light beam having a wavelength required for exciting thespecimen 3. The light beam selected by its wavelength is reflected toward theobjective lens 4 by thedichroic mirror 212, and subsequently projected onto the emission pupil of theobjective lens 4. It is to be noted that the image of theslit 15 projected on the emission pupil surface of theobjective lens 4, that is, a slit image will be described later. - All the light beams transmitted through the opening 43 a of the
slit 15 form totally reflected light beams within a total reflection region. - As shown in
FIG. 2 , the light beam transmitted through the emission pupil surface of theobjective lens 4 passes through a peripheral edge portion of theobjective lens 4, and reaches the cover glass 7 via the oil charged between theobjective lens 4 and the cover glass 7. Here, the total reflection occurs in the boundary surface between thespecimen 3 and the cover glass 7, and the evanescent light is generated in a range of about 50 to 200 nm on a specimen side of the boundary surface. The fluorescent dyestuff with which thespecimen 3 is dyed by the evanescent light is excited to emit the fluorescence. - In this state, a surveyor moves the
stage 2 to search for a desired observation range on thespecimen 3, vertically moves therevolver 5 along the observationoptical axis 6 by the operation of the focusing handle (not shown), and changes the relative distance between thespecimen 3 and theobjective lens 4 to focus thespecimen 3. - The fluorescence emitted from the
specimen 3 passes through thedichroic mirror 212, and the fluorescent wavelength required for the observation is selected by theabsorption filter 213. Moreover, the image of thespecimen 3 formed by theobjective lens 4 is relayed to theeyepiece lens 232 via theoptical relay system 231, and visual observation is possible. - To change the fluorescent wavelength to be observed of the fluorescent dyestuff dyed on the
specimen 3, thecassette 19 is rotated around therotation axis 20 and, for example, themirror unit 18 b may be switched on the observationoptical axis 6 instead of themirror unit 18 a. To change the observation magnification of thespecimen 3, therevolver 5 may be rotated to position theobjective lens 4 having a desired magnification on the observationoptical axis 6. - A relation between the slit image and the emission pupil surface of the
objective lens 4 by the crescent opening 43 a of theslit 15 will be described later. - In the above-mentioned description, the crescent opening 43 a is formed in the
slit 15, but the small-diameter opening 43 b may also be formed in a predetermined position on theslit 15 plane, for example, as shown inFIG. 3B , theannular opening 43 c may also be formed along the peripheral edge portion of theslit 15 surface as shown inFIG. 3C , or an elliptic opening (not shown) may also be formed. A relation between the slit image by the small-diameter opening 43 b, theannular opening 43 c, or the elliptic opening and the emission pupil surface of theobjective lens 4 will be described later. - A case where the total reflection fluorescent observation is switched to usual fluorescent observation to perform the observation will be described.
- In this case, as shown in
FIG. 4 , the wedge-shapedplane plate 41 and slit 15 are removed from the optical path of the optical illumination system, and an aperture stop (AS) 29 is inserted instead of theslit 15. Since theslit 15 is used for transmitting the illuminative light of the light source image through atotal reflection region 27 of theobjective lens 4, theslit 15 is removed from the optical path, and the aperture stop (AS) 29 is inserted as a diaphragm for adjusting brightness instead. The wedge-shapedplane plate 41 is used to project thelight source image 11 a in the periphery of the emission pupil of theobjective lens 4. Especially, when theobjective lens 4 has a high magnification, and the emission pupil diameter is small, the illuminative light is reflected by theobjective lens 4, the region is darkened, and illumination unevenness sometimes occurs. Therefore, the wedge-shapedplane plate 41 is removed from the optical path. - From this state, visual sample observation is possible using a generally known usual fluorescent observation method.
- As described above, since the wedge-shaped
plane plate 41 is disposed as an optical device capable of projecting thelight source image 11 a on an optical path between thelight source 11 and theslit 15, the optical axis of the optical path is decentered and moved, for example, to theopening 43 a of theslit 15. Therefore, since the illuminative light can be efficiently taken into thetotal reflection region 27 of theobjective lens 4 having an emission pupil diameter, the total reflection fluorescent observation by sufficient brightness can be realized. In the observation in the usual fluorescent illumination, when the wedge-shapedplane plate 41 and slit 15 are removed from the optical path, the light source image is projected on the optical axis. Therefore, also in this case, since the illuminative light can be efficiently taken in, the usual fluorescent observation by the sufficient brightness can be realized. - Moreover, the
slit 15 for performing the total reflection illumination has thesector opening 43 b for transmitting the light beam only in a part of the total reflection region. Therefore, even when the position or the size of the slit image changes by eccentricity of the optical illumination system or magnification error, the slit image can be prevented from deviating from the total reflection region of the emission pupil surface of theobjective lens 4. Therefore, the slit image does not enter the fluorescent illumination region where the total reflection is not performed, the eccentricity error can be prevented, and the total reflection fluorescent observation can be realized with good contrast. - The opening 43 a is formed in a crescent shape to enlarge an opening area of a portion which is not easily influenced by contrast deterioration with respect to the eccentricity. Conversely, an opening area of a portion easily influenced by the contrast deterioration with respect to the eccentricity can be reduced. Therefore, since the influence of the contrast deterioration by the eccentricity error of the optical system cannot be easily exerted, and additionally a middle portion of the opening 43 a has a maximum necessary opening area, the illuminative light from the
light source 11 can be efficiently taken. Therefore, the total reflection fluorescent observation can be realized with a sufficient brightness and with good contrast and balance. - The
slit 15 having the small-diameter opening 43 c is strong especially against the deterioration of the contrast by the eccentricity of the optical illumination system. When the slit is combined with thelight source 11 having sufficient luminance, it is easy to apply the slit even to an optical system which does not have good accuracy. Since theslit 15 is easily worked, the slit is inexpensive. Furthermore, since the shape of the opening matches that of the luminescent spot of a general high-luminance arc light source, the illuminative light can be efficiently taken in, and the total reflection fluorescent observation by the sufficient brightness can be realized. - The annular and elliptic openings will be explained in the second embodiment.
- Furthermore, the wedge-shaped
plane plate 41 is disposed between thecollector lens 12 which projects thelight source 11 as the parallel light beam and thecondenser 14 which condenses the parallel light beam to form thelight source image 11 a on theslit 15 plane, and each parallel light beam in alight flux 22 is refracted by the same angle by the wedge-shapedplane plate 41. Therefore, little aberration is caused by the wedge-shapedplane plate 41 or thecondenser 14, and the satisfactorylight source image 11 a can be projected on theslit 15 plane. Accordingly, the illuminative light can be efficiently taken in, and bright total reflection fluorescent observation can be performed. - Next, a second embodiment of the present invention will be described.
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FIG. 5 is a diagram showing a schematic configuration of a main part of the second embodiment, and the same components as those ofFIG. 1 are denoted with the same reference numerals. - In the second embodiment, a
wedge prism 13 is disposed as an optical device for decentering the optical axis in the optical path of the parallel light beam from thecollector lens 12. Thewedge prism 13 refracts the parallel light beam emitted from thecollector lens 12 in two directions including upward and downward directions as shown inFIG. 5 , and emits thelight flux 22 having a vertically linearly symmetric shape with respect to the illuminativelight axis 18.FIG. 6 is a diagram concretely showing thelight flux 22 refracted by thewedge prism 13 in two vertical directions. In this case, thelight flux 22 refracted in two vertical directions by thewedge prism 13 is kept to be parallel. - The
condenser 14 condenses thelight flux 22 having two directions from the wedge prism 13 (parallel light beam inclined at a predetermined angle with respect to the illuminative light axis 18) on different places (two places of upper and lower places) on theslit 15 plane to project the image of thelight source 11. - The
slit 15 has three types ofopenings FIGS. 7A, 8A , 9A in the same manner as in the first embodiment. - Since the configuration other than the above-described configuration is similar to that of the first embodiment, detailed description is omitted.
- In this case, when the
slit 15 is moved, the position of the slit image can be adjusted on the emission pupil surface of theobjective lens 4. That is, when the slit image on the emission pupil surface is moved into afluorescent illumination region 28 shown inFIG. 7B , usual fluorescent observation is possible. When the slit image is moved into thetotal reflection region 27, the total reflection fluorescent observation is possible. Furthermore, when the slit image is moved in thetotal reflection region 27, an incidence angle of the illuminative light upon the specimen from the objective lens can be finely adjusted, and an oozing depth of the evanescent light may also be controlled in accordance with an observation position of the specimen. - The similar effect and advantage can be obtained to the first embodiment even when the opening is formed at only either of upper or lower portion.
- The
wedge prism 13 refracts the parallel light beam from thecollector lens 12 in two vertical directions as described above to emit the light flux 22 (light source image) having a vertically linearly symmetric shape with respect to the illuminativelight axis 18. Thelight flux 22 is condensed onto two places of upper and lower places on theslit 15. - In this case, as the
slit 15, it is preferable to use the slit in whichcrescent openings 23 are formed in two places of upper and lower places having point symmetry with respect to a center of theslit 15 on theslit 15 plane as shown inFIG. 7A . - Moreover, the
light source images 11 a are projected as the image of thelight source 11 in thecrescent openings 23 of theslit 15 as shown inFIG. 7C . -
FIG. 7B shows the image of theslit 15 projected on the emission pupil surface of theobjective lens 4, that is, aslit image 23 a. InFIG. 7B , an orbicular portion shown by meshes in apupil diameter 4 a of theobjective lens 4 shows thetotal reflection region 27 where the light is totally reflected by the boundary surface between thespecimen 3 and the cover glass 7. A shown white circular portion inside the orbicular portion shows thefluorescent illumination region 28 where the total reflection is not performed. - Accordingly, all the light beams transmitted through the
openings 23 of theslit 15 fall in thetotal reflection region 27 to form the totally reflected light beams. Since the subsequent operation is similar to that of the first embodiment, detailed description is omitted. - A relation between the
slit image 23 a by thecrescent openings 23 of theslit 15 and the emission pupil surface of theobjective lens 4 will be described in further detail with reference toFIG. 7B . -
FIG. 7B shows a state in which the center of aslit image 23 a by thecrescent opening 23 deviates from that of apupil diameter 4 a of theobjective lens 4. As causes for the deviation, a shift of an optical axis of the optical illumination system to theFS projection lens 17 from thelight source 11, inclination error of a reflection surface in thedichroic mirror 212, mechanical eccentricity of theobjective lens 4 and the like are considered. - In consideration of a case where the
slit image 23 a has the same size as that of thetotal reflection region 27, when theslit image 23 a is eccentric even slightly, a part of theslit image 23 a enters thefluorescent illumination region 28, light leak occurs, and accordingly a drop of contrast is sometimes caused. However, when the size of theslit image 23 a is set to be smaller than that of thetotal reflection region 27, theslit image 23 a constantly stays in thetotal reflection region 27 even with slight movement of theslit image 23 a for the above-described causes. Therefore, there is not fear that the light beam enters thefluorescent illumination region 28 and leaks, and the observation with good contrast is possible. Therefore, thecrescent opening 23 shown inFIG. 7A is formed in a smaller shape so as not to protrude on afluorescent illumination region 28 side even when theslit image 23 a slightly moves by magnification errors of the optical illumination system to theFS projection lens 17 from thelight source 11 and the magnification error of theobjective lens 4. - Moreover, when the
cassette 19 is rotated to insert or remove themirror units optical axis 6, a positioning reproduction accuracy of a rotation direction of thecassette 19 sometimes results in the error of the inclination of thedichroic mirror 212 or the inclination error of thedichroic mirror 212 for each of themirror units slit image 23 a in the objective lens emission pupil surface. In this case, when theslit image 23 a is configured to change its direction in a left-to-right direction with respect to these errors inFIG. 7B , theslit image 23 a by thecrescent opening 23 long in the horizontal direction constantly stays in thetotal reflection region 27. Accordingly, the influence of deterioration of contrast can be eliminated with respect to vibration of theslit image 23 a in the horizontal direction. Even when the position of theopening 23 of theslit 15 slightly shifts with respect to thelight source image 11 a shown inFIG. 7C , much light can be taken in from thelight source 11, because thecrescent openings 23 has a crescent shape long in the horizontal direction. - It is to be noted that the
slit 15 in which thecrescent openings 23 are formed has been described above in detail. However, the present invention is not limited to this. As described in the first embodiment, for example, a slit in which small-diameter openings 24 are formed in two positions of upper and lower positions having point symmetry with respect to the center of theslit 15 on theslit 15 plane as shown inFIG. 8A , a slit in whichannular openings 25 are formed along the peripheral edge portion of theslit 15 plane as shown inFIG. 9A , a slit in which elliptic opening (not shown) are formed and the like are considered. - In the
slit 15 having the small-diameter openings 24, as shown inFIG. 8C , thelight source images 11 a by thelight source 11 are projected with respect to theopenings 24. As shown inFIG. 8B , slitimages 24 a by the small-diameter openings 24 are projected on the emission pupil surface of theobjective lens 4. In theslit 15 having the small-diameter openings 24, thelight source images 11 a sometimes shift from the small-diameter openings 24 and are darkened by deviation of the positions of theopenings 24 and thelight source images 11 a by the above-described factors. However, if thelight source images 11 a greatly decentered, because theopening 24 is hardly extended to the fluorescent illumination area, the contrast can be prevented being degraded. Since the shape of theslit 15 is simple, the slit is characterized in that the slit is easily worked and is inexpensive. - In the
slit 15 having theannular openings 25, as shown inFIG. 9C , thelight source image 11 a by thelight source 11 is projected with respect to theopenings 25. As shown inFIG. 9B , aslit image 25 a by theopenings 25 is projected on the emission pupil surface of theobjective lens 4. When theslit image 25 a shifts to the right/left in theslit 15 having theannular openings 25, an inner diameter of theslit image 25 a easily overlaps with thefluorescent illumination region 28, and the contrast easily drops. However, even when theannular openings 25 and thelight source image 11 a slightly shift, a ratio at which thelight source image 11 a deviates from theopenings 25 is small. Therefore, this is effective means for securing the brightness in a case where the eccentricity of the optical illumination system is small. - Since the shape of the opening can be matched with that of the luminescent spot of the general arc light source in the
slit 15 having the elliptic openings shown inFIG. 8D andFIG. 8E , it is possible to efficiently take in the illuminative light. - Next, the present embodiment is similar to the first embodiment in a case where the total reflection fluorescent observation is changed to the usual fluorescent observation to perform the observation, and therefore the description is omitted.
- As described above, according to the second embodiment, an effect similar to that of the first embodiment can be obtained.
- It is to be noted that the size or shape of the
light source 11 is not described in the first and second embodiments, but the size or shape of thelight source 11 can be set as follows. - When the
slit 15 has thecrescent openings 23 shown inFIG. 7A or theannular openings 25 shown inFIG. 9A , a light source for obtaining an ellipticlight source image 11 b as shown inFIGS. 7D and 9D is used as thelight source 11. Moreover, the ellipticlight source image 11 b is projected on each slit 15 in a state in which the longitudinal direction is positioned transversely as shown inFIGS. 7D and 9D . Then, since thelight source image 11 b is projected on a broad range of the opening 23 (25), an illumination efficiency can further be improved. To embody this, awhole lamp house 30 in which thelight source 11 is stored may be configured so as to be rotatable around the illuminativelight axis 18 in accordance with the lamp shape of thelight source 11. At this time, thewhole lamp house 30 may be rotatably supported, rotated by a predetermined angle in this state, and fixed via screws. Needless to say, instead of rotating thelamp house 30, thelight source 11 itself may be rotated in thelamp house 30. - Next, a modification of the second embodiment will be described.
- The modification of the second embodiment is an example in which the illumination efficiency is raised without using the wedge prism, and will be described with reference to
FIGS. 10A and 10B . - As shown in
FIG. 10A , thelight source 11 is movable vertically in an arrow direction along the plane crossing the illuminativelight axis 18 at right angles. Moreover, thelight source 11 can be positioned in two positions including a position on the illuminativelight axis 18 and a lower position deviating slightly from the illuminativelight axis 18. - To perform the total reflection fluorescent observation, the
light source 11 is set in a position denoted withreference numeral 11′ slightly deviating from the illuminativelight axis 18. Then, as shown inFIG. 10B , the light beams from thelight source 11′ are formed into the parallel light beam having a predetermined angle with respect to the illuminativelight axis 18 by thecollector lens 12, and are projected as alight source image 11 a′ in theopening 23 in the upper part of theslit 15. Accordingly, the illumination efficiency can be raised without using the wedge prism. To return to the usual fluorescent illumination, thelight source 11 may be positioned on the illuminativelight axis 18. - In the present modification, the
light source 11 may be moved in the vertical direction of thelight source 11 with one touch. However, thelight source 11 usually has a centering function. Therefore, when the centering function is used, the illumination efficiency can be enhanced simply and inexpensively. It is to be noted that with the use of the wedge prism, less light is rejected by thecollector lens 12 andcondenser 14. Therefore, the illumination efficiency is better that that of the present modification, but brightness is to be enhanced inexpensively. In this case, the present modification is effective means. - Next, a third embodiment of the present invention will be described.
- In the third embodiment, means for further reducing the illumination unevenness is added to the configuration of the second embodiment.
-
FIG. 11 is a diagram showing a schematic configuration of the third embodiment. - In the third embodiment, a
conical prism 31 is used instead of thewedge prism 13 described in the second embodiment. In theconical prism 31, a conicalconcave portion 31 a is formed in the surface on alight source 11 side, and a surface on aspecimen 3 side is formed in aflat surface 31 b. Moreover, theconical prism 31 is disposed in such a manner that a vertex of the illuminativelight axis 18 matches that of the conicalconcave portion 31 a on the optical path of the parallel light beam from thecollector lens 12. - The
conical prism 31 refracts the parallel light beam from thecollector lens 12 while keeping a parallel light flux toward the outside from the illuminativelight axis 18 to emit alight flux 32.FIG. 12 is a diagram concretely showing thelight flux 32 refracted toward the outside from the illuminativelight axis 18 by theconical prism 31. Unlike thewedge prism 13, an inner diameter of thelight flux 32 is conical. - The
condenser 14 and slit 15 are disposed in the optical path of thelight flux 32 reflected by theconical prism 31. As theslit 15, a slit is used in which theannular opening 25 is formed along the peripheral edge portion as shown inFIG. 13 . - In the configuration, the parallel light beam emitted from the
collector lens 12 is refracted toward the outside from the illuminativelight axis 18 by theconical prism 31. The refracted parallel light beam is condensed in theannular opening 25 of theslit 15 by thecondenser 14, and projected as thelight source image 11 a in theopening 25 of theslit 15. In this case, an infinite number of thelight source images 11 a projected on theannular opening 25 of theslit 15 are projected along a circumferential direction of theopening 25 around the illuminativelight axis 18 as shown inFIG. 13 . - Moreover, the light transmitted through the
slit 15 is projected as a slit image on the emission pupil surface of theobjective lens 4 via theFS projection lens 17. Accordingly, the total reflection fluorescent observation is possible in the same manner as in the second embodiment. - Thereafter, the
light flux 32 refracted toward the outside from the illuminativelight axis 18 is generated by theconical prism 31, and accordingly thelight source image 11 a can be projected along theannular opening 25 of theslit 15. Accordingly, in addition to the effect of the second embodiment, since theannular opening 25 can be uniformly illuminated, the illumination unevenness can be largely reduced. - Next, a modification of the third embodiment will be described
- The modification of the third embodiment is an example including another means for reducing the illumination unevenness, and will be described with reference to
FIGS. 14A and 14B . - In this case, the
wedge prism 13 is disposed in the optical path between thecollector lens 12 and thecondenser 14 in the same manner as in the second embodiment. Moreover, a slit in which theannular opening 25 is formed along the peripheral edge portion as shown inFIG. 14B is used as theslit 15. Further-more, in this state, thewedge prism 13 is rotated at a high speed in a direction of anarrow 33 using the illuminativelight axis 18 which is a rotational center. In this case, the prism is rotated once at about 30 msec in the visual observation, or rotated at a rotation number higher than a scanning speed of photo-detection, when photo-detection means such as CCD. Accordingly, as shown inFIG. 14B , thelight source image 11 a rotates along theannular opening 25 around the illuminativelight axis 18. Therefore, when a time average of the rotation is taken, an effect similar to that with the use of theconical prism 31 described in the third embodiment is obtained. In this case, the rotation means of thewedge prism 13 can be realized using a known motor and bearing. - Moreover, the light transmitted through the
slit 15 is projected on the slit image on the emission pupil surface of theobjective lens 4 via theFS projection lens 17. Accordingly, the total reflection fluorescent observation is possible in the same manner as in the second embodiment. - Therefore, when the
wedge prism 13 is configured so as to be rotatable around the illuminativelight axis 18 of the optical illumination system at a high speed, thelight source image 11 a can be projected along theannular opening 25, and therefore the total reflection fluorescent observation is realized with the illumination without any directionality or unevenness. The cost can also be reduced without using the expensiveconical prism 31. - A fourth embodiment of the present invention will be described.
-
FIG. 15 is a diagram showing a schematic configuration of the fourth embodiment, and the same components as those ofFIG. 1 are denoted with the same reference numerals. - In the fourth embodiment, six
LEDs 34 having micro luminescent spots are disposed instead of thelight source 11. In this case, sixLEDs 34 are disposed in the position of the point symmetry with respect to the illuminativelight axis 18 on the plane crossing the illuminativelight axis 18 at right angles. In the fourth embodiment, thewedge prism 13 is not required. As theslit 15, a slit is used in which thecrescent openings 23 are formed in two positions of upper and lower positions of the point symmetry with respect to the center of theslit 15 on theslit 15 plane as shown inFIG. 16 . - In the above-described configuration, the illuminative lights emitted from six
LEDs 34 are formed into parallel light beams having a predetermined angle with respect to the illuminativelight axis 18 by thecollector lens 12, and are projected asLED images 35 in the upper/lower crescent openings 23 of theslit 15 by thecondenser 14 as shown inFIG. 16 . - Moreover, the light transmitted through the
slit 15 is projected as the slit image on the emission pupil surface of theobjective lens 4 via theFS projection lens 17. Accordingly, the total reflection fluorescent observation is possible in the same manner as in the second embodiment. - Therefore, since the
respective LED images 35 of sixLEDs 34 can be projected on accordance with the upper/lower crescent openings 23 of theslit 15, the illumination efficiency can be further enhanced. - It is to be noted that when the slit having the
annular openings 25 as shown inFIG. 17 is used as theslit 15, a large number ofLEDs 34 are arranged in an annular form around the illuminativelight axis 18. Moreover, the lights from theLEDs 34 arranged in the annular form are projected as theLED images 35 in theannular openings 25 of theslit 15 via thecollector lens 12 andcondenser 14. - Since the
LED images 35 from the annularly arrangedLEDs 34 can be uniformly projected along theannular openings 25 of theslit 15 in this manner, the illumination unevenness can be reduced. - It is to be noted that when the number of
LEDs 34 is further increased and a large number of LEDs are arranged around the illuminativelight axis 18, theLEDs 34 can only be selectively lit in accordance with the shapes of the openings of theslit 15 to project theLED images 35 in accordance with various openings. All theLEDs 34 may be lit in the usual fluorescent illumination observation. - According to the fourth embodiment, by the use of the light source having a plurality of micro luminescent spots arranged to fill the openings 23 (25) of the
slit 15, the light source image is projected only in a range passing through the openings 23 (25), and the illuminative light is not introduced except the total reflection region. Therefore, the illuminative light can be efficiently taken in, and the total reflection fluorescent observation is possible with the sufficient brightness and good contrast. Since the expensive wedge prism or conical prism is not used, the microscope is inexpensive. Especially, when a slit having theannular opening 25 is used as theslit 15, the total reflection fluorescent illumination having remarkably little unevenness may also be obtained. - A fifth embodiment of the present invention will be described.
-
FIG. 18 is a diagram showing a schematic configuration of the fifth embodiment, and the same components as those ofFIG. 1 are denoted with the same reference numerals. - In the fifth embodiment, an
afocal converter 36 is disposed as magnification varying means for raising light source magnification in the optical path between thecollector lens 12 and thewedge prism 13. Theafocal converter 36 comprises aconvex lens 36 a andconcave lens 36 b. By theafocal converter 36, the parallel light beam from thecollector lens 12 is condensed onto theconvex lens 36 a, and diverted by theconcave lens 36 b. Accordingly, the parallel light beam whose light source magnification has been raised can be emitted. Theafocal converter 36 is detachably inserted together with thewedge prism 13 and slit 15 with respect to the optical path. - In the
slit 15, a slit in which thecrescent openings 23 are formed in the two positions of upper and lower positions of the point symmetry with respect to the center of theslit 15 on theslit 15 plane as shown inFIGS. 19A and 19B , or a slit in which theannular openings 25 are formed along the peripheral edge portion of theslit 15 as shown inFIGS. 20 and 20 B are used. - When the total reflection fluorescent illumination is performed, the illuminative light emitted from the
light source 11 is projected as alight source image 37 on theslit 15 plane via thecollector lens 12, theconvex lens 36 a andconcave lens 36 b configuring theafocal converter 36, thewedge prism 13, and thecondenser 14. In this case, in thelight source image 37, since the light source magnification is raised by theafocal converter 36, thelight source image 37 projected on theslit 15 plane spreads sufficiently in a broad range on therespective openings FIG. 19B or 20B.FIG. 19A or 20A shows a case where theafocal converter 36 is not disposed, and thelight source image 37 projected on theslit 15 plane overlaps with a part of theopenings - Therefore, to perform the total reflection fluorescent illumination, when the
convex lens 36 a andconcave lens 36 b configuring theafocal converter 36 are inserted in the optical path to raise the magnification of thelight source image 37, more openings 23 (25) of theslit 15 can be filled with thelight source images 37, and therefore the illumination efficiency can be further enhanced. - The light transmitted through the
slit 15 is projected as the slit image on the emission pupil surface of theobjective lens 4 via theFS projection lens 17, and the total reflection fluorescent observation is possible in the same manner as in the second embodiment. - When the small-
diameter openings 24 are formed in two positions of upper and lower positions of the point symmetry with respect to the center of theslit 15 in theslit 15 as shown inFIG. 21 , even thelight source images 37 projected onto theslit 15 plane sufficiently fill theopenings 24 of theslit 15 in a state free of theafocal converter 36. Therefore, even when the light source magnification is raised particularly using theafocal converter 36, an effect of enhancement of the illumination efficiency is little. - Next, a case where usual fluorescent observation is performed will be described.
- As shown in
FIG. 22 , theafocal converter 36,wedge prism 13, and slit 15 are removed from the optical path of the optical illumination system, and the aperture stop (AS) 29 is inserted instead of theslit 15. When thewedge prism 13 or theslit 15 enters the optical path, the illumination efficiency drops or the illumination unevenness increases in the same manner as in the second embodiment. Therefore, to perform the usual fluorescent observation, the prism or the slit is removed from the optical path, and the aperture stop (AS) 29 for adjusting the brightness is inserted instead of theslit 15. This can be realized by the use of an inserting/detaching mechanism such as a known slider. Theconvex lens 36 a andconcave lens 36 b configuring theafocal converter 36 are effective for enhancing the illumination efficiency. However, on the contrary, the illumination field is narrowed, and an observable range is narrowed. Therefore, theafocal converter 36 is not required at the time of the usual fluorescent observation with the sufficient brightness, and is also removed from the optical path. - It is to be noted that the
afocal converter 36 is used to changing the light source magnification. Therefore, even when the converter is removed from the optical path, a projection plane of thelight source 11 is unchanged. Therefore, even when the aperture stop (AS) 29 is inserted in the optical path instead of theslit 15, the light source image is projected on the aperture stop (AS) 29 plane. Therefore, there is no fear that the illumination unevenness occurs also at the usual fluorescent observation time, the illumination efficiency does not drop, the illumination is bright, and therefore optimum microscopic inspection can be performed. - In this state, specimen can be visually observed by the generally known usual fluorescent observation method.
- It is to be noted that another variable magnification lens is also usable as means for raising the light source magnification in addition to the
afocal converter 36. - In the fifth embodiment, the
afocal converter 36 is disposed as magnification varying means for raising a projection magnification of thelight source 11 between theslit 15 and thelight source 11. Accordingly, in the total reflection illumination, the magnification of the light source image is raised, and even a portion incapable of filling theopenings 23 of theslit 15 is filled with thelight source image 37. Accordingly, the total reflection fluorescent observation is possible by brighter illumination. Even when theafocal converter 36 is inserted or removed with respect to the optical path, the projection position of thelight source image 37 in the optical-axis direction does not change. Therefore, the illumination efficiency at a total reflection fluorescent observation time does not drop. Moreover, the illumination unevenness does not easily occur at a usual fluorescent observation time, and optimum microscopic inspection can be performed in each observation. - A sixth embodiment of the present invention will be described.
-
FIG. 23 is a diagram showing a schematic configuration of a main part of a sixth embodiment, and the same components as those ofFIG. 1 are denoted with the same reference numerals. - In this case, the
collector lens 12,condenser 14, and slit 15 are disposed on the illuminativelight axis 18 of the light from thelight source 11. Aconvex lens 44 having a large diameter is disposed as a lens having a weak refractive power between thecollector lens 12 and thecondenser 14. - The
convex lens 44 is disposed while acentral axis 44 a is largely shifted from the illuminativelight axis 18, and the parallel light beam from thecollector lens 12 is refracted by a predetermined angle with respect to the illuminativelight axis 18. Thecondenser 14 condenses alight flux 45 refracted by theconvex lens 44 is condensed on theslit 15 plane, and thelight source image 11 a is projected. Also in this case, a slit is used in which acrescent opening 43 is formed on the slit plane is used in the same manner as inFIG. 3A . Thelight source image 11 a is projected on the crescent opening 43 a via thecondenser 14. - Also in the sixth embodiment, the
convex lens 44 and slit 15 can be detachably inserted with respect to the optical path of the illuminative light by known switching mechanisms such as a slider. Theslit 15 inserted in the optical path is movable further along the plane crossing the illuminativelight axis 18 at right angles in a known arrow direction. - The other configuration is similar to that of
FIG. 1 . - Therefore, the effect similar to that of the first embodiment can be expected.
- A seventh embodiment of the present invention will be described.
-
FIG. 24 is a diagram showing a schematic configuration of a main part of the seventh embodiment, and the same components as those ofFIG. 1 are denoted with the same reference numerals. - In
FIG. 24 , thecollector lens 12,condenser 14, and slit 15 are disposed on the illuminativelight axis 18 of the light from thelight source 11. Aparallel plane plate 46 is disposed between thecondenser 14 and theslit 15. - The
parallel plane plate 46 is inclined at a predetermined angle with respect to the illuminativelight axis 18 and disposed to move the light beam from thecondenser 14 in parallel with the illuminativelight axis 18, and condenses the light on theslit 15 plane to project thelight source image 11 a. Also in this case, a slit in which thecrescent opening 43 is formed on the slit plane is used as theslit 15 in the same manner as inFIG. 22 . Thelight source image 11 a is projected on thecrescent opening 43 via thecondenser 14. - Also in this case, the
parallel plane plate 46 and slit 15 are detachably inserted with respect to the optical path of the illuminative light by known switching mechanisms such as the slider. Moreover, the inclination angle of theparallel plane plate 46 inserted in the optical path is adjustable in the arrow direction. Therefore, even when the total reflection region differs with the type of the objective lens, the inclination angle of theparallel plane plate 46 can be adjusted to adjust thelight source image 11 a in an optimum position in the total reflection region. Furthermore, even theslit 15 inserted in the optical path can move along the plane crossing the illuminativelight axis 18 at right angles in the arrow direction. - The other configuration is similar to that of
FIG. 1 . - Therefore, the effect similar to that of the first embodiment can be expected.
- An eighth embodiment of the present invention will be described.
-
FIG. 25 is a diagram showing a schematic configuration of a main part of the eighth embodiment, and the same components as those ofFIG. 1 are denoted with the same reference numerals. - In
FIG. 25 , thecollector lens 12,condenser 14, and slit 15 are disposed on the illuminativelight axis 18 of the light from thelight source 11.Mirrors condenser 14 and theslit 15. - The light beam from the
condenser 14 is reflected by themirror 47, and the reflected light is reflected by themirror 48. Accordingly, the optical path of the optical illumination system is moved in parallel, that is, the light beam from thecondenser 14 is moved in parallel with the illuminativelight axis 18 and condensed onto theslit 15 plane to project thelight source image 11 a. Even in the eighth embodiment, the slit in which thecrescent opening 43 is formed on the slit plane as described with reference toFIG. 22 is used as theslit 15. Thelight source image 11 a is projected on thecrescent opening 43 via thecondenser 14. - Even in this case, the
mirrors slit 15 can be detachably inserted with respect to the optical path of the illuminative light by the known switching mechanisms such as the slider. In themirrors mirror 48 is movable in the arrow direction, and the distance from themirror 47 can be adjusted. Accordingly, even when the total reflection region differs with the type of the objective lens, the distance between themirrors light source image 11 a in an optimum position in the total reflection region. Furthermore, even theslit 15 inserted in the optical path can move along the plane crossing the illuminativelight axis 18 at right angles in the arrow direction. - The other configuration is similar to that of
FIG. 1 . - Therefore, the effect similar to that of the first embodiment can be expected.
- The present invention is not limited to the above-described embodiments, and can be variously modified in a range in which the scope is not changed. For example, in the embodiments, the optical device for decentering the optical axis has been described. Alternatively, a plurality of optical devices having different eccentricities of the optical axis are prepared, and may also be selectively used in accordance with the type of the objective lens (the total reflection region differs). The plane of the
slit 15 on thelight source 11 side may also be formed in a reflection plane or an irregular reflection plane. In this case, degradation by heat or the like in a portion which interrupts the light beam of theslit 15, that is, a portion irradiated with the light beam can be reduced. Furthermore, in the embodiments, the inverted microscope has been described by which the observation is performed by the objective lens disposed under the specimen, but a transmission illumination type may also be used in which the total reflection fluorescent illumination is performed using a condenser lens, or an erected microscope may also be used. - Furthermore, the embodiments include various stages of inventions, and various inventions can be extracted by an appropriate combination of a plurality of configuring elements. For example, even when several configuring elements are removed from all the configuring elements described in the embodiments, the described problems to be solved by the present invention can be resolved, and the described effects of the present invention are obtained. In this case, the configuration from which the configuring elements are removed can be extracted as the invention.
- It is to be noted that the above-described embodiments also include the following inventions.
- A fluorescence microscope according to a first aspect of the present invention is characterized by comprising a light source; an optical illumination system which forms an optical path to irradiate a specimen with a light beam from the light source; an objective lens which condenses the light beam of the optical illumination system onto the specimen; an optical device which is disposed on the optical path of the optical illumination system and which decenters the light beam by decentering an optical axis of the optical path; and a slit which passes the light beam decentered by the optical device through a total reflection illumination region on an emission pupil surface of the objective lens. In the first aspect, the following manners are preferable.
- (1) The total reflection illumination is illumination using an evanescent light oozing by a predetermined amount on a specimen side in a boundary surface between glass on which the specimen is laid and the specimen.
- (2) The optical device and the slit are movable along a plane vertical to the optical axis of the optical path of the optical illumination system.
- (3) The opening has at least one of a crescent shape, a circular shape, a half-ring shape, and an elliptic shape.
- (4) The optical device is a prism.
- (5) In (4), the prism is either a wedge prism or a conical prism.
- (6) In (4), the prism is a wedge-shaped plane plate.
- (7) The optical device is a lens having a small refractive index.
- (8) The optical device is a parallel plane plate disposed with a predetermined angle with respect to the optical axis.
- (9) The optical device comprises a pair of mirrors which move the optical path of the optical illumination system.
- (10) The optical device includes a plurality of optical devices, and the plurality of optical devices are selectively inserted in the optical path of the optical illumination system.
- (11) In (5) or (6), the wedge prism is rotatable around the optical axis of the optical illumination system.
- (12) The light source has a luminescent spot in which a projected image on an emission pupil surface of the objective lens is smaller than an emission pupil diameter of the objective lens.
- (13) In (12), the light source is either a micro arc type light source or a light source comprising a plurality of micro luminescent spots.
- (14) In (13), the light source comprising the plurality of micro luminescent spots includes a plurality of light emitting diodes.
- (15) In (14), the plurality of light emitting diodes are arranged on a circumference having a predetermined diameter.
- (16) An optical magnification varying system which is disposed in the optical path of the optical device on the light source side to raise a projection magnification of the light source is further provided.
- (17) In (16), the optical magnification varying system includes an afocal converter.
- (18) The slit has a reflection surface or an irregular reflection surface formed on a plane on a light source side.
- A fluorescence microscope according to a second aspect of the present invention is characterized by comprising: a light source; an optical illumination system which forms an optical path to irradiate a specimen with a light beam from the light source; an objective lens which condenses the light beam of the optical illumination system onto the specimen; and a slit which passes the light beam from the light source through a total reflection illumination region on an emission pupil surface of the objective lens, in which an emission position of the light beam emitted from the light source is movable between an optical axis of the optical illumination system and a position shifting from the optical axis by a predetermined distance.
- According to the embodiments of the present invention, the optical device for decentering the optical axis of the optical path is disposed between the light source and the slit. Accordingly, the illuminative light can be efficiently taken in the total reflection region having an emission pupil diameter of the objective lens by the eccentricity of the optical axis of the optical path.
- Moreover, the optical device and slit are detachably inserted in the optical path. Therefore, when the states are only selected, the illuminative light can be efficiently taken into the total reflection region of the emission pupil surface of the objective lens or the fluorescent illumination region where the total reflection is not performed.
- Furthermore, the slit for performing the total reflection illumination has the crescent opening which passes the light beam only through a part of the total reflection region. Accordingly, even when the position or the size of the slit image changes by the eccentricity or the magnification error of the optical illumination system, the slit image can be prevented from deviating from the total reflection region of the emission pupil surface of the objective lens.
- Moreover, since the wedge prism or the conical prism is used as the optical device, the respective parallel light beams in the light flux can be refracted by an equal angle, little aberration is generated by the prism or the condenser, and a satisfactory light source image can be projected on the slit plane.
- Furthermore, since a light source having a plurality of micro luminescent spots is used as the light source, the light source image is projected only in a range passing through the slit. Since the illuminative light is not introduced into a region other than the total reflection region, the illuminative light can be efficiently taken.
- Moreover, since the wedge prism is rotatable around the optical axis of the optical illumination system, the light source image can be projected on the annular shape, and the illumination is obtained without any directionality or unevenness.
- Furthermore, since an optical magnification varying system for changing a projection magnification is disposed in the optical path of the optical device on the light source side, in the total reflection illumination, the magnification of the light source image is raised, and even the portion of the slit that cannot be filled is filled with the light source image, and the total reflection fluorescent observation by brighter illumination is possible.
- As described above, according to the embodiments of the present invention, there can be provided a total reflection fluorescent microscope in which use efficiency of the illuminative light is raised, and the total reflection fluorescent observation is possible with the sufficient brightness and good contrast.
- Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general invention concept as defined by the appended claims and their equivalents.
Claims (5)
1. A fluorescent microscope comprising:
a light source;
an optical illumination system which forms an optical path to irradiate a specimen with a light beam from the light source;
an objective lens which condenses the light beam from the light source, which has traveled through the optical illumination system, onto the specimen; and
a slit which is located in the optical illumination system to pass the light beam from the light source through a total reflection illumination region on an emission pupil surface of the objective lens,
wherein an emission position of the light beam emitted from the light source is movable between an optical axis of the optical illumination system and a position shifted from the optical axis by a predetermined distance.
2. The fluorescent microscope according to claim 1 , wherein the optical illumination system comprises:
a collector lens which parallelizes the light beam from the light source;
a condenser which condenses the light beam from the light source that has passed through the collector lens; and
a projection lens which projects an image formed by the light beam condensed by the condenser onto an emission pupil surface of the objective lens.
3. The fluorescent microscope according to claim 1 , wherein the slit is removed from the optical illumination system, and the emission position of the light beam emitted from the light source is moved onto the optical axis of the optical illumination system, to perform fluorescent illumination.
4. A fluorescent microscope comprising:
an objective lens which is used in observation of a specimen;
a movable light source which irradiates the specimen with a light beam;
a collector lens which parallelizes the light beam from the light source;
a condenser which condenses the light beam from the light source that has passed through the collector lens; and
a slit which is located at a position onto which the light beam is condensed by the condenser;
a projection lens which projects an image formed by the light beam from the light source through the slit onto an emission pupil surface of the objective lens,
wherein when the light source is moved to a position displaced at least from an optical axis of the collector lens, the image formed by the light beam from the light source is projected onto the slit, and the light beam from the light source is caused to pass through a total reflection illumination region of the emission pupil surface of the objective lens.
5. The fluorescent microscope according to claim 4 , wherein the light source is moved onto the optical axis of the collector lens and the slit is removed from a path of the light beam from the light source to perform fluorescent illumination.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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US11/734,208 US20070183030A1 (en) | 2003-05-21 | 2007-04-11 | Total reflection fluorescent microscope |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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JP2003-143382 | 2003-05-21 | ||
JP2003143382A JP2004347777A (en) | 2003-05-21 | 2003-05-21 | Total reflection fluorescence microscope |
US10/848,626 US7224524B2 (en) | 2003-05-21 | 2004-05-18 | Total reflection fluorescent microscope |
US11/734,208 US20070183030A1 (en) | 2003-05-21 | 2007-04-11 | Total reflection fluorescent microscope |
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US10/848,626 Division US7224524B2 (en) | 2003-05-21 | 2004-05-18 | Total reflection fluorescent microscope |
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US20070183030A1 true US20070183030A1 (en) | 2007-08-09 |
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US11/734,208 Abandoned US20070183030A1 (en) | 2003-05-21 | 2007-04-11 | Total reflection fluorescent microscope |
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US10/848,626 Active 2024-11-24 US7224524B2 (en) | 2003-05-21 | 2004-05-18 | Total reflection fluorescent microscope |
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Also Published As
Publication number | Publication date |
---|---|
JP2004347777A (en) | 2004-12-09 |
US20040246573A1 (en) | 2004-12-09 |
DE102004024837A1 (en) | 2005-01-05 |
US7224524B2 (en) | 2007-05-29 |
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