Total internal reflection fluorescence microscope Number:7,385,758 from the United States Patent and Trademark Office (PTO) owispatent

Title: Total internal reflection fluorescence microscope

Abstract: A condenser lens is disposed in a position facing an objective lens via a specimen, a reflection mirror is movably disposed in the vicinity of an outermost part of a transmitted illuminative light path on the side of a transmitted illuminative light source from the condenser lens, and a laser beam output from a laser oscillation unit is reflected by the reflection mirror and is incident upon the condenser lens.

Patent Number: 7,385,758 Issued on 06/10/2008 to Aono,   et al.

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Inventors:	Aono; Yasushi (Yokohama, JP), Mochizuki; Tsuyoshi (Hachioji, JP), Osa; Kazuhiko (Hachioji, JP)
Assignee:	Olympus Corporation (Tokyo, JP)
Appl. No.:	10/816,489
Filed:	March 31, 2004

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Foreign Application Priority Data

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Apr 04, 2003 [JP]			2003-101346

Current U.S. Class:	359/390 ; 359/368; 359/385
Field of Search:	359/368,385,390

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References Cited [Referenced By]

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U.S. Patent Documents

4972258	November 1990	Wolf et al.
5866911	February 1999	Baer
6055097	April 2000	Lanni et al.
2003/0086163	May 2003	Aono et al.

Foreign Patent Documents

	3093145		Jul., 2000		JP
	2001-013413		Jan., 2001		JP

Other References

D Axelrod, 5. Total Internal Reflection Fluorescence at Biological Surfaces, Noninvasive Techniques in Cell Biology, pp. 93-127 (1990). cited by other.

Primary Examiner: Pritchett; Joshua L
Attorney, Agent or Firm: Frishauf, Holtz, Goodman & Chick, P.C.

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Claims

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What is claimed is:

1. A total internal reflection fluorescence microscope comprising: at least one objective lens which takes light from a specimen; an image pick-up device which picks up an image of the light taken into the objective lens; an observation optical path via which the light taken into the objective lens is condensed onto the image pick-up device; a condenser lens, which is disposed in a position facing the objective lens via the specimen, which has a numerical aperture that makes possible total internal reflection illumination, and which guides a transmitted illuminative light, which is emitted by a light source, into the specimen; a base including an upper portion that holds the condenser lens; a laser oscillation unit which outputs a laser beam; an optical fiber which transmits the laser beam output from the laser oscillation unit; a reflection mirror provided at a lower portion of the base to reflect the laser beam output from the optical fiber along a path substantially parallel to a light path of the transmitted illuminative light from the light source, so as to introduce the laser beam into a vicinity of an outermost side of the condenser lens; a condensing lens which condenses the laser beam output from the optical fiber, such that the laser beam is condensed at a condensing position in a vicinity of a front focal position of the condenser lens; and a mirror moving section which moves the reflection mirror in a translatory manner, with respect to the condensing lens, in a direction that is substantially perpendicular to the light path of the transmitted illuminative light from the light source, such that when the mirror moving section moves the reflection mirror, the path of the laser beam reflected by the reflection mirror remains substantially parallel to the light path of the transmitted illuminative light, wherein when the mirror moving section moves the reflection mirror with respect to the condensing lens, an incidence angle, at a boundary of the specimen, of the laser beam emitted from the condenser lens is changed, thereby changing a leak-out depth of evanescent light that illuminates the specimen.

2. The total internal reflection fluorescence microscope according to clam 1, further comprising a conversion lens unit which converts a numerical aperture of the laser beam incident upon the condensing position without changing the condensing position of the laser beam.

3. The total internal reflection fluorescence microscope according to claim 2, wherein the conversion lens unit is removably inserted between an emission end of the optical fiber and the condensing lens.

4. The total internal reflection fluorescence microscope according to claim 2, wherein the conversion lens unit includes a lens group which converts a numerical aperture of the laser beam incident upon the condensing position.

5. The total internal reflection fluorescence microscope according to claim 2, wherein the conversion lens unit comprises: a convex lens which converts the numerical aperture of the laser beam diverged and emitted from an emission end of the optical fiber; and a concave lens which diverges the laser beam having the numerical aperture converted by the convex lens.

6. The total internal reflection fluorescence microscope according to claim 5, wherein the concave lens is movable in an optical path direction of the laser beam between the convex lens and the condensing lens.

7. The total internal reflection fluorescence microscope according to claim 3, wherein the at least one objective lens comprises a plurality of objective lenses having different observation magnifications, and the microscope further comprises: an objective lens switching section which selectively disposes one of the plurality of objective lenses on the observation optical path; and a control section which controls inserting and removing of the conversion lens unit between the emission end of the optical fiber and the condensing lens in accordance with the observation magnification of the objective lens disposed on the observation optical path.

8. The total internal reflection fluorescence microscope according to claim 7, wherein the plurality of objective lenses include at least one objective lens for high-magnification observation and at least one objective lens for low-magnification observation, and wherein the control section inserts the conversion lens unit between the emission end of the optical fiber and the condensing lens when the objective lens for high-magnification observation is disposed on the observation optical path, and the control section removes the conversion lens unit from between the emission end of the optical fiber and the condensing lens when the objective lens for low-magnification observation is disposed on the observation optical path.

9. The total internal reflection fluorescence microscope according to claim 8, wherein an irradiation range of the laser beam with respect to the specimen is caused to agree with an observation range of the objective lens for high-magnification observation when the conversion lens unit is inserted between the emission end of the optical fiber and the condensing lens, and the irradiation range of the laser beam with respect to the specimen is caused to agree with an observation range of the objective lens for low-magnification observation when the conversion lens unit is removed from between the emission end of the optical fiber and the condensing lens.

10. The total internal reflection fluorescence microscope according to claim 1, further comprising a zoom lens unit which adjusts the condensing position of the laser beam in the vicinity of the front focal position of the condenser lens.

11. The total internal reflection fluorescence microscope according to claim 10, wherein the zoom lens unit comprises a lens group which adjusts the condensing position of the laser beam in the vicinity of the front focal position of the condenser lens.

12. The total internal reflection fluorescence microscope according to claim 10, wherein the zoom lens unit comprises: a convex lens which converts the numerical aperture of the laser beam diverged and emitted from an emission end of the optical fiber; and a concave lens which diverges the laser beam having the numerical aperture converted by the convex lens.

13. The total internal reflection fluorescence microscope according to claim 12, wherein the convex lens is movable in an optical path direction of the laser beam between the emission end of the optical fiber and the condensing lens.

14. The total internal reflection fluorescence microscope according to claim 12, wherein the concave lens is movable in an optical path direction of the laser beam between the convex lens and the condensing lens.

15. The total internal reflection fluorescence microscope according to claim 12, further comprising: a control section which determines a moving position of the concave lens to adjust the condensing position of the laser beam in the vicinity of the front focal position of the condenser lens in accordance with positional movement of the convex lens, and which controls movement of the convex lens and the concave lens based on information of the determined moving position of the concave lens.

16. The total internal reflection fluorescence microscope according to claim 11, wherein the at least one objective lens comprises a plurality of objective lenses having different observation magnifications, and the microscope further comprises: an objective lens switching section which selectively disposes one of the plurality of objective lenses on the observation optical path; and a control section which determines a relative positional relation of the lens group disposed in the zoom lens unit in each optical axis direction in accordance with an observation magnification of the objective lens disposed on the observation optical path.

17. The total internal reflection fluorescence microscope according to claim 16, wherein the lens group of the zoom lens unit comprises: a convex lens which converts the numerical aperture of the laser beam diverged and emitted from an emission end of the optical fiber; and a concave lens which diverges the laser beam having the numerical aperture converted by the convex lens, and wherein the control section determines a moving position of the concave lens to adjust the condensing position of the laser beam in the vicinity of the front focal position of the condenser lens in accordance with positional movement of the convex lens, and the control section controls movement of the convex lens and the concave lens based on information of the determined moving position of the concave lens.

18. The total internal reflection fluorescence microscope according to claim 1, wherein the laser oscillation unit, the optical fiber, the reflection mirror, the mirror moving section, and the condensing lens form at least a part of a laser introduction section, and the microscope comprises a plurality of said laser introduction sections, each of which emits a laser beam that is condensed at a corresponding condensing position in a vicinity of corresponding front focal positions of the condenser lens; and wherein the microscope further comprises: at least one additional image pick-up device which picks up an image of the light taken into the objective lens; at least one additional observation optical path via which the light taken into the objective lens is condensed onto the additional image pick-up device; an optical dividing system which divides the light taken into the objective lens onto respective ones of the optical paths toward the image pick-up devices depending on optical characteristics of the light.

19. The total internal reflection fluorescence microscope according to claim 18, wherein each of the plurality of laser introduction sections comprises a conversion lens unit which converts a numerical aperture of the laser beam incident upon the condensing position without changing the condensing position of the laser beam.

20. The total internal reflection fluorescence microscope according to claim 19, each said conversion lens unit is removably inserted between an emission end of the optical fiber and the condensing lens in the corresponding one of the laser introduction sections.

21. The total internal reflection fluorescence microscope according to claim 19, wherein each said conversion lens unit includes a lens group which converts a numerical aperture of the laser beam incident upon the corresponding condensing position.

22. The total internal reflection fluorescence microscope according to claim 19, each said conversion lens unit comprises: a convex lens which converts the numerical aperture of the laser beam diverged and emitted from an emission end of the optical fiber in the corresponding one of the laser introduction sections; and a concave lens which diverges the laser beam having the numerical aperture converted by the convex lens.

23. The total internal reflection fluorescence microscope according to claim 22, wherein each said concave lens is movable in an optical path direction of the laser beam between the convex lens and the condensing lens in the corresponding one of the laser introduction sections.

24. The total internal reflection fluorescence microscope according to claim 20, wherein the at least one objective lens comprises a plurality of objective lenses having different observation magnifications, and the microscope further comprises: an objective lens switching section which selectively disposes one of the plurality of objective lenses to take the light from the specimen; and a control section which controls inserting and removing of the conversion lens unit in each of the plurality of laser introduction sections between the emission end of the optical fiber and the condensing lens in accordance with the observation magnification of the objective lens disposed to take the light from the sample.

25. The total internal reflection fluorescence microscope according to claim 24, wherein a plurality of objective lenses include at least one objective lens for high-magnification observation and at least one objective lens for low-magnification observation, and wherein the control section inserts the conversion lens unit in each of the plurality of laser introduction sections between the emission end of the optical fiber and the condensing lens when the objective lens for high-magnification observation is disposed to take the light from the sample, and removes the conversion lens unit in each of the plurality of laser introduction sections between the emission end of the optical fiber and the condensing lens when the objective lens for low-magnification observation is disposed to take the light from the sample.

26. The total internal reflection fluorescence microscope according to claim 25, wherein, for each of the laser introduction sections, an irradiation range of the laser beam with respect to the specimen is caused to agree with an observation range of the objective lens for high-magnification observation when the conversion lens unit is inserted between the emission end of the optical fiber and the condensing lens, and the irradiation range of the laser beam with respect to the specimen is caused to agree with an observation range of the objective lens for low-magnification observation when the conversion lens unit is inserted between the emission end of the optical fiber and the condensing lens.

27. The total internal reflection fluorescence microscope according to claim 18, wherein each of the plurality of laser introduction sections further comprises a zoom lens unit which adjusts the condensing position of the laser beam in the vicinity of the front focal position of the condenser lens.

28. The total internal reflection fluorescence microscope according to claim 27, wherein each said zoom lens unit comprises a lens group which adjusts the condensing position of the laser beam in the vicinity of the front focal position of the condenser lens.

29. The total internal reflection fluorescence microscope according to claim 27, wherein the lens group of each said zoom lens unit comprises: a convex lens which converts the numerical aperture of the laser beam diverged and emitted from an emission end of the optical fiber in the corresponding laser introduction section; and a concave lens which diverges the laser beam having the numerical aperture converted by the convex lens.

30. The total internal reflection fluorescence microscope according to claim 29, wherein each said convex lens is movable in an optical path direction of the laser beam between the emission end of the optical fiber and the condensing lens in the corresponding one of the laser introduction sections.

31. The total internal reflection fluorescence microscope according to claim 29, wherein each said concave lens is movable in an optical path direction of the laser beam between the convex lens and the condensing lens in the corresponding one of the laser introduction sections.

32. The total internal reflection fluorescence microscope according to claim 29, further comprising: a control section which determines, for each said zoom lens unit, a moving position of the concave lens to adjust the condensing position of the laser beam in the vicinity of the front focal position of the condenser lens in accordance with positional movement of the convex lens, and which controls movement of the convex lens and the concave lens based on information of the determined moving position of the concave lens.

33. The total internal reflection fluorescence microscope according to claim 28, wherein the at least one objective lens comprises a plurality of objective lenses having different observation magnifications, and the microscope further comprises: an objective lens switching section which selectively disposes one of the plurality of objective lenses to take the light from the specimen; and a control section which determines a relative positional relation of the lens groups disposed in the zoom lens units in each optical axis direction in accordance with an observation magnification of the objective lens disposed on the observation optical path.

34. The total internal reflection fluorescence microscope according to claim 33, wherein the lens group of each said zoom lens unit comprises: a convex lens which converts the numerical aperture of the laser beam diverged and emitted from an emission end of the optical fiber in the corresponding laser introduction section; and a concave lens which diverges the laser beam having the numerical aperture converted by the convex lens, and wherein, for each of the zoom lens units, the control section determines a moving position of the concave lens to adjust the condensing position of the laser beam in the vicinity of the front focal position of the condenser lenses in accordance with positional movement of the convex lens, and the control section controls movement of the convex lens and the concave lens based on information of the determined moving position of the concave lens.

35. The total internal reflection fluorescence microscope according to claim 18, wherein the plurality of laser introduction sections are disposed radially around the transmitted illuminative light path and extend in directions that are substantially perpendicular to a path of the transmitted illuminative light.

36. The total internal reflection fluorescence microscope according to claim 18, further comprising: at least one optical path length adjustment section which is disposed on at least one divided observation optical path among the plurality of divided observation optical paths divided by the optical dividing system and which extends and contracts an optical path length.

37. The total internal reflection fluorescence microscope according to claim 36, wherein the optical path length adjustment section comprises: a fixed prism group fixed/disposed on the divided observation optical path; and a movable prism which is movable away from and toward the fixed prism group.

38. The total internal reflection fluorescence microscope according to claim 36, further comprising: a control section which calculates/processes an extension/contraction of the optical path length by the optical path length adjustment section.

39. The total internal reflection fluorescence microscope according to claim 18, further comprising: a plurality of shutters disposed in the plurality of laser introduction sections; and a control section which controls opening and closing of the plurality of shutters to control introducing and blocking of the laser beams from the laser introduction sections.

40. The total internal reflection fluorescence microscope according to claim 18, wherein the plurality of laser introduction sections includes at least two laser introduction sections which output laser beams a same wavelength.

41. A total internal reflection fluorescence microscope comprising: at least one objective lens which takes light from a specimen; an image pick-up device which picks up an image of the light taken into the objective lens; an observation optical path via which the light taken into the objective lens is condensed onto the image pick-up device; a condenser lens, which is disposed in a position facing the objective lens via the specimen, which has a numerical aperture that makes possible total internal reflection illumination, and which guides a transmitted illuminative light, which is emitted by a light source, into the specimen; a base including an upper portion that holds the condenser lens; a laser oscillation unit which outputs a laser beam; a laser introduction section which comprises a reflection mirror provided at a lower portion of the base to reflect the laser beam output from the laser oscillation unit along a path substantially parallel to a light path of the transmitted illuminative light from the light source, so as to introduce the laser beam into a vicinity of an outermost side of the condenser lens; and a mirror moving section which moves the reflection mirror in a translatory manner in a direction that is substantially perpendicular to the light path of the transmitted illuminative light from the light source, such that when the mirror moving section moves the reflection mirror, the path of the laser beam reflected by the reflection mirror remains substantially parallel to the light path of the transmitted illuminative light; wherein when the mirror moving section moves the reflection mirror, an incidence angle, at a boundary of the specimen, of the laser beam emitted from the condenser lens is changed, thereby changing a leak-out depth of evanescent light that illuminates the specimen.

42. A total internal reflection fluorescence microscope comprising: at least one objective lens which takes light from a specimen; an image pick-up device which picks up an image of the light taken into the objective lens; an observation optical path via which the light taken into the objective lens is condensed onto the image pick-up device; a condenser lens, which is disposed in a position facing the objective lens via the specimen, which has a numerical aperture that makes possible total internal reflection illumination, and which guides a transmitted illuminative light, which is emitted by a light source, into the specimen; a laser oscillation unit which outputs a laser beam; a laser introduction section which comprises a reflection mirror provided integrally at a lower portion of the condenser lens to reflect the laser beam output from the laser oscillation unit along a path substantially parallel to a light path of the transmitted illuminative light from the light source, so as to introduce the laser beam into a vicinity of an outermost side of the condenser lens; and a mirror moving section which moves the reflection mirror in a translatory manner in a direction that is substantially perpendicular to the light path of the transmitted illuminative light from the light source, such that when the mirror moving section moves the reflection mirror, the path of the laser beam reflected by the reflection mirror remains substantially parallel to the light path of the transmitted illuminative light; wherein when the mirror moving section moves the reflection mirror, an incidence angle, at a boundary of the specimen, of the laser beam emitted from the condenser lens is changed, thereby changing a leak-out depth of evanescent light that illuminates the specimen.

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Description

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2003-101346, filed Apr. 4, 2003, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a total internal reflection fluorescence microscope for performing fluorescence observation by use of an evanescent light generated by total internal reflection illumination.

2. Description of the Related Art

In recent years, a total internal reflection fluorescence microscopy (hereinafter referred to as TIRFM) has attracted attentions as a microscope for fluorescence observation of a living thing. In this TIRFM, an illuminative light is totally reflected by a boundary surface between a cover glass and a specimen, and a fluorescent substance is excited using a light called an evanescent light which leaks into a small region having a size of several hundreds nm or less on a specimen side. In this TIRFM, only the fluorescence of the small region in the vicinity of the cover glass is observed. An observed image of TIRFM provides a very dark background. Accordingly, it is possible to observe fluorescence having a high contrast and faint fluorescence.

Additionally, in a site of biological research using TIRFM, there are a case where a shallow plane is to be observed with good contrast in the vicinity of the boundary surface between the cover glass and the specimen, and a case where the evanescent light is extended to a certain degree of depth to observe a broad range. Therefore, it is desirable to change a leak-out depth of the evanescent light in accordance with the specimen.

The leak-out depth of the evanescent light from the boundary surface is described, for example, in D. Axelrod's document "Total Internal Reflection Fluorescence at Biological Surfaces". Accordingly, the following equation is established. d=.lamda./4.pi. {square root over ((n.sub.1.sup.2sin .theta..sub.1.sup.2-n.sub.2.sup.2))} (1), where d denotes the leak-out depth of the evanescent light, .lamda. denotes a wavelength of the light, n.sub.1 denotes a refractive index on the incidence side, .theta..sub.1 denotes an incidence angle, and n.sub.2 denotes a refractive index on an emission side.

Therefore, when the incidence angle .theta..sub.1 of the illuminative light with respect to the boundary surface, that is, an inclination angle of the illuminative light with respect to a normal to the boundary surface increases, the leak-out depth d of the evanescent light becomes shallow. In actual TIRFM, a laser beam having a high coherent property is used, and the incidence angle of the illuminative light is adjusted. Accordingly, the incidence angle of the laser beam onto the boundary surface changes, and the leak-out depth of the evanescent light is adjusted.

FIGS. 17A to 17C are diagrams showing a function of TIRFM described in Jpn. Pat. No. 3093145. An objective lens 1 has a numerical aperture with which total internal reflection illumination is possible. A specimen 3 is laid on a cover glass 2. A mirror 4 is movable in a direction crossing an optical axis direction of the objective lens 1 at right angles.

A laser beam 5 for use as the illuminative light is incident upon the mirror 4. In this state, as shown in FIGS. 17A to 17C, the mirror 4 moves in the direction crossing the optical axis direction of the objective lens 1 at right angles. Accordingly, an incidence position of the laser beam incident upon the objective lens 1 moves in a direction distant from an optical axis of the objective lens 1. By the movement of the incidence position of the laser beam, the incidence angle of the laser beam 5 emitted toward the boundary surface between the cover glass 2 and the specimen 3 from the objective lens 1 changes. The laser beam 5 emitted from the objective lens 1 is totally reflected by the boundary surface between the cover glass 2 and specimen 3 via an immersion oil 6 as shown in FIG. 17C.

FIG. 18 is a constitution diagram of TIRFM described in Jpn. Pat. Appln. KOKAI Publication No. 2001-013413. The TIRFM includes a laser illuminating device 7 which outputs the laser beam 5. In the TIRFM, the laser beam 5 is incident upon a side surface 10 of a point ball lens 9 of a condenser lens for transmission illuminating 8 and the total internal reflection illumination is possible. The laser illuminating device 7 is rotatable with respect to a microscope main body 11. The laser illuminating device 7 rotates centering on an intersection of the boundary surface of the cover glass 2 and specimen 3 and the observation optical axis. Accordingly, the laser beam 5 changes its incidence angle with respect to the boundary surface between the cover glass 2 and specimen 3.

Additionally, the incidence angle of the laser beam 5 needs to be inclined by a critical angle or more, at which the total internal reflection occurs. Here, assuming that a refractive index on a cover glass 2 side via the boundary surface between the cover glass 2 and specimen 3 is n.sub.1, and a refractive index on a specimen 3 side is n.sub.2, a critical angle .theta.c is represented by the following equation (2). sin .theta.c=n.sub.2/n.sub.1 (2)

Therefore, conditions of the incidence angle .theta..sub.1 for realizing the total internal reflection illumination is represented by the following equation (3). n.sub.1sin .theta..sub.1>n.sub.2 (3)

On the other hand, to incline an incident light of the laser beam 5 passing through the objective lens 1 as in the Jpn. Pat. No. 3093145 shown in FIGS. 17A to 17C, a maximum incidence angle .theta.max that can be set depends on the numerical aperture (NA) of the objective lens 1, and is represented by the following equation (4). n.sub.1sin .theta.max=NA (4)

Therefore, for the conditions for realizing the total internal reflection illumination, the NA of the objective lens 1 needs to be larger than the refractive index n.sub.2.

In general, a refractive index of a living cell is about 1.37 to 1.38. The NA of the objective lens 1 for use needs to be about 1.4 at minimum.

At present, a magnification of the objective lens having an NA of 1.4 or more is limited to a high magnification of 60 times or more. To realize a high NA by the objective lens 1 having a low magnification, an effective diameter of the objective lens 1 needs to be increased. However, it is difficult to increase the effective diameter of the objective lens 1 while keeping a standard diameter of an attaching screw of the objective lens 1. Therefore, in the TIRFM shown in FIGS. 17A to 17C, total internal reflection fluorescence observation at a magnification of about 20 or 40 times is impossible.

In the TIRFM shown in FIG. 18, when the laser beam 5 is incident from a condenser lens for transmission illuminating 8 side, an illuminative range can be set without depending on the objective lens 1. Accordingly, the total internal reflection fluorescence observation using the objective lens 1 having a low magnification is possible.

However, in the TIRFM shown in FIG. 18, the laser illuminating device 7 is disposed right beside the point ball lens 9 of the condenser lens for transmission illuminating 8. Additionally, the laser illuminating device 7 itself needs to be rotated. Therefore, a considerable space is necessary including a holding section of a rotary mechanism and a space of a track of the rotating laser illuminating device 7. Consequently, a space in which the specimen 3 is laid is compressed. It is supposed that operation properties are remarkably impaired.

An irradiation range of the laser beam is set in such a manner that an observation range of the objective lens 1 having the low magnification can be illuminated. Then, in the observation with the objective lens 1 having the high magnification, only a part of the irradiation range of the laser beam is observed. Therefore, the laser beam with which another part is irradiated is useless.

An energy density of the laser beam on the surface of the specimen 3 is in inverse proportion to an irradiation area. In the observation with the objective lens 1 having the high magnification, the irradiation range of the laser beam is condensed so as to illuminate only a range required for the observation, and the energy density of the laser beam is preferably enhanced.

Especially, there is an experiment for the purpose of detection of very weak fluorescence such as a single molecule. In this experiment, the irradiation energy density of the laser beam is required to be as high as possible. On the other hand, in the TIRFM, the leak-out depth of the evanescent light is changeable. In recent years, the TIRFM has been spread in the site of the biological research. Furthermore, there has started to be a demand for the simultaneous illuminating of a plurality of optional wavelengths in optional depths.

This background has the following actual circumstances. Improvement of fluorescent protein such as GFP has been advanced, and it becomes easy to observe a dynamic state or a function of the living cell with multicolored fluorescence. Moreover, as seen also from Axelrod formula (equation (1)), the leak-out depth of the evanescent light depends also on the wavelength of the light. Therefore, there is a principle problem that a range to be observed differs, when the wavelength differs even at the equal laser beam incidence angle. There is also a realistic problem that a depth position of a tissue in a cell corresponding to each wavelength differs.

In the TIRFM, it is possible to switch the incidence angle or the wavelength of the laser beam at a high speed using mechanical means or electric driving means such as a motor. However, in cases where a simultaneous property in a strict meaning is required such as a case where a fast phenomenon is traced, there is a restriction on a high-speed switch. In this case, it is necessary to simultaneously-illuminate introductory portions of the laser beams disposed in a plurality of places.

However, in the TIRFM shown in FIG. 17A, a dichroic mirror is used in order to reflect the laser beam on a objective lens 1 side and to transmit the fluorescence on an observation side.

Additionally, when there are a plurality of wavelengths to be illuminated, the dichroic mirror needs to have wavelength characteristics of the corresponding multi-band. The dichroic mirror of the multi-band has a high difficulty in manufacturing, and is expensive. Furthermore, the dichroic mirror of the multi band has a bad separation level of the wavelength, and brightness and SN ratio of a fluorescent image are deteriorated. When the illuminative wavelengths are to be further increased halfway, the dichroic mirror needs to be newly prepared again.

On the other hand, to prevent the dichroic mirror from being used, as shown in FIG. 19, it is possible to dispose a total internal reflection mirror 12 in a position of an outermost portion of a pupil of the objective lens 1. However, it is necessary to dispose another total internal reflection mirror 13 also in the outermost portion of the pupil of the objective lens 1 on an opposite side in order to prevent the laser beam 5 totally reflected by the boundary surface between the cover glass and the specimen 3 from passing on an observation side.

Therefore, a considerable part of the pupil of the objective lens 1, which should have been originally used 100% for observation, is lost by the respective total internal reflection mirrors 12, 13. Therefore, a capability of the objective lens 1 is deteriorated.

In the TIRFM shown in FIG. 18, as described above, the laser illuminating device 7 is disposed right beside the point ball lens 9 of the condenser lens for transmission illuminating 8, and additionally the laser illuminating device 7 itself needs to be rotated. Therefore, a considerable space is required including the space of the track of the holding section of the rotation mechanism or the rotating laser illuminating device 7. In this constitution, when the laser illuminating device 7 including an emission angle adjustment section of independent laser beams is to be disposed, two laser illuminating devices at maximum can be disposed on opposite sides of the condenser lens for transmission illuminating 8.

Moreover, each TIRFM has a common problem. There is a case where a plurality of wavelengths are observed by the use of the evanescent lights having different depths. For example, when a shallow region is observed with B excitation, and a deep region is observed with G excitation, the B and G excitations can be simultaneously observed only in the shallow region. In this case, the image of the G excitation can only be observed as a defocused background image. Therefore, for example, the whole cell film is dyed by a fluorescent reagent, and the image of the TIRFM in the deep region can be used only in limited applications such as grasping of an approximate size of the cell.

While the objective lens is fixed, the surfaces in the different depths can be simultaneously observed. Then, the application of multi-wavelength TIRFM can further be broadened to simultaneous observation of forms of small organs in the vicinity of the cell film and inside the cell.

BRIEF SUMMARY OF THE INVENTION

According to a major aspect of the present invention, there is provided a total internal reflection fluorescence microscope comprising: at least one objective lens which takes light from a specimen; an image pick-up device which picks up an image of the light taken into the objective lens; an observation optical path via which the light taken into the objective lens is condensed onto the image pick-up device; a condenser lens which is disposed in a position facing the objective lens via the specimen and which has a numerical aperture making possible total internal reflection illumination and which guides a transmitted illuminative light into the specimen; and a laser introduction section which allows a laser beam to be incident upon a direction crossing the optical path of the transmitted illuminative light at right angles and which introduces the incident laser beam on a condenser lens side in the vicinity of an outermost part of the transmitted illuminative light path.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a constitution diagram showing a first embodiment of TIRFM according to the present invention;

FIG. 2 is a constitution diagram showing a second embodiment of the TIRFM of the present invention;

FIG. 3 is an enlarged top plan view of a conversion lens unit in the TIRFM;

FIG. 4 is an enlarged top plan view of the conversion lens unit in the TIRFM;

FIG. 5 is a constitution diagram showing a third embodiment of the TIRFM of the present invention;

FIG. 6 is a diagram showing each objective lens switch mechanism in the TIRFM;

FIG. 7A is a constitution diagram showing the conversion lens unit in a fourth embodiment of the TIRFM of the present invention;

FIG. 7B is a diagram showing an operation of the conversion lens unit;

FIG. 8 is a constitution diagram showing a modification of the fourth embodiment of the present invention;

FIG. 9 is a constitution diagram showing a fifth embodiment of the TIRFM of the present invention;

FIG. 10 is a diagram showing an arrangement of each laser introduction section in the TIRFM;

FIG. 11 is a diagram showing a relation between wavelengths separated by each dichroic mirror and emission filter in the TIRFM;

FIG. 12 is a constitution diagram showing a modification of the TIRFM;

FIG. 13 is a constitution diagram of a laser combiner in the TIRFM;

FIG. 14 is a constitution diagram showing a fifth embodiment of the TIRFM of the present invention;

FIG. 15 is an enlarged view of the inside of the specimen and an observation optical path in the TIRFM;

FIG. 16 is a diagram showing a state in which an image forming position of fluorescence emitted from a fluorescent substance agrees with an image pick-up surface of an image pick-up device in the TIRFM;

FIG. 17A is a diagram showing a function of a conventional TIRFM;

FIG. 17B is a diagram showing the function of the conventional TIRFM;

FIG. 17C is a diagram showing the function of the conventional TIRFM;

FIG. 18 is a constitution diagram of the conventional TIRFM; and

FIG. 19 is a constitution diagram of the conventional TIRFM.

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the present invention will be described hereinafter with reference to the drawings.

FIG. 1 is a constitution diagram of an erected type total internal reflection fluorescence microscopy (TIRFM). An image forming lens 21, emission filter 22, and image pick-up device 23 are disposed on an observation optical path Q of an objective lens 20. The emission filter 22 is a band pass filter which passes a light only of a specific wavelength band .lamda..sub.E1 longer than a wavelength .lamda..sub.L1 of a laser beam output from a laser oscillation unit 32 described later. The image pick-up device 23 is disposed in a focal position of the image forming lens 21.

A slide glass for observation 24 is disposed under the objective lens 20. A specimen 25 is placed on the slide glass 24. The specimen 25 is covered with a cover glass 26.

A condenser lens 27 is disposed in a position facing the objective lens 20 via the slide glass 24. The condenser lens 27 is disposed on a base 28. An immersion oil 29 is dotted/attached to a tip of the condenser lens 27. Accordingly, the immersion oil 29 is charged between the condenser lens 27 and slide glass 24.

A numerical aperture (NA) of the condenser lens 27 is designed to be larger than a refractive index of the specimen 25. That is, assuming that a refractive index of the immersion oil 29 or the slide glass 24 is n.sub.1, and a refractive index of the specimen 25 is n.sub.2, the following relation is established from above equations (3) and (4): NA=n.sub.1sin .theta.max>n.sub.2 (5), where .theta.max corresponds to a maximum incidence angle at which the incidence is possible through the immersion oil 29 and slide glass 24 from the condenser lens 27.

In general, the refractive index of the living cell is about 1.37 to 1.38. Accordingly, the numerical aperture of the condenser lens 27 has a value larger than the refractive index of the living cell of 1.37 to 1.38, and concretely has a value of about 1.65 to 1.45.

A collector lens 30 and transmitted illuminative light source 31 are disposed on a transmitted illuminative light path T of the condenser lens 27. The collector lens 30 introduces the illuminative light output from the transmitted illuminative light source 31 into the condenser lens 27.

A laser introduction section L.sub.1 is disposed under a base 28. The laser introduction section L.sub.1 includes the laser oscillation unit 32. For example, a laser diode is used in the laser oscillation unit 32. An oscillation wavelength of the laser oscillation unit 32 has a single wavelength .lamda..sub.L1.

A laser output end of the laser oscillation unit 32 is connected to an optical fiber 33. One end of the optical fiber 33 is preferably a single mode fiber. A fiber emission end 34 is disposed on the other end of the optical fiber 33. The fiber emission end 34 emits the laser beam transmitted through the optical fiber 33 as a divergent ray. The fiber emission end 34 is connected, for example, to one end of a support section 35 having a hollow structure.

The support section 35 is fixed/supported, for example, on the bottom surface of the base 28. A condensing lens 36 converts a divergent ray emitted from the fiber emission end 34 into a convergent ray, and condenses the light in the vicinity of a front focal position of the condenser lens 27.

A downward support portion 37 is disposed integrally with a lower part of the support section 35. A hole for movement 38 and a hole for support 39 are disposed in the downward support portion 37. The hole for movement 38 and hole for support 39 are disposed in a direction substantially crossing the transmitted illuminative light path T at right angles.

A mirror holding section 40 is movably disposed in the hole for movement 38. For the mirror holding section 40, a mirror support section 41 and a movement guide section 42 are integrally formed. The movement guide section 42 is inserted in the hole for movement 38, and is movable in the hole for movement 38. The mirror support section 41 is disposed substantially in a direction crossing the movement guide section 42 at right angles.

A micrometer 43 is disposed in the hole for support 39. The micrometer 43 includes a micrometer head 44 and a micrometer operation section 45. The micrometer head 44 abuts on the lower part of the mirror support section 41. The micrometer operation section 45 directly moves the micrometer head 44 by rotation. A translatory direction of the micrometer head 44 agrees with the direction crossing the transmitted illuminative light path T substantially at right angles.

A spring 46 is disposed between the mirror support section 41 and the downward support portion 37. The spring 46 has a tensile force to urge the mirror support section 41 on a micrometer head 44 side. Accordingly, the micrometer head 44 abuts on and is kept by the mirror support section 41.

A reflective mirror 47 is disposed on an upper end of the mirror support section 41. Therefore, when the micrometer operation section 45 is rotated, the micrometer head 44 moves in a translatory manner in a direction crossing the transmitted illuminative light path T substantially at right angles. In response to this movement, the mirror support section 41 moves in the translatory manner in the direction crossing the transmitted illuminative light path T substantially at right angles. As a result, the reflective mirror 47 moves in the translatory manner in the direction crossing the transmitted illuminative light path T substantially at right angles. It is to be noted that the mirror holding section 40, micrometer 43, and spring 46 constitute a mirror movement section.

The reflective mirror 47 is disposed in the vicinity of an outermost side of the opening diameter of the condenser lens 27. The reflective mirror 47 reflects upwards the laser beam introduced from the direction crossing the transmitted illuminative light path T substantially at right angles upwards substantially at right angles.

Next, an operation of the TIRFM constituted as described above will be described.

The laser oscillation unit 32 oscillates the laser beam having a wavelength .lamda..sub.L1 The laser beam having the wavelength .lamda..sub.L1 is introduced into the optical fiber 33, and emitted as a divergent ray from the fiber emission end 34. The laser beam emitted as the divergent ray is converted to the convergent ray through the condensing lens 36 and is incident upon the reflective mirror 47.

The laser beam incident upon the reflective mirror 47 is reflected on the condenser lens 27 side in the vicinity of the outermost side of the transmitted illuminative light path T. The laser beam reflected by the reflective mirror 47 is once condensed in the vicinity of the front focal position of the condenser lens 27 by the condensing lens 36. Moreover, the laser beam is incident upon the condenser lens 27, and is emitted as a parallel ray advancing in an oblique direction from the condenser lens 27. The laser beam emitted from the condenser lens 27 is transmitted through the immersion oil 6 and is incident upon the boundary surface between the slide glass 24 and the specimen 25.

When the incidence angle .theta..sub.L1 of the laser beam upon the boundary surface between the slide glass 24 and the specimen 25 is larger than the critical angle of the total internal reflection, the laser beam is totally reflected by the boundary surface. Accordingly, the evanescent light leaks on a specimen 25 side.

The specific fluorescent substance existing in the specimen 25 is excited by the evanescent light having the wavelength .lamda..sub.L1 By this excitation, the fluorescent substance emits the fluorescence such that a maximum luminance wavelength of the fluorescence is in a transmission wavelength band .lamda..sub.E1 of the emission filter 22. The fluorescence is incident upon the objective lens 20 through the cover glass 26. Furthermore, the fluorescence is transmitted through the emission filter 22, and is incident upon the image pick-up device 23. The image pick-up device 23 picks up a fluorescent image of the wavelength band .lamda..sub.E1.

On the other hand, when the micrometer operation section 45 is rotated, the reflective mirror 47 moves in the translatory manner in the direction crossing the transmitted illuminative light path T at right angles. When the position of the reflective mirror 47 moves in the direction crossing the transmitted illuminative light path T substantially at right angles, the incidence position of the laser beam upon the condenser lens 27 moves. Accordingly, an emission angle of the laser beam emitted from the condenser lens 27, that is, an incidence angle .theta..sub.L1 of the laser beam upon the boundary surface between the slide glass 24 and specimen 25 changes.

The leak-out depth of the evanescent light in the total internal reflection illumination changes with the incidence angle .theta..sub.L1 of the laser beam upon the boundary surface between the slide glass 24 and specimen 25. Therefore, the micrometer operation section 45 is rotated to slightly move the reflective mirror 47 in the direction crossing the transmitted illuminative light path T-substantially at right angles. That is when the reflective mirror 47 is brought close to or far from the transmitted illuminative light path T, the leak-out depth d.sub.L1 of the evanescent light can be optionally changed.

It is to be noted that when the transmission illuminating observation is performed using the illuminative light output from the transmitted illuminative light source 31, the reflective mirror 47 is completely retreated from the transmitted illuminative light path T.

As described above, according to the first embodiment, the condenser lens 27 is disposed in the position facing the objective lens 20 via the specimen 25, the reflective mirror 47 is movably disposed in the vicinity of the outermost part of the transmitted illuminative light path T on the transmitted illuminative light source 31 side from the condenser lens 27, and the laser beam output from the laser oscillation unit 32 is reflected by the reflective mirror 47 and is incident upon the condenser lens 27. That is, the laser beam which excites the fluorescent substance dyed by the specimen 25 is not transmitted through the objective lens 20, and is transmitted through the condenser lens 27 positioned so as to face the objective lens 20.

Accordingly, a maximum incidence angle .theta.max of the laser beam incident upon the boundary-surface between the slide glass 24 and the specimen 25 depends only on the NA of the condenser lens 27 represented by the above equation (5). Therefore, fluorescence observation by the total internal reflection illumination is possible regardless of the NA or the magnification of the objective lens 20.

The laser introduction section L.sub.1 is disposed apart from the specimen 25 and the condenser lens 27. Accordingly, there is not any structure disposed in the vicinity of the specimen 25, and a space in the vicinity of the specimen 25 is not compressed.

The laser introduction section L.sub.1 includes a structure in which the reflective mirror 47 is moved by the micrometer 43. The laser introduction section L.sub.1 has a simple structure and also has a narrow operation range. Therefore, for the laser introduction section L.sub.1, the TIRFM itself can be compact, and the TIRFM can have a superior operation property.

The first embodiment may also be modified as follows.

A glass bottom dish may also be used in the slide glass 24. Accordingly, the cover glass 26 may also be omitted. In this case, when an operating distance of the objective lens 20 is short, an immersion objective lens is used in the objective lens 20.

For example, an electromotive motor, piezo-actuator or the like may also be used in the micrometer 43 for moving the reflective mirror 47.

The reflective mirror 47 is fixed. The fiber emission end 34 and condensing lens 36 are integrally moved in a direction parallel to the transmitted illuminative light path T. Even with this constitution, a function similar to that of the first embodiment is obtained. In this case, the fiber emission end 34 and condensing lens 36 are moved, for example, using a micro-motor, electromotive motor, piezo-actuator, or the like.

The first embodiment is applicable not only to the erected type but also to an inverted microscope.

An optical observation system is not limited to the constitution by the image forming lens 21, emission filter 22, and image pick-up device 23, and can be optionally constituted as long as the fluorescence wavelength with respect to the excited wavelength of the laser introduction section L.sub.1 is selected and the image can be picked up.

In the first embodiment, the laser beam emitted from the fiber emission end 34 is converted to the convergent ray by the single condensing lens 36, and the convergent ray is reflected by the reflective mirror 47 and condensed in the vicinity of the front focal position of the condenser lens 27. The present invention is not limited to this. For example, the laser beam emitted from the fiber emission end 34 is converted to a parallel light flux by a single lens or a plurality of lenses, the condensing lens is disposed in an optional position on the optical path from when the parallel light flux is reflected by the reflective mirror 47 and is incident upon the vicinity of the front focal position of the condenser lens 27, and the light may be condensed by the condensing lens.

A second embodiment of the present invention will be described with reference to the drawings. It is to be noted that the same part as that of FIG. 1 is denoted with the same reference numerals and detailed description thereof is omitted.

FIG. 2 is a constitution diagram showing the erected type total internal reflection fluorescence microscopy (TIRFM). A conversion lens unit 50 is disposed between the fiber emission end 34 and the condensing lens 36. The conversion lens unit 50 is integrally detachably inserted with respect to a laser introductory optical path between the fiber emission end 34 and the condensing lens 36.

FIGS. 3 and 4 are enlarged top plan views of the conversion lens unit 50. A shelter space portion 51 is disposed in the side surface of the support section 35 including a hollow structure. The conversion lens unit 50 is provided with a knob 52. The knob 52 protrudes out of the shelter space portion 51 through the wall of the shelter space portion 51. The conversion lens unit 50 is detachably inserted between the laser introductory optical path in the support section 35 and the inside of the shelter space portion 51 by the operation of the knob 52. For example, when the knob 52 is pulled out as shown in FIG. 3, the conversion lens unit 50 comes out of the laser introductory optical path. When the knob 52 is pushed in as shown in FIG. 4, the conversion lens unit 50 is inserted into the laser introductory optical path.

The conversion lens unit 50 has, for example, a hollow structure. A convex lens 53 and concave lens 54 are disposed in the conversion lens unit 50. The convex lens 53 converts the NA of a divergent laser beam emitted from the fiber emission end 34.

The concave lens 54 is disposed movably in an optical axis direction of the laser introductory optical path between the convex lens 53 and condensing lens 36. The concave lens 54 diverges the laser beam NA-converted by the convex lens 53. Accordingly, when the concave lens 54 is moved in the optical axis direction of the laser introductory optical path, the laser beam can be condensed in the vicinity of the front focal position of the condenser lens 27 through the condensing lens 36. Therefore, the focal distance of the condensing lens 36 is adjustable by the concave lens 54.

Next, the operation of the TIRFM constituted as described above will be described.

When the knob 52 is pushed inwards as shown in FIG. 4, the conversion lens unit 50 is inserted into the laser introductory optical path. The NA of the laser beam diverged from the fiber emission end 34 is converted by the convex lens 53 in this state.

Thereafter, the laser beam is condensed in the vicinity of the front focal position of the condenser lens 27 through the concave lens 54 and condensing lens 36 At this time, the incidence NA of the laser beam upon the condenser lens 27 is reduced as compared with a case where the convex lens 53 and concave lens 54 deviate from the laser introductory optical path. Accordingly, the laser beam is condensed in the vicinity of the front focal position of the condenser lens 27 at a small incidence NA.

As a result, a ray flux diameter of the parallel ray advancing in an oblique direction after passing through the condenser lens 27, that is, a laser beam irradiation range in the specimen 25 is condensed. Accordingly, energy density of the laser beam increases.

As described above, according to the second embodiment, the conversion lens unit 50 including the convex lens 53 and concave lens 54 is detachably attached onto the laser introductory optical path. Accordingly, the irradiation range of the laser beam on the specimen 25 can be condensed. That is, the energy density of the laser beam with which the specimen 25 is irradiated can be converted, for example, to be high.

Therefore, when the weak fluorescence is to be observed with a strong power, the conversion lens unit 50 is inserted into the laser introductory optical path. In the fluorescence observation of the broad range, the conversion lens unit 50 is detached from the laser introductory optical path. The specimen 25 is selectively used in accordance with the application of the observation in this manner.

The second embodiment may also be modified as follows.

Inserting/detaching means of the conversion lens unit 50 is not limited to the knob and may also be, for example, the electromotive motor and can be freely constituted.

The conversion lens unit 50 is not limited to a combination of the convex lens 53 and concave lens 54, and may also be a combination of the convex and concave lenses, from which the same function is obtained.

For the conversion lens unit 50, one type of unit is constituted to be detachably inserted between the fiber emission end 34 and condensing lens 36, but the present invention is not limited to this. For example, the conversion lens unit 50 is converted to a different incidence NA, and is selectively detachably inserted between the fiber emission end 34 and condensing lens 36. With the constitution, the specimen 25 can be irradiated with the laser beams having three or more different incidence NA. Accordingly, a finer irradiation range of the laser beam, and further the energy density are adjustable.

A third embodiment of the present invention will be described with reference to the drawing. It is to be noted that the same part as that of FIG. 2 is denoted with the same reference numerals, and detailed description is omitted.

FIG. 5 is a constitution diagram of the erected type total internal reflection fluorescence microscopy (TIRFM). An objective lens for high-magnification observation 61 and an objective lens for low-magnification observation 62 are attached to an objective lens switching section 60.

FIG. 6 is a diagram showing a switch mechanism for the respective objective lenses 61, 62. A member for switching 63, such as a rack, is disposed on the side surface of the objective lens switching section 60. The member for switching 63 is connected to a magnification switch driving section 64 vi