JP2009181088A - Confocal unit, confocal microscope, and confocal diaphragm - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enhance a confocal effect more easily. <P>SOLUTION: A confocal diaphragm 33 is disposed on an optical system which is common to an illuminating optical system for illuminating a specimen, which is a target for confocal observation, and an imaging optical system for imaging observation light from the specimen. The confocal diaphragm 33 horizontally includes an aperture line formed from apertures 81L arranged at intervals vertically, an aperture line formed from apertures 81C arranged at intervals vertically, and an aperture line formed from apertures 81R arranged at intervals vertically. Thus, since the apertures 81 are arranged at intervals vertically and horizontally, cross-talk components of observation light in horizontal and vertical directions are removed, and the confocal effect is improved. The present invention is applied to a confocal microscope. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a confocal unit, a confocal microscope, and a confocal stop that can enhance the confocal effect more easily.

  In a confocal microscope having a slit-like opening in a confocal stop, illumination light condensed in a slit shape is irradiated onto a specimen to be observed, and the observation light generated on the observation surface of the specimen is thereby converted into a line sensor or 2 A two-dimensional image of the observation surface is obtained by detection by a photodetector such as a dimension sensor.

  In such a confocal microscope of the slit scanning method, since the illumination light only needs to be scanned in one direction, it is a two-dimensional image of the specimen as compared with a pinhole scanning confocal microscope that requires scanning in two directions. Can be obtained in a short time.

  However, the confocal microscope of the slit scanning type can obtain the confocal effect in the short direction (width direction) of the slit-shaped illumination light, but the confocal effect is inferior due to the occurrence of crosstalk in the longitudinal direction of the illumination light. It has the principle drawback. Therefore, as a method of removing the crosstalk component in the longitudinal direction, a pixel signal obtained by the pixels on the light receiving surface of the photodetector receiving observation light is used as a pixel signal obtained in pixels around the pixel. It has been proposed to use and correct (see, for example, Patent Document 1).

  Note that the confocal effect is obtained when a confocal microscope can obtain a higher sectioning resolution in the optical axis direction of the confocal microscope, or in a two-dimensional image of the specimen, a higher contrast in the plane direction of the observation surface. Is obtained.

International Publication No. WO 2007/055082 Pamphlet

  However, the above-described technique can enhance the confocal effect, but complicated arithmetic processing is required to obtain the final two-dimensional image of the sample, and the software for executing the arithmetic processing is complicated and large-scale. There was a problem of becoming.

  The present invention has been made in view of such a situation, and makes it possible to enhance the confocal effect more easily.

  The first confocal unit of the present invention is disposed on an optical system common to an illumination optical system that irradiates illumination light onto a specimen to be observed and an imaging optical system that forms an image of the observation light from the specimen. A confocal stop provided with a plurality of aperture rows arranged in a second direction different from the first direction, the aperture array including a plurality of apertures arranged at intervals in the first direction; and the illumination light A beam shaping means for condensing the light and entering the aperture of the confocal stop; and a reflective surface for reflecting the illumination light that has passed through the confocal stop and irradiating the specimen, and driving the reflective surface A scanner that scans the illumination light on the sample, and the confocal stop has all the openings in the plurality of aperture rows adjacent to each other when viewed from the second direction. Arrange so that the virtual opening row aligned in the first direction cannot be formed Is such that said end of the aperture when the virtual opening row is formed is substantially overlap with an end of the adjacent opening, said opening characterized in that it is arranged.

  The second confocal unit of the present invention is arranged in an illumination optical system that irradiates illumination light to a specimen to be observed, and an aperture row composed of a plurality of apertures arranged at intervals in the first direction is A plurality of first confocal stops arranged side by side in a second direction different from the first direction, and beam shaping for condensing the illumination light and making it incident on the opening of the first confocal stop And a scanner having a reflection surface that reflects the illumination light that has passed through the first confocal stop and irradiates the sample, and drives the reflection surface to scan the illumination light on the sample. And a second confocal stop provided in an imaging optical system for forming an image of the observation light from the specimen and provided with an aperture in the same array as the first confocal stop. When viewed from the second direction, the focus stop includes all of the plurality of aperture rows. The openings are arranged adjacent to each other so that a virtual opening row arranged in a row in the first direction cannot be formed, and when the virtual opening row is formed, the end of the opening is substantially the same as the end of the adjacent opening. The openings are arranged so as to overlap each other.

  The confocal stop of the present invention is a confocal stop used for confocal observation of a specimen, and an aperture row composed of a plurality of apertures arranged at intervals in the first direction is defined as the first direction. A plurality of openings are arranged in different second directions, and when viewed from the second direction, all the openings in the plurality of opening rows are adjacent to each other and arranged in a row in the first direction. The apertures are arranged such that the aperture rows are not formed, and the openings are arranged so that the ends of the apertures substantially overlap with the ends of the adjacent apertures when the virtual aperture row is formed. .

  According to the present invention, the confocal effect can be enhanced more easily.

  Embodiments to which the present invention is applied will be described below with reference to the drawings.

  FIG. 1 is a diagram showing a configuration example of an embodiment of a confocal microscope system to which the present invention is applied. In the figure, the arrow S indicates the direction in which the illumination light applied to the specimen to be observed travels, and the arrow K indicates the direction in which the observation light travels from the specimen.

  The confocal microscope system 11 includes a light source unit 21, a confocal microscope 22, a confocal unit 23, and a computer 24, and the confocal unit 23 is attached to the confocal microscope 22.

  The light source unit 21 includes a light source that emits illumination light. The illumination light emitted from the light source unit 21 passes through an illumination optical system provided in the confocal unit 23 and the confocal microscope 22 and is an observation target. The specimen 25 is irradiated. For example, the light source provided in the light source unit 21 is a white light source such as an ultra-high pressure mercury lamp, a xenon lamp, or a metal hydride lamp, or a laser light source. When white light is used as the illumination light, an excitation filter (not shown) is disposed between the light source unit 21 and the confocal unit 23.

  The confocal unit 23 is detachable from the confocal microscope 22, and the confocal unit 23 and the confocal microscope 22 are illuminated with an illumination optical system for irradiating the specimen 25 with illumination light and observation from the specimen 25. Optical components constituting an imaging optical system that forms an image of light are provided.

  Illumination light that has entered the confocal unit 23 from the light source unit 21 is collected by a beam shaping optical system 31 including a cylindrical microlens and the like, and enters a confocal stop 33 through a beam splitter 32. The confocal stop 33 is a stop provided with slits having a plurality of openings, and is disposed at a position optically conjugate with the observation surface Ta of the sample 25. In the light incident on the confocal stop 33, only the component incident on the opening of the confocal stop 33 passes through the confocal stop 33, and other components are shielded.

  The illumination light that has passed through the confocal stop 33 is converted into parallel light by the illumination system collimating lens 34, enters the reflection mirror 35, is further reflected by the reflection mirror 35, and enters the scanner 36. The scanner 36 has a scan mirror 36a that scans illumination light on the observation surface Ta, and reflection surfaces 36a-s and reflection surfaces 36a-r are provided on both surfaces of the scan mirror 36a.

  The scan mirror 36a rotates in the direction of arrow A about a straight line that passes through the center of the scan mirror 36a and is perpendicular to the optical path of the illumination light. When the scan mirror 36a rotates, the optical path of the illumination light that is incident from the reflection mirror 35 and reflected by the reflection surface 36a-s is changed, and as a result, the illumination light irradiated on the sample 25 is scanned in the direction of arrow B. Is done.

  The illumination light reflected by the reflection surface 36a-s of the scan mirror 36a is condensed by the scanner lens 37 and the second objective lens 38 and enters the objective lens 39. Then, the illumination light is collected by the objective lens 39 and irradiated onto the specimen 25 placed on the stage 40. At this time, the objective lens 39 condenses (images) the illumination light from the second objective lens 38 on the observation surface Ta of the sample 25.

  The specimen 25 is a fluorescently stained biological sample, for example, and expresses fluorescence as observation light when irradiated with illumination light. Observation light from the specimen 25 travels in the reverse direction of the optical path of the illumination light and enters the confocal stop 33. That is, the observation light enters the scan mirror 36a via the objective lens 39 to the scanner lens 37, is reflected by the reflection surface 36a-s, and enters the reflection mirror 35. Further, the observation light is reflected by the reflection mirror 35, collected by the illumination system collimating lens 34, and enters the confocal stop 33.

  Part of the observation light incident on the confocal stop 33 is shielded, and only the component that has passed through the opening of the confocal stop 33 enters the beam splitter 32. The observation light incident on the beam splitter 32 is reflected by the beam splitter 32, passes through the filter 41, and enters the imaging system collimating lens 42.

  Here, when an epi-fluorescence optical system is assumed, that is, when the specimen 25 is observed with fluorescence, for example, the beam splitter 32 is a dichroic mirror that transmits illumination light and reflects observation light, and the filter 41 is observation light. The barrier filter removes components other than the fluorescent component. When observing the illumination light reflected from the specimen 25, that is, when observing the specimen 25 in a bright field, for example, the beam splitter 32 is a half mirror that reflects a part of the illumination light and the observation light, and the filter 41 is Not particularly provided.

  The observation light incident on the imaging system collimating lens 42 is converted into parallel light by the imaging system collimating lens 42, further reflected by the reflection mirror 43, and then incident on the scan mirror 36 a. The observation light incident on the scan mirror 36 a is reflected on the reflection surfaces 36 a-r, collected by the condenser lens 44, and incident on the photodetector 45.

  Here, the photodetector 45 is arranged so that the light receiving surface thereof is in the vicinity of the imaging surface of the observation light, more specifically, the light receiving surface is positioned optically conjugate with the observation surface Ta. The photodetector 45 is a two-dimensional photodetector such as a CCD camera equipped with a two-dimensional CCD (Charge Coupled Devices), for example, and receives the observation light from the condensing lens 44 and photoelectrically converts it, and is thus obtained. The captured image is supplied to the computer 24 as two-dimensional luminance distribution data of the observation light.

  In FIG. 1, the beam shaping optical system 31 to the scanner lens 37 and the filter 41 to the condenser lens 44 are provided in the confocal unit 23, the second objective lens 38 to the stage 40, and the photodetector 45. Is provided in the confocal microscope 22. The illumination optical system is composed of optical parts arranged on the optical path of illumination light, and the imaging optical system is composed of optical parts arranged on the optical path of observation light.

  The computer 24 includes an operation unit 46, a control unit 47, and a display unit 48, and controls the entire confocal microscope system 11.

  The operation unit 46 includes, for example, a mouse and a keyboard, and supplies an operation signal corresponding to the operation of the observer to the control unit 47. The control unit 47 performs processing according to an operation signal from the operation unit 46. For example, the control unit 47 controls the light source unit 21 to emit illumination light to the light source unit 21, or controls the scanner 36 to rotate the scan mirror 36a. In addition, the control unit 47 acquires two-dimensional luminance distribution data from the photodetector 45, generates a two-dimensional image of the observation surface Ta based on the acquired two-dimensional luminance distribution data, and displays the display unit 48. The generated two-dimensional image is controlled and displayed on the display unit 48. The display unit 48 includes, for example, an LCD (Liquid Crystal Display) and the like, and displays the two-dimensional image supplied from the control unit 47.

  By the way, in the confocal stop 33 arranged on the imaging plane of the illumination light, for example, as shown in FIG. 2, an aperture row in which apertures are arranged at equal intervals in the vertical direction in the figure is arranged in the horizontal direction. A plurality are arranged side by side.

  In FIG. 2, the confocal stop 33 includes square openings 81L-1 to 81L-n, openings 81C-1 to 81C-n, and openings 81R-1 to 81R- each having a length of D1 on one side. n is provided.

  The n openings 81L-1 to 81L-n are arranged at predetermined intervals in the vertical direction in the drawing to form an opening row. Similarly, the n openings 81C-1 to 81C-n are arranged at predetermined intervals in the vertical direction to form an opening row, and the n openings 81R-1 to 81R-n are also arranged in the vertical direction. Are arranged at predetermined intervals to form an aperture row. In addition, these three opening rows are arranged at a predetermined interval in the horizontal direction in the figure.

  Hereinafter, when it is not necessary to distinguish each of the openings 81L-1 to 81L-n, they are simply referred to as the openings 81L. Further, when it is not necessary to individually distinguish each of the openings 81C-1 to 81C-n, they are simply referred to as an opening 81C, and when it is not necessary to individually distinguish each of the openings 81R-1 to 81R-n, This is simply referred to as the opening 81R. Further, hereinafter, when it is not necessary to individually distinguish each of the opening 81L, the opening 81C, and the opening 81R, they are simply referred to as the opening 81.

  In the confocal stop 33, in the drawing, a row of openings 81L, a row of openings 81C, and a row of openings 81R are arranged in order from the left side to the right side. Here, the row of the openings 81C is positioned above the row of the openings 81L by a distance D1 in the drawing, and the row of the openings 81R is positioned above the row of the openings 81C by a distance D1 in the drawing. ing.

  In the drawing in each opening row, the distance between the ends of the two openings 81 adjacent to each other in the vertical direction is 2D1. That is, in the opening row, the openings 81 are arranged with a distance of 2D1. Therefore, for example, the distance from the lower end in the drawing of the opening 81L-1 to the upper end in the drawing of the opening 81L-2 is 2D1.

  Further, in the figure, the distance between the ends of the openings 81 in the two rows of openings adjacent to each other in the horizontal direction is D2. That is, the aperture rows are arranged with a distance of D2. Therefore, for example, the distance from the right end in the drawing of the opening 81L-1 to the left end in the drawing of the opening 81C-1 is D2.

  For example, as shown in FIG. 3, the slit formed of the opening 81 provided on the confocal stop 33 divides the slit-like opening, and a part of the opening is divided in the short direction of the slit-like opening. It has been moved to.

  Specifically, a slit-like (rectangular) region 121 surrounded by a dotted line is divided into 3n square regions each having a D1 side, and among the divided regions, 3 (i -1) The + 1st (where 1 ≦ i ≦ n) region is moved to the right in the figure by (D1 + D2). Then, openings are provided in the n regions after the movement, and these openings are referred to as openings 81R.

  In addition, among the square areas formed by dividing the area 121, openings are provided in the 3 (i−1) + second (where 1 ≦ i ≦ n) area from the top in the drawing, and these openings are defined. The opening is 81C. Furthermore, among the square areas formed by dividing the area 121, the 3 (i−1) + 3rd (where 1 ≦ i ≦ n) area from the top is only (D1 + D2) on the left side in the figure. Move. Then, openings are provided in the n regions after the movement, and these openings are referred to as openings 81L.

  Accordingly, the aperture 81 of an arbitrary aperture row among the plurality of aperture rows provided in the confocal stop 33 is moved in the horizontal direction in the figure so that the aperture 81 is positioned on any other aperture row. In this case, the end (upper or lower end) of the moved opening 81 overlaps the end of the opening 81 adjacent in the vertical direction in the drawing of the opening row to be moved. In other words, the openings 81 are arranged in the horizontal direction (second direction) so that all the openings 81 of the plurality of opening rows provided in the confocal stop 33 are aligned in the vertical direction (first direction) in the figure, for example, in the region 121. In the vertical direction), the openings 81 are arranged in a row (virtual opening row) in the vertical direction adjacent to each other.

  In this state, when the confocal stop 33 is viewed from the lateral direction that is the second direction, all the apertures 81 of the aperture row including the plurality of apertures 81 are adjacent to each other in the longitudinal direction that is the first direction. The openings 81 are arranged in a row so that the ends of the openings 81 substantially overlap the ends of the adjacent openings 81.

  Furthermore, the illumination light and the observation light that have passed through the confocal stop 33 become light having the same pattern as the arrangement of the openings 81. That is, the illumination light and the observation light (spot light) that have passed through the openings 81 are arranged in the same manner as the arrangement of the openings 81. In other words, the illumination light and the observation light that have passed through the confocal stop 33 become a slit-shaped light beam formed by arranging spot lights.

  Thus, in the confocal stop 33, a rectangular slit is formed by the openings 81 arranged at intervals. In the confocal stop 33, when the slits are viewed from the short side, that is, from the lateral direction (second direction) in FIG. 2, all the openings 81 of the plurality of aperture rows are in the first direction of the slits. The openings 81 are arranged in a row adjacent to each other in a certain longitudinal direction (vertical direction in FIG. 2) so that the ends of the openings 81 overlap the ends of the adjacent openings 81.

  Therefore, when the slit-shaped illumination light irradiated to the observation surface Ta of the specimen 25 is scanned in the slit-shaped short direction by the scan mirror 36a, the observation surface Ta is filled with the illumination light without a gap. It is scanned (illuminated) by the illumination light.

  Although the confocal stop 33 looks like a multi-pinhole type confocal stop at first glance, it actually functions as a slit type confocal stop. That is, when a multi-pinhole type confocal stop is used, scanning of illumination light in two directions is required, whereas when the confocal stop 33 is used, illumination light is scanned in one direction. You can do it.

  When the confocal stop 33 is configured as shown in FIG. 2, the beam shaping optical system 31 includes, for example, as shown in FIG. 4, cylindrical microlenses 151-1 to 151-11 that are long in the depth direction in the drawing. -3. In the figure, arrow S indicates the direction in which the illumination light travels, and arrow K indicates the direction in which the observation light travels.

  In the drawing, the illumination light incident on the beam shaping optical system 31 from the upper side is condensed by each of the cylindrical microlenses 151-1 to 151-3, and each of the rows of the openings 81 </ b> L to 81R. It is shaped into three light beams having substantially the same shape (slit shape). The illumination light collected by the cylindrical microlenses 151-1 to 151-3 is transmitted through the beam splitter 32 and passes through the rows of the openings 81L to 81R.

  Here, the illumination light condensed by the cylindrical microlenses 151-1 to 151-3 is condensed at a position conjugate with the observation surface Ta, that is, a position where the opening 81 is provided. At this time, the confocal stop 33 functions as a confocal stop on the illumination optical system side.

  Furthermore, since the observation light from the specimen 25 passes through the optical path of the illumination light in the reverse direction and enters the confocal stop 33, the observation light is shaped into three slit-shaped light beams, and an opening 81 is provided. The light is condensed at the position and passes through the opening 81 from the bottom to the top in the drawing. The observation light that has passed through the opening 81 is reflected leftward in the drawing by the beam splitter 32 and enters the filter 41. At this time, the confocal stop 33 functions as a confocal stop on the imaging optical system side.

  In the example of FIG. 4, it has been described that the illumination light is collected by the cylindrical microlenses 151-1 to 151-3 corresponding to the rows of the openings 81L to 81R. The light may be collected by a lens corresponding to each of the openings 81.

  In such a case, the beam shaping optical system 31 includes, for example, a fly-eye lens (lens array) in which lenses corresponding to the openings 81 are arranged. Then, the illumination light is condensed by each of the lenses to be spot light, and is incident on each of the openings 81 corresponding to the lenses.

  Further, the illumination light is condensed into a slit shape (rectangular shape) by the beam shaping optical system 31, and only the light incident on the opening 81 among the slit-shaped illumination light passes through the confocal stop 33. Also good.

  Next, the operation of the confocal microscope system 11 will be described.

  When the light source unit 21 emits illumination light under the control of the control unit 47, the beam shaping optical system 31 condenses the illumination light from the light source unit 21 and slits the illumination light in the depth direction in FIG. Shaped into a line-shaped light beam.

  Then, the illumination light shaped into the slit shape enters the scan mirror 36 a through the confocal stop 33 to the reflection mirror 35. The scan mirror 36a rotates under the control of the control unit 47 and reflects the illumination light incident from the reflection mirror 35 on the reflection surfaces 36a-s. Here, the scan mirror 36a rotates about a straight line passing through the center thereof and parallel to the longitudinal direction of the slit-shaped illumination light incident on the reflection surface 36a-s.

  The illumination light reflected by the reflecting surfaces 36 a-s is condensed on the observation surface Ta of the sample 25 by the scanner lens 37 to the objective lens 39.

  For example, as shown in FIG. 5, it is assumed that a region Q of the observation surface Ta is illuminated with illumination light. In FIG. 5, an observation field F indicates a field region on the observation surface Ta observed by the confocal microscope 22.

  In FIG. 5, each of the squares in the region Q represents illumination light (spot light) irradiated to the observation surface Ta through the opening 81, and the region Q is represented by rectangular illumination light composed of spot light. Illuminated. In addition, each of the squares representing the spot light is arranged in three rows in the vertical direction in the drawing, similarly to the arrangement of the openings 81 provided in the confocal stop 33.

  When the scan mirror 36a rotates during the confocal observation of the specimen 25, the rectangular illumination light is scanned in a direction substantially perpendicular to the longitudinal direction thereof, that is, in the horizontal direction (direction of arrow B) in the drawing, and the observation field of view. The illumination area E including F is illuminated with illumination light without gaps.

  In this way, when the observation surface Ta of the sample 25 is illuminated, the observation light from the observation surface Ta enters the confocal stop 33 through the objective lens 39 through the illumination system collimating lens 34. Further, the observation light incident on the confocal stop 33 passes through the opening 81 and enters the beam splitter 32.

  Here, the confocal stop 33 includes not only light from the observation surface Ta but also light from a position shifted in the vertical direction in FIG. 1 from the observation surface Ta in the specimen 25, that is, out-of-focus diffused light (cross). Talk component) also enters as observation light. In the confocal unit 23, since the confocal stop 33 is disposed at a position conjugate with the observation surface Ta, most of the crosstalk component of the observation light incident on the confocal stop 33 passes through the opening 81. Will be shielded.

  For example, as shown in FIG. 6A, light incident on a hatched area near the opening 81 in the crosstalk component of the observation light is blocked by the confocal stop 33 without passing through the opening 81. In FIG. 6A, a region with a diagonal line in the lower left direction in the vicinity of the opening 81, that is, a region located on the left and right in the drawing of each opening 81 is not only the confocal stop 33 but also a confocal stop of a conventional slit scanning method. This is the region where the crosstalk component that could be shielded by the incident is incident.

  On the other hand, the area hatched in the lower right direction in the vicinity of the opening 81, that is, the area located above and below in the drawing of each opening 81 is an area where a crosstalk component shielded by the confocal stop 33 is incident, This crosstalk component is a component that cannot be shielded by the conventional confocal stop of the slit scanning method.

  In other words, the conventional slit scanning confocal stop has a slit-like opening, so that the crosstalk component can be shielded in the short direction of the opening, but the crosstalk component in the longitudinal direction. Can not be shielded.

  On the other hand, in the confocal stop 33 according to the present invention, since the openings 81 are arranged at intervals of the length 2D1 in each opening row, the crosstalk component in the longitudinal direction of the slit-like observation light is also generated. Can be shielded.

  For example, as shown in FIG. 6B, the light incident on the portion between the opening 81R and the other adjacent opening 81R out of the observation light incident on the confocal stop 33 is shielded. FIG. 6B shows the confocal stop 33 as viewed from the right to the left in FIG. 6A, and the hatched portion represents the out-of-focus observation light.

  In FIG. 6B, the out-of-focus observation light from a portion farther from the objective lens 39 than the observation surface Ta in the specimen 25 is condensed on the front side (right side in FIG. 6B) of the confocal stop 33 to be collected. Is incident. In FIG. 6B, most of the observation light incident on the vicinity of each of the openings 81R-1 to 81R-3 from the right side is shielded by the confocal stop 33, and only a small amount of light enters the opening 81R.

  Thus, with the conventional confocal stop, only the crosstalk component in the short direction of the slit-like observation light can be shielded. However, according to the confocal stop 33 of the present invention, the short observation light is short. Not only the direction but also the crosstalk component in the longitudinal direction can be shielded, and the confocal effect can be further enhanced.

  The observation light that has passed through the confocal stop 33 in this way enters the condenser lens 44 through the filter 41 to the scanner 36, is condensed by the condenser lens 44, and is received by the photodetector 45. The light detector 45 receives the observation light from the condensing lens 44 and supplies a captured image obtained as a result to the control unit 47 as two-dimensional luminance distribution data of the observation light.

  Then, the control unit 47 generates a two-dimensional image of the observation surface Ta based on the two-dimensional luminance distribution data from the photodetector 45 and supplies the two-dimensional image to the display unit 48 for display. For example, the control unit 47 generates a two-dimensional image for one sheet (one frame) using two-dimensional luminance distribution data obtained while the illumination light is scanned from end to end of the illumination area E. .

  Thereby, the observer can observe the observation visual field F, that is, the observation surface Ta of the sample 25 by viewing the two-dimensional image displayed on the display unit 48. Further, the observer can observe an arbitrary part of the specimen 25 as the observation surface Ta by operating the operation unit 46 and the like to move the stage 40 in a direction parallel to the optical axis of the objective lens 39. .

  As described above, by using the confocal stop 33 in which the aperture rows composed of a plurality of apertures are arranged, not only the short direction of the observation light but also the crosstalk component in the longitudinal direction can be shielded easily. Confocal effects such as sectioning resolution and contrast can be increased.

  That is, according to the confocal microscope system 11, it is not necessary to perform an arithmetic process for correcting the pixel signal, that is, an arithmetic process for removing the crosstalk component from the two-dimensional image, and before receiving the observation light by the photodetector 45. Since the crosstalk component in the longitudinal direction of the observation light can be physically removed, the confocal effect can be easily enhanced. In addition, since a calculation process for correcting the pixel signal is not required, a two-dimensional image can be obtained quickly, and the high speed characteristic of the slit scanning method is not impaired. Further, the cost and time for developing a program for executing arithmetic processing are not required.

  In the above description, the beam splitter 32 and the sample 25 in the illumination optical system and the imaging optical system are a common optical system, and the confocal stop 33 is a confocal stop on the illumination optical system side. Although described as functioning as a confocal stop on the image optical system side, a plurality of confocal stops may be provided.

  In such a case, the confocal stop 33 is a confocal stop on the illumination optical system side, and the confocal stop on the imaging optical system side is another confocal point different from the confocal stop 33. A diaphragm is placed. The observation light enters the other confocal stop through an optical path different from that of the illumination light, and the observation light that has passed through the other confocal stop collects the filters 41 to 41 arranged at positions different from those in FIG. The light is received by the photodetector 45 through the lens 44. In this case, the other confocal stop is the same as the confocal stop 33, that is, the other confocal stop is provided with the same array of apertures as the confocal stop 33. It is arranged at a position conjugate with the observation surface Ta.

  In FIG. 1, the scan mirror 36 a is provided with the reflective surfaces 36 a-s and the reflective surfaces 36 a-r, but only the reflective surface 36 a-s is provided on the scan mirror 36 a and observation from the reflective mirror 43 is performed. Another scan mirror that reflects light may be provided separately.

  Further, when the confocal stop 33 is viewed from the short direction of the slit formed of the opening 81, it has been described that the opening 81 is provided so that the ends of the openings 81 adjacent to each other in the longitudinal direction overlap each other. It suffices if the ends of 81 are substantially overlapped.

  That is, when (a part of) the openings 81 overlap with each other when viewed from the short side direction, a white line resulting from an increase in the amount of received light enters a portion corresponding to the boundary of the openings 81 in the two-dimensional image, and the openings 81 If they are separated from each other, a black line resulting from a decrease in the amount of received light enters a portion corresponding to the boundary of the opening 81 of the two-dimensional image. The thicknesses of the white lines and black lines on the two-dimensional image change depending on the overlapping state and the separating state of the openings 81. For this reason, the distance between the ends of the adjacent openings 81 viewed from the short side direction may be a distance in which the thickness of the line on the two-dimensional image is an allowable thickness for the observer to observe the sample 25. .

  For example, as shown in FIG. 7, it is assumed that the opening provided in the confocal stop 33 is circular, and the opening 181 and the opening 182 that are adjacent to each other in the longitudinal direction overlap with each other when viewed from the short direction of the slit formed by the opening. Suppose that That is, when the opening 181 on a predetermined opening row of the confocal stop 33 is moved onto another opening row, the opening 182 and the opening 181 on the opening row overlap each other. Further, assume that the diameter of each of the opening 181 and the opening 182 is X, and the length in the short direction of the slit of the portion where the opening 181 and the opening 182 overlap is P.

  When the specimen 25 is scanned with the illumination light, the part with the longest scanning time in the specimen 25 is a part scanned with the illumination light that passes through the diameter of each opening. When the value 2P, which is twice the length P of the portion where the opening 181 and the opening 182 overlap, exceeds the diameter X, the unevenness of brightness on the two-dimensional image increases, and the thickness of the white line increases. Therefore, in order to illuminate the specimen 25 as uniformly as possible, it is desirable that, for example, 2P ≦ X is satisfied.

  When 2P = X, the distance from the lower end to the upper end in the drawing of the opening 182 in the drawing of the opening 181 where the openings overlap is 0.14X.

  In the above description, an example in which three aperture rows each including the square-shaped apertures 81 are arranged on the confocal diaphragm 33 has been described. However, any number of aperture rows may be used. The shape of the opening 81 is not limited to a square shape, and may be another shape such as a circular shape.

  8 and 9 are diagrams showing another configuration example of the confocal stop 33. FIG.

  For example, in the confocal stop 33 shown in FIG. 8A, square openings 81 having a side length D1 are spaced apart by 2D1 in the vertical direction and spaced apart by D2 in the horizontal direction. Are lined up. That is, a row of n openings 81L, a row of n openings 81C, and a row of n openings 81R are arranged in the horizontal direction in the drawing. Further, the row of openings 81L is positioned lower than the row of openings 81C by a distance 2D1, and the row of openings 81R is positioned lower than the row of openings 81C by a distance D1. .

  Further, in the example shown in FIG. 8B, the confocal diaphragm 33 has square openings 81 each having a length D1 that are spaced apart by 2D1 in the vertical direction and are adjacent in the horizontal direction. Are listed. That is, a row of n openings 81L, a row of n openings 81C, and a row of n openings 81R are provided adjacent to each other without being spaced apart from each other in the horizontal direction in the figure. Further, the row of openings 81L is positioned lower than the row of openings 81C by a distance D1 in the drawing, and the row of openings 81C is positioned lower than the row of openings 81R by a distance D1. .

  Further, in the example shown in FIG. 8C, the confocal stop 33 includes circular openings 211L-1 to 211L-n having a diameter D1, openings 211C-1 to 211C-n, and openings 211R-1 to 211R. -N are arranged in the same arrangement as each of the openings 81L-1 to 81L-n, the openings 81C-1 to 81C-n, and the openings 81R-1 to 81R-n in FIG. That is, the example shown in FIG. 2 and the example shown in FIG. 8C differ only in the shape of the opening provided in the confocal stop 33.

  In the example shown in FIG. 9A, the confocal stop 33 has square openings 231L-1 to 231L-n each having a length D1 of one side spaced apart by a distance D1 in the vertical direction in the figure. Are listed. In the confocal stop 33, square-shaped openings 231R-1 to 231R-n each having a side length D1 are arranged in the vertical direction in the figure at an interval of a distance D1.

  Hereinafter, when it is not necessary to individually distinguish the openings 231L-1 to 231L-n, they are simply referred to as openings 231L, and it is not necessary to individually distinguish the openings 231R-1 to 231R-n. In this case, it is simply referred to as opening 231R.

  Further, in the confocal stop 33 shown in FIG. 9A, the rows of the apertures 231L and the rows of the apertures 231R are arranged in the horizontal direction in the drawing with a distance D2. Further, the row of the openings 231L is positioned below the row of the openings 231R in the drawing by the distance D1.

  Further, in the example shown in FIG. 9B, in the confocal stop 33, the distance D2 between the row of the openings 231L and the row of the openings 231R shown in FIG. 9A is set to 0, and the row of the openings 231L and the row of the openings 231R Are arranged adjacent to each other without being spaced apart from each other.

  As described above, the number of aperture rows provided in the confocal stop 33, the shape of the aperture, the interval between adjacent aperture rows, and the like can be changed as appropriate.

  For example, if the number of aperture rows (for example, 3 rows in the example of FIG. 2) is increased, the distance between adjacent apertures in the aperture row can be increased, thereby reducing the crosstalk component. Become more advantageous. On the other hand, since the length in the short direction of the illumination light shaped into the slit shape becomes long, the power (intensity) per unit area of the illumination light becomes weak, and in order to observe a predetermined part of the specimen 25 The distance for scanning the illumination light is increased by the increase in the number of aperture rows. Therefore, the number of aperture rows may be determined by balancing both the removal of the crosstalk component and the scanning distance of the illumination light and the intensity of the illumination light.

  Further, the crosstalk component can be reduced as the distance D2 between the opening rows is increased. However, if the distance D2 is increased, the length in the short direction of the illumination light shaped into the slit shape is increased correspondingly, so that the distance over which the illumination light is scanned in order to observe a predetermined part of the specimen 25 is increased. Become. Moreover, it is necessary to further widen the width of the illumination light incident on the confocal stop 33 by beam shaping. Therefore, the distance D <b> 2 between the opening rows may be determined in consideration of the performance and practicality of the confocal unit 23.

  Furthermore, for example, when the distance D2 between the aperture row of the confocal stop 33 is 0 as in the example shown in FIG. 8B or FIG. 9B, the distance between the aperture rows is short. Although this is disadvantageous in terms of removing the crosstalk component, the scanning range of the illumination light is small, and the intensity per unit area of the illumination light shaped into a slit shape becomes stronger.

  Furthermore, the confocal unit 23 may be provided with a plurality of confocal stops 33 having different sizes and arrangements of the openings 81. In such a case, the confocal unit 23 selects an appropriate one of the plurality of confocal stops 33 according to the objective lens 39 and the sample 25 used at the time of confocal observation, and the light of the illumination light and the observation light Place on the road. In this case, a plurality of beam shaping optical systems 31 suitable for each confocal stop 33 are provided, and the confocal unit 23 is appropriate for the confocal stop 33 arranged on the optical path of the illumination light and the observation light. A suitable beam shaping optical system 31 may be selected and arranged on the optical path of the illumination light.

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

It is a figure which shows the structural example of one Embodiment of the confocal microscope system to which this invention is applied. It is a figure which shows the structural example of a confocal stop. It is a figure explaining the arrangement | sequence of the opening in a confocal stop. It is a figure which shows the structural example of a beam shaping optical system. It is a figure explaining the scanning of illumination light. It is a figure explaining the crosstalk component shielded with a confocal stop. It is a figure explaining the distance of opening. It is a figure which shows the other structural example of a confocal stop. It is a figure which shows the other structural example of a confocal stop.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 11 Confocal microscope system, 21 Light source part, 22 Confocal microscope, 23 Confocal unit, 24 Computer, 25 Specimen, 31 Beam shaping optical system, 33 Confocal stop, 36 Scanner, 36a Scan mirror, 39 Objective lens, 45 Light Detector, 81L-1 to 81L-n, 81C-1 to 81C-n, 81R-1 to 81R-n Opening, 211L-1 to 211L-n, 211C-1 to 211C-n, 211R-1 to 211R- n opening, 231L-1 to 231L-n, 231R-1 to 231R-n opening

Claims (6)

Arranged on an optical system common to an illumination optical system for irradiating illumination light to the specimen to be observed and an imaging optical system for forming an image of the observation light from the specimen, and arranged in the first direction at intervals. A confocal stop provided with a plurality of aperture rows formed of a plurality of apertures arranged in a second direction different from the first direction;
Beam shaping means for condensing the illumination light and making it incident on the aperture of the confocal stop;
A reflection surface that reflects the illumination light that has passed through the confocal stop and irradiates the sample; and a scanner that drives the reflection surface to scan the illumination light on the sample,
In the confocal stop, when viewed from the second direction, it is possible to form a virtual aperture row in which all the apertures of the plurality of aperture rows are adjacent to each other and aligned in the first direction. The confocal units are arranged such that, when the virtual opening row is formed, the openings are arranged so that the ends of the openings substantially overlap with the ends of the adjacent openings. The beam shaping means includes lenses corresponding to each of the aperture rows, and the lens condenses the illumination light in substantially the same shape as the aperture rows and enters the corresponding aperture rows. The confocal unit according to claim 1. The said beam shaping means consists of each of the lens corresponding to each of the said opening, The said lens condenses the said illumination light, and makes it enter into the said corresponding opening. Confocal unit.   A confocal microscope comprising the confocal unit according to any one of claims 1 to 3. A second direction different from the first direction is an aperture row that is arranged in an illumination optical system that irradiates illumination light to the specimen to be observed and is arranged with a plurality of apertures arranged at intervals in the first direction. A plurality of first confocal stops arranged side by side,
Beam shaping means for condensing the illumination light and making it incident on the opening of the first confocal stop;
A scanner that reflects the illumination light that has passed through the first confocal stop and irradiates the sample, and drives the reflection surface to scan the illumination light on the sample;
A second confocal stop disposed in an imaging optical system that forms an image of observation light from the specimen, and provided with openings in the same array as the first confocal stop;
In the first confocal stop, when viewed from the second direction, a virtual aperture row is formed in which all the apertures of the plurality of aperture rows are adjacent to each other and aligned in the first direction. The confocal units are arranged such that the openings are arranged so that the ends of the openings substantially overlap with the ends of the adjacent openings when the virtual opening row is formed. A confocal stop used for confocal observation of a specimen,
A plurality of aperture rows each having a plurality of apertures arranged at intervals in the first direction are arranged in a second direction different from the first direction,
When viewed from the second direction, all the openings of the plurality of opening rows are arranged adjacent to each other so as to form a virtual opening row arranged in a row in the first direction, The confocal stop, wherein the apertures are arranged so that the ends of the apertures substantially overlap with the ends of the adjacent apertures when the aperture row is formed.

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