JP2010160371A - Confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To change the sectioning resolution with a simple constitution. <P>SOLUTION: When illumination light from a light source 22 is radiated to a sample 13, observation light appears from the sample 13, and the observation light passes an optical image forming system 51 and is detected by an optical detector 52. The optical detector 52 is arranged at a position substantially conjugate with an observation surface 13s, so that the observation light from a near region close to the observation surface 13s and the observation light from a peripheral region around the near region can be individually detected. An image processing section 54 generates a near image from the detected result of the observation light from the near region, and generates a peripheral image from the detected result of the observation light from the peripheral region. A personal computer 12 generates, from the near image and peripheral image, an observation image of a sectioning resolution different from that of these images. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a confocal microscope in which sectioning resolution can be changed with a simpler configuration.

  Conventionally, by irradiating a sample to be observed with illumination light, among the observation light expressed from the sample, a light beam that has passed through a pinhole at a position substantially conjugated with the sample is detected by a photodetector. A confocal microscope that obtains a two-dimensional image is known. By providing a pinhole in the confocal microscope, it is possible to detect only the observation light from only a thin layer in the sample in substantially the same direction as the direction of illumination light to be irradiated, and obtain an image of that portion. .

  In particular, the ability to adjust the observation range in the thickness direction of the sample to be observed, more specifically, the ability to adjust the observation range in the thickness direction of the sample is called sectioning resolution. It is said that the sectioning resolution is higher. For example, in a confocal microscope, the smaller the pinhole opening, the thinner the observation range in the thickness direction of the sample to be detected by the observation light, so the sectioning resolution increases.

  In addition, the conventional confocal microscope allows the observation light from the sample to pass through a pinhole having a small opening diameter, and at the same time, reflects the observation light that has failed to pass through the pinhole to provide a pinhole having a larger opening diameter. In some cases, the sectioning resolution is changed by passing the signal (for example, see Patent Document 1).

  In this confocal microscope, a plurality of images with different sectioning resolutions are generated by detecting each of the observation light that has passed through the pinhole with a photodetector, and further, the target is obtained by performing arithmetic processing using these images. An image with sectioning resolution is obtained. Therefore, according to such a confocal microscope, the sectioning resolution can be easily changed by arithmetic processing (for example, Patent Document 2).

JP 2005-274591 A International Publication No. 2007/010697 Pamphlet

  However, in the technique described above, the confocal microscope has a plurality of pinholes, and it is necessary to provide a photodetector for each member that separates the observation light and each pinhole through which the observation light passes. The configuration becomes complicated.

  The present invention has been made in view of such a situation, and makes it possible to change the sectioning resolution of a confocal microscope with a simpler configuration.

  The confocal microscope of the present invention includes an illumination optical system that condenses illumination light on an observation surface of a sample to be observed, a scanner that is included in the illumination optical system and that scans the illumination light on the observation surface, and An imaging optical system that condenses observation light from a sample, a light detection means that is disposed at a position that is substantially conjugate with the light collection position of the illumination light in the sample, and a light detection means that detects the observation light. Based on the detection result, the first image data based on the observation light from the region near the condensing position on the sample, and the second image based on the observation light from the peripheral region of the vicinity region on the sample. And a third image having a sectioning resolution different from that of the first image data and the second image data based on the first image data and the second image data. De Characterized in that it comprises a calculating means for generating data.

  According to the present invention, the sectioning resolution can be changed with a simpler configuration.

It is a figure which shows the structural example of one Embodiment of the confocal observation system to which this invention is applied. It is a figure explaining the scanning by illumination light. It is a figure explaining the area | region where the observation light from each site | part of a sample injects in the light-receiving surface of a photodetector. It is a figure explaining the observation light from the site | part of a sample which injects into each area | region of the light-receiving surface of a photodetector.

  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 observation system to which the present invention is applied. This confocal observation system includes a confocal microscope 11 and a personal computer 12, and the confocal observation of the sample 13 is performed by the confocal observation system.

  The sample 13 to be observed is placed on the stage 21 of the confocal microscope 11, and the sample 13 is irradiated with illumination light emitted from the light source 22 and guided by the illumination optical system 23 of the confocal microscope 11. .

  That is, the optical fiber 31 is connected to the light source 22, and the illumination light incident on the optical fiber 31 from the light source 22 is guided to the collimating lens 32 by the optical fiber 31. The illumination light incident on the collimating lens 32 from the optical fiber 31 is converted into parallel light by the collimating lens 32, and further converted into line-shaped light converged in the depth direction in the figure by the cylindrical lens 33 to be converted into a microlens array. 34 is incident.

  In addition, the microlens array 34 includes a plurality of lenses arranged in the vertical direction in the drawing, and the illumination light incident on the microlens array 34 is perpendicular to the traveling direction of the illumination light, that is, by these lenses, that is, In the figure, the light beam is separated into a plurality of light beams arranged in the vertical direction. That is, each lens constituting the microlens array 34 collects the incident illumination light and makes it incident on the lens 35.

  Each light beam of the illumination light collected by the microlens array 34 is converted into parallel light by the lens 35 and passes through the galvano scanner 36, pupil projection lens 37, second objective lens 38, dichroic mirror 39, and objective lens 40. The sample 13 is irradiated. At this time, the galvano scanner 36 rotates a mirror (not shown) to scan the illumination light on the observation surface 13 s of the sample 13. Specifically, illumination light is scanned in the depth direction and the lateral direction in the drawing.

  As described above, the illumination light from the light source 22 is guided to the sample 13 by the illumination optical system 23 including the optical fiber 31 to the objective lens 40 and is condensed on the observation surface 13 s of the sample 13.

  When the sample 13 is irradiated with illumination light, fluorescence as observation light appears from the sample 13, and this observation light (fluorescence) is guided to the photodetector 52 by the imaging optical system 51. The imaging optical system 51 includes an objective lens 40, a dichroic mirror 39, and a second objective lens 53. The imaging optical system 51 has an optical system that is common to the illumination optical system 23.

  That is, the observation light from the sample 13 passes through the objective lens 40 and enters the dichroic mirror 39, is reflected by the dichroic mirror 39, and enters the second objective lens 53. Here, the dichroic mirror 39 transmits the illumination light from the second objective lens 38 as it is and separates the illumination light and the observation light by reflecting the observation light from the objective lens 40.

  Further, the observation light from the dichroic mirror 39 is condensed on a light receiving surface (not shown) of the photodetector 52 by the second objective lens 53. The light receiving surface of the photodetector 52 is disposed at a position where the illumination light is condensed on the sample 13, that is, a position substantially conjugate with the observation surface 13 s.

  The light detector 52 is composed of, for example, a CCD (Charge Coupled Devices) and the like. Among the observation light from the sample 13, the observation light from the vicinity region in the vicinity of the light collection position of the illumination light in the sample 13 and the vicinity in the sample 13. The observation light from the peripheral area around the area is detected.

  Here, the vicinity region refers to a region in the thin observation range in the thickness direction of the sample 13 including the observation surface 13s on which the illumination light is collected. Further, the peripheral region includes a thin layer region adjacent to the upper side in the drawing in the vicinity region and a thin layer region adjacent to the lower side in the drawing in the vicinity region in the sample 13 centered on the observation surface 13s. Become. Hereinafter, the longitudinal direction in the drawing of the sample 13 is also referred to as the thickness direction of the sample 13.

  The photodetector 52 receives and photoelectrically converts the observation light from the neighboring region and the observation light from the neighboring region, respectively, detects the observation light, and outputs the electric signal obtained by the photoelectric conversion. The observation result is supplied to the image processing unit 54 as a result of detection. The value of the electrical signal indicates the amount of received observation light, that is, the luminance of the observation light.

  Based on the electrical signal supplied from the photodetector 52, the image processing unit 54 generates nearby image data and surrounding image data having different sectioning resolutions, and supplies them to the personal computer 12.

  That is, the image processing unit 54 generates neighborhood image data that is image data of an image in the vicinity region of the sample 13 based on the electrical signal obtained by the observation light from the vicinity region. Similarly, the image processing unit 54 generates peripheral image data that is image data of an image of the peripheral region of the sample 13 based on an electrical signal obtained from observation light from the peripheral region. Hereinafter, an image based on the neighborhood image data is also referred to as a neighborhood image, and an image based on the neighborhood image data is also referred to as a neighborhood image.

  The personal computer 12 generates observation image data having a sectioning resolution different from that of the neighborhood image data and the surrounding image data based on the neighborhood image data and the surrounding image data supplied from the image processing unit 54, and performs observation based on the observation image data. Display an image.

  The sectioning resolution of the observation image data, that is, the portion of the sample 13 displayed in the observation image is a layer thinner in the thickness direction of the sample 13 than the region composed of the vicinity region and the peripheral region in the sample 13 with the observation surface 13s as the center. It is considered as an area. Further, the sectioning resolution is appropriately changed by the personal computer 12. Therefore, the thickness in the thickness direction of the portion of the sample 13 displayed in the observation image may be thinner than the thickness of the neighboring region.

  By the way, in the confocal microscope 11, since the illumination light is separated into a plurality of light beams by the micro lens array 34, when observing the sample 13, for example, as shown in FIG. A plurality of light beams L11 to L14 are scanned. 2 shows a view of the observation surface 13s of the sample 13 as viewed from the upper side to the lower side in FIG.

  In FIG. 2, light beams L11 to L14 as illumination light are arranged in the vertical direction at a predetermined interval in the drawing and are condensed on the observation surface 13s. These light beams L11 to L14 are simultaneously scanned by the galvano scanner 36 in the horizontal direction and the vertical direction in the drawing. Thereby, illumination light is irradiated to substantially all areas of the observation surface 13s.

  On the other hand, the light detector 52 detects a plurality of light beams expressed from the sample 13 as observation light by irradiating the sample 13 with the light beams L11 to L14.

  For example, as illustrated in FIG. 3, the light beam expressed from the sample 13 when the sample 13 is irradiated with the light beam L <b> 11 enters the region R <b> 11 and the region R <b> 12 of the light receiving surface of the photodetector 52. Further, the light beam expressed from the sample 13 when the sample 13 is irradiated with the light beam L12 enters the region R13 and the region R14 of the light receiving surface of the photodetector 52. In FIG. 3, one square represents one pixel constituting the light receiving surface of the photodetector 52.

  More specifically, out of the observation light expressed by irradiating the sample 13 with the light beam L11, the component expressed from the nearby region is incident on the region R11 including one pixel. Similarly, of the observation light that is expressed by irradiating the sample 13 with the light beam L12, a component that is expressed from a nearby region is incident on a region R13 including one pixel.

  In addition, among the observation light expressed by irradiating the sample 13 with the light beam L11, a component expressed from the peripheral region enters a region R12 including a plurality of pixels surrounding the region R11. Further, among the observation light expressed by irradiating the sample 13 with the light beam L12, a component expressed from the peripheral region enters a region R14 including a plurality of pixels surrounding the region R13.

  Actually, as shown in the upper side of FIG. 4, the region R <b> 11 is a pixel on which the observation light expressed from the sample 13 is incident by irradiating the sample 13 with the focus component of the light beam L <b> 11, that is, the sample 13. Is determined in advance so as to be composed of pixels (a plurality of pixels) on which the observation light from the vicinity region of the light enters.

  In the region R12, as shown on the lower side in FIG. 4, the sample 13 is irradiated with the defocused component of the light beam L11, so that the observation light expressed from the sample 13 is incident, that is, the sample 13. Is determined in advance so as to be composed of pixels (a plurality of pixels) on which the observation light from the peripheral region is incident.

  In FIG. 4, the vertical axis indicates the position in the thickness direction of the sample 13, and the horizontal axis indicates the luminance (light quantity) of the observation light that appears from each position of the sample 13. Further, the position Z0 indicates the position of the observation surface 13s, and the area from the position Z1 to the position Z2 indicates the vicinity area.

  As shown in the upper side of FIG. 4, most of the observation light incident on the region R11 is light that is expressed from the position Z1 to the position Z2, that is, from the vicinity region. Therefore, by using the electrical signal obtained from the pixels constituting the region R11, it is possible to extract only the information of the portion irradiated with the light beam L11 in the region near the sample 13.

  Therefore, the image processing unit 54 displays a region near the sample 13 irradiated with the light beam L11 from an electrical signal obtained by photoelectrically converting light incident on the pixels constituting the region R11 by the photodetector 52. The pixel value of the pixel on the neighboring image is obtained.

  Similarly, the image processing unit 54 displays a region near the sample 13 irradiated with the light beam L12 from an electrical signal obtained by photoelectrically converting light incident on the pixels constituting the region R13 by the photodetector 52. The pixel value of the pixel on the neighboring image is obtained.

  As described above, the image processing unit 54 generates the neighborhood image data by obtaining the pixel value of each pixel on the neighborhood image using the electrical signal indicating the amount of observation light received from the neighborhood region.

  Further, as shown in the lower side of FIG. 4, most of the observation light incident on the region R12 in the sample 13 is a region above the position Z1 and a region below the position Z2 in the drawing, that is, the region below the position Z2. Light is emitted from a substantially peripheral region. Therefore, by using the electrical signal obtained by the pixels constituting the region R12, it is possible to extract only the information of the portion irradiated with the light beam L11 in the peripheral region of the sample 13.

  Therefore, the image processing unit 54 detects the peripheral region of the sample 13 irradiated with the light beam L11 from each electrical signal obtained by photoelectrically converting the light incident on each pixel constituting the region R12 by the photodetector 52. The pixel value of the pixel on the peripheral image to be displayed is obtained. That is, the pixel value of one pixel on the peripheral image is obtained based on the sum of the values of the electrical signals obtained at the pixels constituting the region R12.

  Similarly, the image processing unit 54 is a peripheral region of the sample 13 irradiated with the light beam L12 from each electrical signal obtained by photoelectrically converting light incident on each pixel constituting the region R14 by the photodetector 52. The pixel value of the pixel on the peripheral image is displayed.

  As described above, the image processing unit 54 generates the peripheral image data by obtaining the pixel value of each pixel on the peripheral image using the electrical signal indicating the amount of the observation light received from the peripheral region.

  The microlens array 34 of the confocal microscope 11 converts the illumination light into a plurality of light beams so that a plurality of light beams as observation light from the sample 13 do not enter one pixel on the light receiving surface of the photodetector 52. To separate. For example, in FIG. 3, the illumination light is separated so that the region R12 and the region R14 do not overlap.

  More specifically, it is desirable that the distance between the light beams constituting the observation light be at least 10 times the distance ED of the air disk of the observation light. For example, when the wavelength of the observation light is λ and the numerical aperture of the second objective lens 53 is NA ′, the diameter ED of the air disk is obtained by the following equation (1).

  ED = 1.22 × (λ / NA ′) (1)

  Note that the numerical aperture NA ′ of the second objective lens 53 in Expression (1) is obtained by the following Expression (2), where NA is the numerical aperture of the objective lens 40 and β is the magnification of the objective lens 40.

  NA ′ = NA / β (2)

  In this way, when the illumination light applied to the sample 13 is separated into a plurality of light beams so that the light beams constituting the observation light are arranged at regular intervals, each light beam of the observation light is reliably detected by the photodetector 52. be able to.

  Further, the personal computer 12 generates observation image data having a predetermined predetermined sectioning resolution using the vicinity image data and the vicinity image data obtained by the image processing unit 54.

  Specifically, the personal computer 12 sequentially sets pixels of the observation image to be generated from now on as the target pixel. Then, the personal computer 12 performs the calculation shown in the following equation (3) from the pixel value Da of the pixel of the neighboring image at the same position as the target pixel and the pixel value Db of the pixel of the peripheral image at the same position as the target pixel. Then, the pixel value D of the target pixel is obtained.

  D = Da + (α × Db) (3)

  Note that the pixels in the vicinity image and the peripheral image at the same position as the target pixel refer to pixels in the vicinity image and the peripheral image in which the part of the sample 13 displayed on the target pixel is displayed. More specifically, the neighboring image pixels and the neighboring image pixels at the same position as the target pixel have different positions in the thickness direction, but the part of the sample 13 irradiated with one light beam constituting the illumination light, that is, the neighboring region and The peripheral area is a pixel to be displayed.

  In Expression (3), α is a variable that determines sectioning resolution, and −1 ≦ α ≦ 1. The smaller the variable α, the higher the sectioning resolution and the thinner the thickness direction of the portion of the sample 13 to be observed.

  For example, when the variable α is 0, D = Da, and an image in the vicinity of the sample 13 is displayed in the observation image. Further, when the variable α is 1, D = Da + Db, so that an image of the vicinity region and the peripheral region of the sample 13 is displayed in the observation image.

  Therefore, if the variable α is changed between 0 and 1 or less, any thickness that is thicker in the thickness direction of the sample 13 than the layer in the vicinity region and thinner than the layer in the region including the vicinity region and the peripheral region. An observation image of the layer region of the sample 13 can be obtained.

  Further, when the variable α is −1, D = Da−Db. Therefore, the observation image is obtained by subtracting a peripheral image containing a lot of information on the peripheral region from a neighborhood image containing a lot of information on the neighborhood region. It becomes. The calculation for obtaining the difference between the neighboring image and the neighboring image is based on the neighboring area information, that is, the component appearing blurred on the neighboring image while maintaining the S / N ratio (Signal to Noise ratio) of the data. This is equivalent to subtraction, and the sectioning resolution becomes higher. That is, in the observation image, an image of an area that is thinner in the thickness direction than the vicinity area of the sample 13 is displayed.

  Therefore, if the variable α is changed between −1 and 0 and 0 or less, the layer of the sample 13 having an arbitrary thickness that is thinner than the layer in the vicinity region in the thickness direction of the sample 13 around the observation surface 13s. An observation image of the area can be obtained.

  In this way, if the observation image is generated while changing the variable α in accordance with the target sectioning resolution, an observation image having an arbitrary sectioning resolution can be easily obtained simply by performing arithmetic processing using the neighboring image and the peripheral image. Obtainable.

  That is, arranging the light detector 52 at a position substantially conjugate with the observation surface 13 s and changing the variable α to generate an observation image is substantially the same as the observation surface 13 s between the sample 13 and the light detector 52. This is equivalent to observing the sample 13 by changing a virtual pinhole provided at a conjugate position.

  Note that, depending on the value of the variable α, the pixel value D of the target pixel may be a negative value or a value exceeding the dynamic range of the observation image. In such a case, the personal computer 12 appropriately determines the pixel value of the pixel of interest by setting the pixel value D to 0 or a predetermined value that does not exceed the dynamic range.

  As described above, according to the confocal microscope 11, the photodetector 52 is arranged at a position substantially conjugate with the observation surface 13s, and the neighborhood image data and the neighborhood image data are generated from the detection result of the observation light. The region of an arbitrary thickness of the sample 13 can be observed with a simple calculation process.

  That is, if the light receiving surface of the photodetector 52 is arranged at a position substantially conjugate with the observation surface 13s, the neighboring image data and the peripheral image data are obtained from the electrical signal output from a predetermined specific pixel on the light receiving surface. Can be obtained individually. Thereby, an observation image of an area of an arbitrary thickness of the sample 13 can be generated from the obtained image data by a simple arithmetic process.

  Therefore, the confocal microscope 11 does not need to be provided with a member for separating the observation light by a plurality of pinholes or a photodetector for each pinhole through which the observation light passes, and can observe the sample 13 with a simpler configuration. The sectioning resolution at the time can be changed.

  In addition, since the confocal microscope 11 separates the illumination light into a plurality of light beams and scans the observation surface 13s with these light beams, information on a plurality of parts of the sample 13 can be obtained simultaneously. Conventionally, in the confocal microscope, scanning is performed by a method called so-called raster scan with one light beam as illumination light. However, since the confocal microscope 11 performs scanning with a plurality of light beams at the same time, the raster scan is performed. As a result, an observation image can be obtained more quickly.

  Further, when the illumination light is separated, the illumination light is separated so that a plurality of light beams are arranged at a constant interval. Therefore, in the photodetector 52, observation light from a plurality of parts of the sample 13 is incident on the same pixel. The information on each part can be reliably extracted without any problem.

  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.

  11 confocal microscope, 12 personal computer, 13 sample, 23 illumination optical system, 34 microlens array, 36 galvano scanner, 39 dichroic mirror, 40 objective lens, 51 imaging optical system, 52 photodetector, 53 second objective lens , 54 Image processing unit

Claims (3)

An illumination optical system that focuses the illumination light on the observation surface of the sample to be observed;
A scanner included in the illumination optical system for scanning the illumination light on the observation surface;
An imaging optical system for condensing the observation light from the sample;
A light detecting means that is disposed at a position substantially conjugate with the condensing position of the illumination light in the sample, and detects the observation light;
Based on the detection result by the light detection means, the first image data based on the observation light from the region near the focusing position in the sample and the observation light from the peripheral region of the vicinity region in the sample. Image generating means for generating second image data based thereon;
Computation means for generating, based on the first image data and the second image data, third image data having a sectioning resolution different from that of the first image data and the second image data. A confocal microscope. 2. The separating apparatus according to claim 1, further comprising a separating unit that constitutes the illumination optical system and separates the illumination light into a plurality of light beams arranged at a predetermined interval in a direction perpendicular to a traveling direction of the illumination light. Confocal microscope. The light detection means detects the observation light incident on a predetermined area on the light receiving surface of the observation light provided on the light detection means as the observation light from the vicinity area, and detects the predetermined light on the light receiving surface. The confocal microscope according to claim 1, wherein the observation light incident on a region surrounding the region is detected as the observation light from the peripheral region.

Images