JP2005140925A - Microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope applying total reflection lighting and polarized oblique lighting to a sample simultaneously. <P>SOLUTION: The microscope comprises an objective lens 11, and an illumination optical system that performs illumination via the objective lens 11. Therein, the illumination optical system applies an illumination luminous flux over both regions of the total reflection lighting region and the polarized oblique lighting region of a pupil 12 of the objective lens 11. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a biological microscope, and more particularly, to a biological microscope capable of simultaneously realizing a total reflection illumination state and a polar oblique illumination state.

Conventionally, in a biological microscope, in order to perform fluorescence observation of cells in the immediate vicinity of a cover glass, a total reflection illumination state is realized through an objective lens, and fluorescence generated by a specimen is observed by an evanescent wave generated at this time. Has been done. At this time, the illumination light flux is switched from the epi-illumination state to the total reflection illumination state (see, for example, Patent Document 1).
JP 2003-98439 A

  The characteristic of total reflection illumination through the objective lens is that an evanescent wave is generated at a depth of 0.1 μm or less of the cell in contact with the cover glass, and the fluorescence excited by this evanescent wave can be observed with good contrast. . However, since the evanescent wave attenuates abruptly at a depth of 0.1 microns or more, it is difficult to observe fluorescence at a portion deeper than the depth reached by the evanescent wave.

  In recent years, in order to analyze various phenomena in cells and the like, it has been desired to observe a change in state from the cell membrane in the vicinity of the cover glass to the inside of the cell.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a microscope capable of simultaneously realizing a total reflection illumination state and a polar oblique illumination state on a specimen.

  To achieve the above object, the present invention includes an objective lens and an illumination optical system that performs illumination via the objective lens, and the illumination optical system includes a total reflection illumination area of a pupil of the objective lens, Provided is a microscope characterized by irradiating an illumination light beam over both regions of a polar oblique illumination region.

  In the microscope of the present invention, it is preferable that the illumination light beam is irradiated in the range of the numerical aperture of the pupil of the objective lens from 1.00 to 1.70.

  In the microscope according to the aspect of the invention, it is preferable that the illumination optical system can change an irradiation area of the illumination light beam.

  In the microscope of the present invention, it is preferable that the illumination light beam is movable in a plane substantially perpendicular to the optical axis.

  In the microscope of the present invention, it is preferable that the illumination light beam is limited by an aperture stop arranged at a position conjugate with the pupil position of the objective lens of the illumination optical system.

  In the microscope of the present invention, it is preferable that the aperture stop is an arc-shaped aperture stop, a circular aperture stop, or a ring-shaped aperture stop.

  ADVANTAGE OF THE INVENTION According to this invention, the microscope which can irradiate a sample simultaneously with a total reflection illumination light beam and a polar oblique illumination light beam can be provided.

  Hereinafter, a microscope according to an embodiment of the present invention will be described with reference to the drawings.

  FIG. 1 is a schematic configuration diagram of a microscope according to the first embodiment of the present invention. FIG. 2 schematically shows the illumination light beam (solid line) in the total reflection illumination state and the illumination light beam (dashed line) in the polar oblique illumination state with respect to the specimen. FIG. 3 shows an illumination state of the arc-shaped aperture stop at the pupil of the objective lens of the microscope according to the first embodiment. FIG. 4A shows the arc portion of the lamp light source in the first embodiment, and FIG. 4B shows a state where the arc portion is positioned at the arcuate opening. FIG. 5 shows the illumination state of the circular aperture stop at the pupil of the objective lens of the microscope of the first embodiment. FIG. 6 shows an illumination state of the ring-shaped aperture stop at the pupil of the objective lens of the microscope according to the first embodiment. FIGS. 7A and 7B are diagrams showing a slider on which an aperture stop used in the first embodiment is arranged. FIG. 7A shows an arc-shaped aperture stop, FIG. 7B shows a circular aperture stop, and FIG. 7C shows a ring-like aperture stop. Respectively. FIG. 8 shows a state where the arc-shaped aperture stop in the pupil of the objective lens of the microscope of the first embodiment is moved in the plane to illuminate only the total reflection illumination area. FIG. 9 shows a schematic configuration diagram of a microscope according to the second embodiment of the present invention.

(First embodiment)
Hereinafter, a first embodiment of a microscope according to the present invention will be described with reference to FIGS.

  1 to 8, the luminous flux of the lamp light source 1 is collected by the collector lens 2 and collected by the first condenser lens 3 at the opening of the aperture stop 4 disposed at a position conjugate with the pupil 12 of the objective lens 11. Lighted. The condensed light flux enters the collimating lens 5 to become parallel light, the field of view is limited by the field stop 6, the direction of travel is changed by the bending mirror 7, and the second condensing lens 8 passes through the excitation filter. 9, reflected by the dichroic mirror 10, condensed by the second condenser lens 8 on both the total reflection illumination area and the polar oblique illumination area of the pupil 12 of the objective lens 11, and is emitted from the objective lens 11 as illumination light. The specimen 14 is irradiated. In this way, the illumination optical system is configured.

  A petri dish 13 is placed on a stage (not shown), and a specimen 14 to be observed and a culture solution 15 for preventing drying of the specimen 14 are contained therein. The specimen 14 is obtained by, for example, staining cultured cell or cell tissue sections with a fluorescent dye. Further, a cover glass 16 is attached to the petri dish 13 on the objective lens 11 side, and a specimen 14 is attached thereon. The objective lens 11 uses a high-resolution, high-magnification objective lens, and a dedicated oil 17 is filled between the tip of the objective lens 11 and the cover glass 16.

  As shown in FIG. 2, an evanescent wave is generated on the surface of the cover glass 16 on the specimen 14 side by the total reflection illumination light beam (solid line), and the cells near the cover glass 16 of the specimen 14 are irradiated. The specimen 14 located at a position where the evanescent wave does not reach away from the cover glass 16 is illuminated with a polar oblique illumination light beam (broken line).

  In FIG. 1, the fluorescence 18 generated from the specimen 14 by both illumination lights is collected by the objective lens 11, passes through the dichroic mirror 10, passes through an emission filter 19 that selects a necessary wavelength, and forms an imaging lens 20. The image is formed on the image forming surface 21, picked up by an image pickup device such as a CCD (not shown) disposed on the image forming surface 21, and observed on a monitor (not shown). In this way, the microscope is configured.

  Note that the intensity of the illumination light may be adjusted by appropriately inserting an ND filter (not shown) in the optical path of the illumination optical system. The position where the ND filter is inserted is preferably between the field stop 6 and the second condenser lens 8, for example.

  In FIG. 1, the optical axis of the objective lens 11 and the optical axis from the lamp light source 1 to the folding mirror 7 are substantially parallel, but in actual arrangement, the optical axis of the objective lens 11 is bent from the lamp light source 1 with respect to the vertical. The optical axis up to the mirror 7 is substantially horizontal.

  In the first embodiment, as shown in FIG. 3, an arcuate aperture stop 4 a is arranged in the aperture stop 4 in order to achieve illumination in both the total reflection illumination region 31 and the polar oblique illumination region 33. The position of the opening of the arcuate aperture stop 4a is the optical axis at a position where the illumination light beam is irradiated to the total reflection illumination region 31 and the oblique illumination region 33 of the pupil 12 of the objective lens 11 in a plane perpendicular to the optical axis of the illumination light beam. Arranged from the center O. Reference numeral 35 denotes NA = 1.38 (in the case where the specimen 14 is a cell) serving as a boundary between the total reflection illumination area and the polar oblique illumination area, and reference numeral 37 denotes the maximum NA of the objective lens 11 used here. NA = 1.45, and reference numeral 39 indicates NA = 1.00, which is the limit NA of polar oblique illumination.

  Further, the maximum numerical aperture that can be realized with generally available high refractive index glass and oil is 1.70. However, the characteristics of the glass and oil are different from those normally used. For example, glass can adversely affect cells cultured from its components. Preferably, the conditions for using a normally used glass or oil objective lens are good. The maximum numerical aperture at that time is 1.50. The maximum numerical aperture of this type of objective lens currently mass-produced is 1.45.

  In the first embodiment, the opening of the arcuate aperture stop 4a opens an area from NA = 1.00 to NA = 1.45 in an arcuate shape (about 120 degrees), and is entirely in the pupil 12 of the objective lens 11. The reflected illumination area 31 and the polar oblique illumination area 33 are simultaneously irradiated with the illumination light flux. By setting NA = 1.00 to 1.30, the polar oblique illumination region 33 is limited to balance the total reflection illumination state and the polar oblique illumination state, and the fluorescence due to both illuminations can be more easily observed. Become.

  Further, in the first embodiment, as shown in FIGS. 4A and 4B, the lamp light source 1 in FIG. 1 is arranged such that the arc portion 1a of the lamp light source 1 is positioned at the opening of the arcuate aperture stop 4a. Are shifted in a plane perpendicular to the optical axis, and the focal position is adjusted in the optical axis direction so that the arc portion 1a of the lamp light source 1 is focused on the opening of the arcuate aperture stop 4a. Thus, by arranging the arc part 1a, it becomes possible to perform illumination efficiently even when the lamp light source 1 is used.

  As a method of aligning the arc part 1a with the opening part of the arcuate aperture stop 4a, a method of moving the lamp light source 1 with respect to the optical axis, a method of moving the lens of the illumination optical system, and a movement of the mirror of the optical system Various methods such as a method for performing the above can be used.

  In addition to the arc-shaped aperture stop 4a shown in FIG. 3, the circular aperture stop 4b shown in FIG. 5, the ring-shaped aperture stop 4c shown in FIG. Can be used. The actions and effects of the circular aperture stop 4b and the ring-shaped aperture stop 4c are the same as those of the arc-shaped aperture stop 4a, and the description thereof is omitted.

  7A to 7C show an arc-shaped aperture stop 4a (FIG. 7A), a circular aperture stop 4b (FIG. 7B), and a ring-shaped aperture stop 4c (FIG. 7B) used in the first embodiment. The sliders 41a, 41b, and 41c on which FIG. 7 (c) is mounted are shown. By inserting these sliders 41a to 41c as the aperture stop 4 in FIG. 1, both the total reflection illumination state and the polar oblique illumination state can be realized.

  In FIG. 7A, the slider 41a is provided with an arcuate aperture stop 4a that can be adjusted in position. A click groove 43 is provided in a part of the outer periphery of the slider 41a, and when inserted into the aperture stop 4 of the microscope shown in FIG. 1, it is fixed and held in the optical path by a spring (not shown) provided in the microscope. The The depressions 43 and 43 formed in the slider 41a are provided so that the slider 41a can be easily picked up with a finger when the slider 41a is inserted into and removed from the optical path of the microscope.

  The arcuate aperture stop 4a is disposed on the stop support 47 and supported by a spring 49 provided at one end of the stop support 47 and screws 51 and 53 provided at the other end. By rotating the screws 51 and 53 with a tool (not shown) or a hex key, the arcuate aperture stop 4a can be moved in the plane of the drawing, and the position of the arcuate aperture stop 4a can be adjusted.

  Further, in the first embodiment, as shown in FIG. 8, the arcuate aperture stop 4a is moved along, for example, the X axis, so that both the total reflection illumination state and the oblique illumination state (see FIG. 3), the illumination state can be varied only in the total reflection illumination state. Accordingly, the position of the arcuate aperture stop 4a can be adjusted from the total illumination state shown in FIG. 3 to the state of only the total reflection illumination state shown in FIG. Further, the illumination light beam in the oblique illumination state can be irradiated onto the specimen 14 while maintaining the total reflection illumination state, and the incident position of the oblique illumination light beam on the specimen 14 (distance from the cover glass 16 to the oblique illumination light beam). Can be changed.

  The position of the arcuate aperture stop 4a can be finely adjusted by using a micrometer head instead of the screws 51 and 53 in addition to the mechanism shown in FIG. If the electric micrometer head is used, the moving position can be stored and the arcuate aperture stop 4a can be moved to a predetermined position by operating one button.

  The slider 41b mounted with the circular aperture stop 4b shown in FIG. 7B and the slider 41c mounted with the ring-shaped aperture stop 4c shown in FIG. 7C are mounted with the arc-shaped aperture stop 4a shown in FIG. 7A. Since the configuration is the same as that of the slider 41a and the same operation and effect as the arcuate aperture stop 4a can be achieved, the same members as those in FIG.

  Hereinafter, a method for adjusting the illumination optical system of the microscope according to the first embodiment will be briefly described.

  In FIG. 1, the position of the lamp light source 1 with respect to the aperture stop 4 moves the lamp light source 1 itself relative to the collector lens 2 in the direction substantially perpendicular to the optical axis, and the focal direction moves the collector lens 2 in the optical axis direction. And adjust. Confirmation of the adjustment is made by focusing on the portion where the objective lens 11 is in close contact with the cover glass 16 of the specimen 14, and then adjusting the position of the pupil 12 of the objective lens 11 with a belt run lens (not shown) or a diopter telescope. Do this while observing. Alternatively, a centering adjustment jig (not shown) having a matte surface at the position of the pupil 12 of the objective lens 11 may be placed in the optical path instead of the objective lens 11 while observing the focus state of the arc portion of the lamp light source 1. good.

  In order to align the aperture of the aperture stop 4 with the pupil 12 of the objective lens 11, the position adjustment mechanism of the slider 41a is used in a direction substantially perpendicular to the optical axis. To adjust the optical axis direction, first, the distance between the collimating lens 5 and the aperture stop 4 is adjusted by moving to one of the predetermined accuracy, and the second condenser lens 8 is moved in the optical axis direction during use. To do.

  The aperture stop 4 is provided so as to be detachable, and various shapes of the aperture stop 4 (for example, FIGS. 7A to 7C) can be inserted into the optical path.

  The aperture stop 4 not only can move in a direction perpendicular to the optical axis but also can be rotated, so that the aperture can be further aligned with the pupil 12 of the objective lens 11.

  Further, when the wavelength of the illumination light beam is changed by switching the fluorescence filter cassette 22 including the excitation filter 9, the dichroic mirror 10, and the emission filter 19, if each lens has chromatic aberration, the condensing position with respect to the pupil 12 is shifted. End up. In order to prevent this, it is necessary to correct the chromatic aberration of each lens. In particular, the collector lens 2, the collimating lens 5, and the second condenser lens 8 are greatly affected by chromatic aberration. Therefore, by correcting the chromatic aberration of each lens, the change in the condensing position with respect to the pupil 12 due to the wavelength is suppressed within an allowable value. Is possible.

(Second Embodiment)
FIG. 9 shows a schematic configuration diagram of a microscope according to the second embodiment of the present invention. The second embodiment shows a case where the light source is an end face of the fiber, the members from the lamp light source 1 to the collimating lens 5 of the first embodiment are different, and the other configurations are the same as those of the first embodiment. . The same components as those in the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  In FIG. 9, the light beam from the output end face 61 a of the fiber 61 is converted into a substantially parallel light beam by the collector lens 62, passes through the filter system FL including the excitation filter 9, and the field stop 6, the bending mirror 7, and the condenser lens 8. The light is condensed on the pupil 12 of the objective lens 11 via the dichroic mirror 10. The exit diameter of the exit end face 61 a of the fiber 61 is enlarged by the focal length of the collector lens 62 and the condenser lens 8. By setting this magnification to a desired value, an illumination light beam corresponding to the circular aperture stop 4b shown in FIG. 5 can be formed on the pupil 12 of the objective lens 11.

  For example, when a single mode fiber is used, the core diameter of the fiber is about 0.004 mm. On the other hand, the diameter of the illumination light beam in the pupil 12 of the objective lens 11 is determined by the focal length of the objective lens 11 (for example, f = 3.33) and the NA of the pupil 12 of the objective lens 11. In order to realize the total reflection illumination state, it is necessary to collect the illumination light beam between NA = 1.45 and NA = 1.38 (in the case of cells), and the diameter of the illumination light beam is 3.33 × It is necessary to collect light within (1.45-1.38) = 0.23 mm. Therefore, if the core diameter of 0.004 mm is expanded to 0.23 mm, it is possible to irradiate the illumination light beam over almost the entire area of the total reflection illumination area, and to perform total reflection illumination efficiently. Therefore, by setting the magnification of the collector lens 62 and the condenser lens 8 to about 60 times, the luminous flux from the core diameter of 0.004 mm can be expanded to about 0.23 mm at the pupil 12 of the objective lens 11, and the efficiency is increased. Total reflection illumination is possible.

  In the second embodiment, the pupil 12 of the objective lens 11 can be in both the total reflection illumination state and the polar oblique illumination state, so that the center of the 0.23 mm illumination light beam is shown in FIG. It is possible to move so as to match the boundary position 35 between 31 and the oblique illumination region 33.

  In the second embodiment, in the case of only the total reflection illumination state, since the illumination light beam has sufficient luminance using a laser beam as the light source, it is not necessary to enter the light beam over the entire total reflection illumination region. Illumination with a luminous flux of about 0.04 mm, which is 10 times the illumination luminous flux from the core diameter, is configured. For this reason, the position of the illumination light beam can be adjusted with a margin with respect to the width of 0.23 mm of the total reflection illuminable region.

  The size of the illumination light beam can be appropriately changed depending on the observation conditions.

  The illumination light beam can be moved by moving the fiber 62, moving the lens of the optical system, moving the mirror, or the like.

  Note that a laser, a mercury lamp light source, or the like can be used as a light source of the illumination light beam introduced into the fiber 62.

  The above-described embodiment is merely an example, and is not limited to the above-described configuration and shape, and can be appropriately modified and changed within the scope of the present invention.

1 is a schematic configuration diagram of a microscope according to a first embodiment of the present invention. The illumination light beam in the total reflection illumination state (solid line) and the illumination light beam in the oblique illumination illumination state (broken line) are schematically shown for the sample. The illumination state of the circular-arc-shaped aperture stop in the pupil of the objective lens of the microscope of 1st Embodiment is shown. In the first embodiment, (a) shows an arc portion of a lamp light source, and (b) shows a state in which the position of the arc portion is adjusted to an arc opening. The illumination state of the circular aperture stop in the pupil of the objective lens of the microscope of 1st Embodiment is shown. The illumination state of the annular aperture stop in the pupil of the objective lens of the microscope of the first embodiment is shown. It is a figure which shows the slider by which aperture stop arrangement | positioning used for 1st Embodiment was arrange | positioned, (a) has shown the circular-arc-shaped aperture stop, (b) has shown the circular aperture stop, (c) has shown the ring-shaped aperture stop, respectively. . The state in which the illumination state at the arc-shaped aperture stop in the pupil of the objective lens of the microscope of the first embodiment is moved in the plane and only the total reflection illumination region is illuminated is shown. The schematic block diagram of the microscope concerning 2nd Embodiment of this invention is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source 2 Collector lens 3 1st condensing lens 4 Aperture stop 4a Circular aperture stop 4b Circular aperture stop 4c Ring-shaped aperture stop 5 Collimate lens 6 Field stop 7 Mirror 8 2nd condensing lens (condensing lens)
DESCRIPTION OF SYMBOLS 9 Excitation filter 10 Dichroic mirror 11 Objective lens 12 Pupil 13 Petri dish 14 Sample 15 Culture solution 16 Cover glass 17 Oil 18 Fluorescence 19 Emission filter 20 Imaging lens 21 Imaging surface 22 Filter box 31 Total reflection illumination region 33 Polar oblique illumination region 35 Boundary 41a, 41b, 41c Slider 43 Click groove 45 Recessed portion 47 Filter holding portion 49 Spring 51, 53 Screw 61 Fiber 62 Collector lens O Optical axis FL Filter group

Claims (6)

An objective lens;
An illumination optical system that performs illumination through the objective lens;
The microscope according to claim 1, wherein the illumination optical system irradiates an illumination light beam over both the total reflection illumination region and the polar oblique illumination region of the pupil of the objective lens.   The microscope according to claim 1, wherein the illumination light beam is irradiated in a range of a numerical aperture of a pupil of the objective lens from 1.00 to 1.70.   The microscope according to claim 1, wherein the illumination optical system has a variable irradiation area of the illumination light beam.   The microscope according to claim 1, wherein the illumination light beam is movable in a plane substantially perpendicular to the optical axis.   The microscope according to any one of claims 1 to 4, wherein the illumination light beam is limited by an aperture stop disposed at a position conjugate with a pupil position of an objective lens of the illumination optical system.   The microscope according to claim 5, wherein the aperture stop is an arc-shaped aperture stop, a circular aperture stop, or a ring-shaped aperture stop.

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