JP5019279B2 - Confocal microscope and method for generating focused color image - Google Patents

Description

  The present invention relates to a confocal microscope capable of acquiring a focused color image of a sample, and a method for generating a focused color image in the confocal microscope.

  Conventionally, as a confocal microscope, light from a point light source is focused on a sample by an objective lens, the focused point is optically two-dimensionally scanned using a scanner, and light from the sample is detected through the objective lens. A confocal scanning microscope is known in which two-dimensional image information is obtained by detecting with a scanner.

  FIG. 7 shows a schematic configuration of an example of such a confocal scanning microscope. A light beam emitted from a light source 101 is transmitted through a beam splitter 102 and then enters a two-dimensional scanning mechanism 103 via a mirror 105. To do. The two-dimensional scanning mechanism 103 scans and outputs a light beam two-dimensionally. The light beam scanned two-dimensionally is guided to the objective lens 106 through the plurality of lenses 104. The light beam incident on the objective lens 106 is emitted as convergent light, and scans the surface of the sample 107. The light reflected from the surface of the sample 107 is again introduced from the objective lens 106 to the beam splitter 102 via the two-dimensional scanning mechanism 103 and the mirror 105, reflected by the beam splitter 102, and formed on the pinhole 109 by the imaging lens 108. Condensate. In this case, since the condensing position by the objective lens 106 is in an optically conjugate position with the pinhole 109, the light from the surface of the sample 107 focused on the surface of the sample 107 passes through the pinhole 109, Light from the sample 107 surface with defocusing hardly passes through the pinhole 109. As a result, only the light passing through the pinhole 109 is detected by the photodetector 110, and a signal corresponding to the intensity of the detected light is output to the computer 111. In this case, the sample 107 is placed on a Z stage 112 that can move in the Z-axis direction, and the Z stage 112 can move in the optical axis direction (Z direction). Further, the two-dimensional scanning mechanism 103, the Z stage 112, and the photodetector 110 are controlled by a computer 111.

  Accordingly, if the two-dimensional scanning mechanism 103 two-dimensionally scans the focal point on the surface of the sample 107 and images the output from the photodetector 110 in synchronization with the scanning by the two-dimensional scanning mechanism 103, Only a specific height is imaged, and an image (confocal image) obtained by optically slicing the sample 107 is obtained. Also, from this state, the sample 107 is moved in the optical axis direction (Z direction) by the Z stage 112 and scanned at each position by the two-dimensional scanning mechanism 103 to obtain a confocal image. Thus, by detecting the position of the Z stage 112 at which the output of the photodetector 110 is maximized, the height information of the sample 107 can also be obtained. The surface shape of the sample 107 obtained in this way is displayed as a three-dimensional image that does not include color information.

  By the way, it is known that the color information of the sample is also important for observing the sample in detail. For this reason, a confocal scanning microscope that can acquire color information of a sample has been conventionally considered. For example, Patent Document 1 includes a confocal optical system that acquires a two-dimensional image using laser light and a non-confocal optical system that acquires a two-dimensional image using white light, and receives the confocal optical system using laser light. An apparatus for creating a color confocal image from luminance information obtained from an element and color information obtained from an imaging element (CCD) of a non-confocal optical system using white light is disclosed.

  However, in Patent Document 1, a laser drive circuit that drives a laser beam and a CCD drive circuit that drives an imaging device (CCD) are alternately arranged at all height positions on the sample 107 for acquiring a two-dimensional image. It is necessary to drive the laser and stop the laser driving every time the image pickup device (CCD) is picked up at each height position, or to shield the laser light by a light shielding means such as a shutter. For this reason, there arises a problem that the number of images taken in the optical axis direction increases and the imaging operation becomes complicated. The acquired color image is created for each pixel by using color information from the non-confocal optical system at the position where the luminance information from the confocal optical system is acquired. The in-focus position may be shifted due to the difference in chromatic aberration between the system and the non-confocal optical system. In particular, when the lens magnification is high, the depth of focus becomes small, so that the influence on the result of defocusing of each optical system cannot be ignored. Therefore, there is a problem that the color image generated by the method of Patent Document 1 may include blur.

Therefore, as disclosed in Patent Document 2, after acquiring confocal image signals at a plurality of stage positions by a confocal optical system, color image data at a plurality of stage positions is acquired by using a CCD camera, and these common images are obtained. An apparatus for generating a colored three-dimensional image using a focus image signal and color image data has been proposed. Patent Document 2 proposes an attempt to reduce the number of images by performing imaging with a CCD camera at an interval wider than the height interval of confocal image acquisition.
Japanese Patent Laid-Open No. 11-14907 JP 2004-29537 A

  However, since the thing of such patent document 2 produces the color information that the color data becomes the maximum about each pixel based on all the color image data acquired by the imaging of the CCD camera, for example, For a position where the brightness is saturated, the color data is maximized, and there is a possibility that the position is in focus. Although the position where the color data is maximized is not necessarily the position where the focus is achieved, it may be erroneously determined as the position where the focus is achieved. There is a problem that it is difficult to obtain a color image that is out of focus and in focus.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a confocal microscope and a method for generating a focused color image that can efficiently generate a high-quality focused color image that does not include blur in a short time. .

The invention according to claim 1 is a method for generating a focused color image applied to a confocal microscope capable of acquiring a focused color image of a sample, wherein the relative position between the focusing position of the objective lens and the sample is determined. A first step of obtaining a confocal image in the direction of the optical axis of the objective lens, and obtaining height information of a focus position for each pixel position of each point of the sample based on the confocal image; A second step of capturing a color image by changing a relative position between the focusing position of the objective lens and the sample in the optical axis direction of the objective lens; and the color image captured by the second step of the sample. With respect to the pixel position of each point, a focus determination is performed based on the height information corresponding to the pixel position, and a focused color image is acquired from the determination result, and the third step Acquired in-focus color It is characterized by comprising a fourth step of generating a three-dimensional color image of the sample using the height information and the image.

  According to a second aspect of the present invention, in the first aspect of the invention, the third step is obtained for the pixel position of each point of the sample by the height information of the pixel position and the first step. The focus determination start is determined based on the height information corresponding to the pixel position.

  According to a third aspect of the present invention, in the first aspect of the invention, the third step is a height corresponding to the pixel position obtained in the first step with respect to the pixel position of each point of the sample. Based on the information, a range to be used for the focus determination in the third step is determined among the color images captured in the second step, and focus determination target regions are determined for a plurality of color images in the range. It is characterized in that the focus determination is performed on these focus determination target areas.

  According to a fourth aspect of the present invention, in the third aspect of the invention, the focus determination in the third step is to obtain a focus evaluation value for each of a plurality of focus determination target regions, and the focus evaluation value. Among the above, the pixel of interest in the focus determination target region having the maximum value is determined as the focus position.

According to a fifth aspect of the present invention, there is provided a confocal image acquisition means for acquiring a confocal image by changing the relative position between the objective lens for condensing the light from the light source on the sample and the sample in the optical axis direction of the objective lens. Height information acquisition means for obtaining height information of the in-focus position for each pixel position of each point of the sample based on the plurality of confocal images, and a relative position between the focusing position of the objective lens and the sample Color image acquisition means for picking up a color image by changing the position in the optical axis direction of the objective lens, and the pixel position of each point of the sample of the color image picked up by the color image acquisition means A focus color image acquisition unit that performs focus determination based on the corresponding height information and acquires a focus color image from the determination result; a focus color image acquired by the focus color image acquisition unit; Combining the height information It is characterized by comprising a three-dimensional color image generation hand handling generating a three-dimensional color image of the sample Te.

  According to a sixth aspect of the present invention, in the fifth aspect of the invention, the focused color image acquisition unit is configured to obtain height information of the pixel position and the height information acquisition unit for the pixel position of each point of the sample. And a focus determination start determining means for determining the start of the focus determination based on the height information corresponding to the pixel position obtained in step (1).

  According to a seventh aspect of the invention, in the fifth aspect of the invention, the focused color image acquisition unit corresponds to the pixel position obtained by the height information acquisition unit with respect to the pixel position of each point of the sample. Based on the height information to be determined, a range to be used for the focus determination among the color images captured by the color image acquisition unit is determined, and a focus determination target region is specified for a plurality of color images within the range. In addition, the image forming apparatus includes a focus determination unit that performs focus determination on these focus determination target areas.

  The invention according to claim 8 is the invention according to claim 7, wherein the focus determination means obtains a focus evaluation value for each of the plurality of focus determination target areas, and the value is the value among the focus evaluation values. It is characterized in that the target pixel in the focus determination target region where the amount of is determined as the focus position is determined.

  According to the present invention, it is possible to provide a confocal microscope and a method for generating a focused color image that can efficiently generate a high-quality focused color image that does not include blur in a short time.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 shows a schematic configuration of a confocal scanning microscope to which a method for generating a focused color image according to an embodiment of the present invention is applied.

  In the figure, 1 is a light source, and the light source 1 is a point light source such as a laser beam. A two-dimensional scanning mechanism 3 is disposed on the optical path of light from the light source 1 via a shutter 2. The shutter 2 enables light from the light source 1 to be blocked and is controlled by a processing device 10 to be described later. The two-dimensional scanning mechanism 3 has two mirrors (not shown) for deflecting light in two orthogonal directions, and two-dimensionally scans light on the surface of the sample 6 to be described later by these mirrors. Yes.

  A beam splitter 4 is disposed on the optical path of the light emitted from the two-dimensional scanning mechanism 3. The beam splitter 4 has such characteristics that it reflects light (laser light) from the light source 1 and transmits light (white light) from a white light source 11 described later.

  An objective lens 5 is disposed on the optical path of the light from the two-dimensional scanning mechanism 3 reflected by the beam splitter 4. The objective lens 5 converges the incident light and causes the convergent light to be two-dimensionally scanned in the plane direction (X and Y directions in the drawing) perpendicular to the optical axis on the surface of the sample 6 by the operation of the two-dimensional scanning mechanism 3.

  The sample 6 is placed on the stage 7. The stage 7 is movable in the optical axis direction (direction orthogonal to the surface of the stage 7), that is, in the Z direction. In this case, the stage 7 is controlled by the processing apparatus 10 so that the relative position of the sample 6 with respect to the objective lens 5 can be continuously changed in the Z-axis direction.

  Reflected light from the sample 6 passes through the objective lens 5, is reflected again by the beam splitter 4, and returns to the two-dimensional scanning mechanism 3. On the optical path of the reflected light returned to the two-dimensional scanning mechanism 3, a photodetector 9 is disposed via a pinhole 8. The pinhole 8 is disposed at a position optically conjugate with the focal point of the objective lens 5 and allows a focused component of light reflected from the sample 6 to pass therethrough but prevents a non-focused component from being transmitted. ing. The photodetector 9 comprises a photomultiplier or the like, detects light that has passed through the pinhole 8, converts the luminance of this light into an electrical signal, and outputs it. In this case, the light detected by the light detector 9 has the maximum luminance when the sample 6 is focused, and conversely, the luminance is extremely small when the sample 6 is out of focus. .

  A processing device 10 is connected to the photodetector 9. An electrical signal corresponding to the luminance of the light detected by the photodetector 9 is input to the processing device 10.

  On the other hand, reference numeral 11 denotes a white light source. As the white light source 11, for example, a light source such as halogen or mercury is used. A beam splitter 12 is disposed on the optical path of light from the white light source 11. The beam splitter 12 has such characteristics that it reflects light (white light) from the white light source 11 and transmits light (white light) reflected from the sample 6.

  On the optical path of the light from the white light source 11 reflected by the beam splitter 12, the above-described beam splitter 4 is disposed via a shutter 13. The shutter 13 can block light from the white light source 11. Further, the shutter 13 has an exclusive relationship with the shutter 2 described above, and the processing device 10 is configured such that the shutter 2 is closed when the shutter 13 is open, and conversely, the shutter 13 is closed when the shutter 2 is open. Controlled by.

  The light from the white light source 11 that has passed through the shutter 13 passes through the beam splitter 4 and the objective lens 5 described above, and is irradiated onto the sample 6 on the stage 7. The white light reflected from the sample 6 is transmitted through the objective lens 5, the beam splitter 4, the shutter 13, and the beam splitter 12. A color camera 14 is disposed on the optical path of the light transmitted through the beam splitter 12 via an imaging lens (not shown). The color camera 14 includes a CCD camera and captures a color image of the sample 6 formed by an imaging lens (not shown).

  A processing device 10 is connected to the color camera 14. The processing apparatus 10 includes a control unit 10a, a data storage unit 10b, and a data processing unit 10c. The control unit 10a controls the two-dimensional scanning mechanism 3, the stage 7, the shutters 2 and 13, and the color camera 14 described above. The data storage unit 10 b stores data such as the luminance of light from the sample 6 detected by the photodetector 9 and the captured image of the color camera 14. The data processing unit 10c obtains luminance information, height information, a focused color image, a three-dimensional focused color image, and the like based on the data stored in the data storage unit 10b.

A display device 15 is connected to the processing device 10. The display device 15 displays various data obtained by the processing device 10.
Next, a description will be given of generation of a focused color image by the confocal scanning microscope configured as described above. In the following description, all expressions relating to movement of the stage indicate movement in the Z direction.

First, acquisition of a confocal image will be described.
In this case, the flowchart shown in FIG. 2 is executed. First, in step 201, the shutter 2 is opened and the shutter 13 is closed according to an instruction from the control unit 10a of the processing apparatus 10, and the operation mode is switched to a confocal image acquisition mode using a confocal optical system. Next, in step 202, the stage 7 is moved to a predetermined start position.

  Next, in step 203, a confocal image is acquired for the first stage position Lm (L0) as the stage position Lm (m = 0, 1, 2,...) Corresponding to the designated start position. In this case, the light from the light source 1 enters the two-dimensional scanning mechanism 3 through the shutter 2. The light beam scanned two-dimensionally by the two-dimensional scanning mechanism 3 is reflected by the beam splitter 4, passes through the objective lens 5, becomes convergent light, and is a surface direction perpendicular to the optical axis on the surface of the sample 6 (X and Y directions in the drawing). Two-dimensionally scanned. The reflected light from the sample 6 returns to the two-dimensional scanning mechanism 3 through the objective lens 5 and the beam splitter 4, is condensed on the pinhole 8, and the light that has passed through the pinhole 8 is detected by the photodetector 9. The The light detected by the photodetector 9 is stored in the data storage unit 10b and then imaged by the data processing unit 10c. This image is acquired as an optically sliced image (confocal image) at the stage position Lm (L0). This confocal image is represented by Limage_Lm, and the luminance value at each pixel position (i, j) is Limage_Lm (i, j). That is, Image_Lm (i, j) represents the luminance value of the pixel position (i, j) of each point on the surface of the sample 6 at the stage position Lm (L0).

  Next, in step 204, the confocal image Image_Lm is compared with the maximum luminance image LMax (initially LMax = 0) stored in the data storage unit 10b of the processing apparatus 10 for each pixel position, and LMax and high The height map HighMap is updated. Specifically, if the value of Lmax_Lm (i, j) is larger than LMax (i, j), the value of LMax (i, j) is replaced with the value of Lage_Lm (i, j), and the height map HighMap (i, j) Replace the value with Lm-L0.

  Next, in step 205, the stage 7 is moved in a predetermined direction at a predetermined interval. Next, the process proceeds to step 206, where it is determined whether or not the stage position has reached a predetermined end position. Here, if it is not the designated end position, the process returns to step 203, and for the newly designated stage positions L1, L2,... Lm, acquisition of the confocal image and height map HighMap (i, j) are performed in the same manner as described above. ) Repeat the update. Thereafter, when the end position is determined in step 206, acquisition of the confocal image is ended.

  Through the series of operations as described above, the position (focusing position) of the stage 7 at which the luminance output of the photodetector 9 is maximized at each point on the sample 6 is obtained by acquiring a confocal image at each stage position Lm. It is detected and stored in the data storage unit 10b as height information (height map HighMap) of each point of the sample 6.

Next, color image acquisition and in-focus color image generation will be described.
In this case, the flow chat shown in FIG. 3 is executed. First, in step 301, the shutter 13 is opened and the shutter 2 is closed in accordance with an instruction from the control unit 10a of the processing apparatus 10, and the operation mode is switched to a non-confocal image acquisition mode in which imaging is performed by the color camera 14.

  Next, in step 302, the height information (height map HighMap) stored in the data storage unit 10b of the processing apparatus 10 is read into the data processing unit 10c. Next, in step 303, the stage 7 is moved to a predetermined start position.

  Next, in step 304, the first stage position C0 is imaged by the color camera 14 as the stage position Cn (n = 0, 1, 2,...) Corresponding to the designated start position. In this case, the light from the white light source 11 is reflected by the beam splitter 12 and irradiated onto the sample 6 via the shutter 13, the beam splitter 4, and the objective lens 5. The white light reflected from the sample 6 passes through the objective lens 5, the beam splitter 4, the shutter 13, and the beam splitter 12, and forms an image on the imaging surface of the color camera 14 through an imaging lens (not shown). Is done. The captured color image is stored in the data storage unit 10b as Image_C0.

  Next, in step 305, with respect to the color image acquired in step 304, the focus determination of each pixel is performed by a focus determination method described later with reference to height information (height map HighMap). Next, in step 306, the stage 7 is moved in a predetermined direction at predetermined intervals. Next, the process proceeds to step 307, where it is determined whether or not the stage position has reached an end position designated in advance. Here, if it is not the designated end position, the process returns to step 304, and the newly designated stage positions C1, C2,... Cn are referred to the color image acquisition and height map HighMap as described above, Repeat the pixel focus determination. Thereafter, when the end position is determined in step 307, in step 308, a single in-focus color image is created using the focus determination result of each pixel so far, and the process is terminated.

Next, the method for obtaining a focused color image will be further described.
In this case, FIG. 4 illustrates the flow from the acquisition of the confocal image to the acquisition of the color image. First, the confocal image is moved while moving the stage 7 upward from the predetermined start position L0 by a predetermined interval. When the end position is reached, the process returns to the start position L0, and this time, a color image is acquired while moving the stage 7 upward from the stage position C0 corresponding to the start position L0 by a predetermined interval. Here, a point a on the sample 6 in the drawing corresponds to a common pixel position (u, v) of each confocal image and each color image.

  Then, the height map High (u, v) of the pixel position (u, v) is obtained by acquiring the confocal image. This map High (u, v) is a distance Lm-L0 between L0 and Lm shown in FIG. 4, and the pixel position (u, v) at this time is a relative distance High (u, v) from the start position L0. ) It is represented by a remote stage position Lm.

  In Patent Document 1 described above, in-focus color data for each pixel is created using color information of a color image captured at the same stage position Lm as the stage position Lm at which the confocal image was captured. However, the focus position of the confocal optical system and the non-confocal optical system may shift due to the influence of chromatic aberration or the like. For example, in the case of FIG. 4, the focus position of the pixel position (u, v) of the non-confocal optical system is not Lm at the relative distance High (u, v) from L0 obtained by the confocal optical system. , Cn at a relative distance Cn−L0 (C0) from L0 (C0). In such a case, the in-focus color data created by the method disclosed in Patent Document 1 includes blur.

  In contrast, in the present invention, the height map High (u, v) of the pixel position (u, v) is obtained from the confocal image, and then the sample 6 is obtained while acquiring the color image by the non-confocal optical system. In-focus determination for each pixel is performed, and one in-focus color image is generated from the in-focus information of each pixel obtained from the in-focus determination. These processes are performed in steps 305 and 308 in FIG. 3 described above.

  FIG. 5 explains the process in step 305 of FIG. 3 in more detail. Here, the pixel position (u, v) corresponding to the point a on the sample 6 is the object of explanation. In this case, all the acquired color images may be used for the focus determination of the pixel position (u, v) of the point a, but the stage position is [L0 + High (u, v) as shown in FIG. Only a plurality of color images acquired within a range from −α] to [L0 + High (u, v) + α] may be used. Then, for each of a plurality of color images within this range, a 3 × 3 pixel area centered on the pixel position (u, v) of the point a is extracted, and these areas A1, A2,. The target of position (u, v) is in focus determination.

  Here, α is a predetermined value in consideration of the chromatic aberration between the confocal optical system and the non-confocal optical system and the mechanical error of the stage movement. If [L0 + High (u, v) −α] is smaller than the start position of the color image acquisition of the stage due to the value of α, the focus determination range is determined from the start position of the color image acquisition of the stage. , [L0 + High (u, v) + α] is larger than the color image acquisition end position, the focus determination range is set to the end color image acquisition end position. The 3 × 3 size of the pixel area described above is not limited to this, and can be arbitrarily changed. Further, the value of α can be set within the range of the focal depth of the non-confocal optical system.

  In FIG. 5, first, in step 501, the height information of the pixel position (u, v) of the point a, that is, the stage position Cn (relative position between the objective lens 5 and the sample 6) and the pixel position (u, v). ) To determine whether or not the focus determination of the pixel at the point a can be started. In this case, as shown in FIG. 6, the current position of the stage 7 representing the height of the pixel position (u, v) at the point a, that is, the stage position Cn reaches [L0 + High (u, v) + α] or the end position. Determine if you did. If this condition is not satisfied, it is determined that the focus determination of the pixel at the point a cannot be started, and the process proceeds to step 506. In step 506, it is determined whether or not it has been determined whether or not the focus determination can be started at a pixel other than the point a at the current position of the stage 7. Here, when it is not determined whether the focus determination can be started in other pixels, the process returns to step 501 and the same processing is repeated for the pixels for which determination has not been performed.

  On the other hand, in step 501, if the condition for processing start is satisfied, the process proceeds to step 502, and calculation of focus determination for this pixel is started. In this case, pixel positions (u, v) are obtained from a plurality of color images already acquired within the range from [L0 + High (u, v) −α] to [L0 + High (u, v) + α] shown in FIG. The 3 × 3 pixel areas A1, A2,... Ak at the center are extracted and read into the data processing unit 10c of the processing apparatus 10.

  Next, in step 503, for the first region A1, the luminance signal is separated from the R, G, and B signals of each pixel. Then, using this luminance signal information, a focus evaluation value for determining focus is obtained. Specifically, using this luminance information, a Laplacian filter process is performed around the pixel position (u, v), and the Laplacian filter process result at this pixel position (u, v) is used as the focus evaluation value of the area A1. To do. Note that Laplacian filter processing is a method of determining a value of a target pixel (the above-described focus evaluation value) based on the target pixel (the above-described pixel position (u, v)) and a pixel adjacent thereto. This is a technique performed in general image processing. Here, the Laplacian filter process is used to obtain the focus evaluation value. However, as other processing methods, for example, a primary differential filter process, a Sobel filter process, a Fourier transform method, or the like may be used.

  Similarly, focusing evaluation values for the respective evaluations are obtained for the other areas A2,. The focus evaluation value obtained in this way can be considered as a contrast value relating to the pixel position (u, v), and the larger the contrast value, the more in-focus position.

  Next, in step 504, a region showing a large amount of contrast value among the focus evaluation values of the plurality of regions A1, A2,... Ak is determined as the in-focus position. The color information of the pixel at the focus position is stored as the color information of this pixel position. Note that pixels for which color information has already been determined are determined to be in focus.

  Then, the process proceeds to step 506, where it is determined whether or not it is possible to start focusing determination with other pixels, and it is determined whether or not focusing determination can be started with other pixels. If not, the process returns to step 501, and it is determined whether or not the focus determination can be started for the pixels that have not been determined, and thereafter the same processing is repeated. Through such a series of operations, the focus determination of each pixel corresponding to the current position of the stage 7 is performed, and the focus determined pixels for which the focus determination has been performed are stored in the data storage unit 10b. After the completion of these processes, the process returns to the flowchart shown in FIG. 3 and the operations in and after step 306 are executed, and each time the stage 7 moves, the color information of the incompletely focused pixels corresponding to each stage position Cn is obtained. get.

  Thereafter, one focused color image is created using the color information of each pixel stored in the data storage unit 10b. Then, the focused color image is combined with height information (height map HighMap) by the data processing unit 10 c, and a three-dimensional color image of the sample 6 is generated and displayed on the display device 15.

  Note that the start position, end position, and movement interval of the stage 7 at the time of confocal image acquisition do not have to coincide with the start position, end position, and movement interval of the stage 7 at the time of color image acquisition, respectively. For example, since the depth of focus of the objective lens 5 of the non-confocal optical system is larger than the depth of focus of the objective lens 5 of the confocal optical system, the stage movement interval for acquiring the color image is set to the stage for acquiring the confocal image. It can be larger than the movement interval. In this case, the stage moving interval may be set to a value smaller than the focal depth of the objective lens 5 of the non-confocal optical system. In this way, the color image acquisition time can be shortened. Further, if the stage moving direction at the time of confocal image acquisition and the stage moving direction at the time of color image acquisition are reversed, only one reciprocation of the stage is required, and the overall operation time can be shortened.

  Therefore, in this way, a confocal image is acquired while changing the relative position between the focusing position of the objective lens 5 and the sample 6 by the confocal optical system, and each point of the sample 6 is obtained based on these confocal images. The height information of the in-focus position is obtained for each pixel position, and then a color image is captured while changing the relative position between the focusing position of the objective lens 5 and the sample 6 by the non-confocal optical system, and these images are captured. Focus determination is performed on the pixel position of each point of the sample 6 of the color image based on height information corresponding to the pixel position, and in-focus color information is obtained from the determination result. The height information is synthesized to generate a three-dimensional color image of the sample 6. Thereby, the focus determination is performed for each pixel position imaged by the non-confocal optical system, and a focus color image is generated based on the focus color information acquired from the result of the focus determination. A strictly focused color image, that is, a high-quality color image that does not include so-called blur can be generated.

  Further, in the in-focus determination of a plurality of color images, the use range of the color image is determined using the height information as reference data, and the evaluation for the in-focus determination is performed on the plurality of color images captured within this range. Therefore, the amount of processing data can be minimized by using only data within a predetermined range for in-focus determination, thereby reducing processing time and generating in-focus color images efficiently in a short time. can do.

  Furthermore, since the focus determination process can be performed while capturing a color image, for example, when the stage movement is completed, the data processing is almost completed, and the entire system can be speeded up.

  In the above-described embodiment, the focused color image generation method in the confocal microscope is described. However, in the confocal microscope to which the focused color image generation method is applied, the confocal image acquisition for acquiring the confocal image is performed. Means, height information acquisition means for obtaining height information of the focus position for each pixel position of each point of the sample 6 based on the confocal image, color image acquisition means for acquiring a color image, and the sample 6 in the color image. A focused color image acquisition unit and a focused color image acquisition unit that perform focusing determination on the pixel position of each point based on the height information corresponding to the pixel position and acquire a focused color image from the determination result A focused color image generating means for generating a three-dimensional focused color image of the sample by synthesizing the focused color image and the height information acquired in step 1, height information of the pixel position for each pixel position of the sample 6 When Focus determination start determining means for determining the start of focus determination based on height information corresponding to the pixel position obtained by the height information acquisition means, and further, the height information acquisition for the pixel position of each point of the sample 6 Based on the height information corresponding to the pixel position obtained by the means, a range to be used for focusing determination is determined among the color images captured by the color image acquisition means, and the plurality of color images in the range are focused. A focus determination unit that specifies a determination target region and performs focus determination on the focus determination target region, obtains a focus evaluation value for each of the plurality of focus determination target regions, and determines the focus evaluation value. Means for determining the focus pixel of the focus determination target area having the maximum value among the focus positions is provided.

  In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary.

  Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.

The figure which shows schematic structure of the confocal scanning microscope to which the production | generation method of the focused color image concerning one embodiment of this invention is applied. The flowchart which shows the acquisition procedure of the confocal image of one embodiment. The flowchart which shows the color image of one Embodiment, and a focus color image production | generation procedure. The figure which shows the example of the shift | offset | difference of the focus position of the confocal image and color image of one Embodiment. The flowchart which shows the procedure which calculates | requires the focus color image of one Embodiment. The figure for demonstrating the focus determination of one Embodiment. The figure which shows schematic structure of the conventional confocal microscope.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Light source, 2 ... Shutter 3 ... Two-dimensional scanning mechanism, 4 ... Beam splitter 5 ... Objective lens, 6 ... Sample 7 ... Stage, 8 ... Pinhole 9 ... Photo detector, 10 ... Processing apparatus 10a ... Control part, 10b Data storage unit 10c Data processing unit 11 White light source 12 Beam splitter 13 Shutter 14 Color camera 15 Display device

Claims (8)

A focused color image generation method applied to a confocal microscope capable of acquiring a focused color image of a sample,
A confocal image is obtained by changing the relative position between the focusing position of the objective lens and the sample in the optical axis direction of the objective lens, and focusing is performed for each pixel position of each point of the sample based on the confocal image. A first step for determining position height information;
A second step of capturing a color image by changing a relative position between the focusing position of the objective lens and the sample in the optical axis direction of the objective lens ;
The focus determination is performed on the pixel position of each point of the sample of the color image captured in the second step based on the height information corresponding to the pixel position, and the focused color image is determined based on the determination result. A third step of obtaining
A fourth step of generating a three-dimensional color image of the sample using the in-focus color image acquired in the third step and the height information;
A method for generating a focused color image in a confocal microscope.   In the third step, for the pixel position of each point of the sample, the focusing is performed based on the height information of the pixel position and the height information corresponding to the pixel position obtained in the first step. The focused color image generation method according to claim 1, wherein the start of the determination is determined.   In the third step, for the pixel position of each point of the sample, based on the height information corresponding to the pixel position obtained in the first step, the color image captured in the second step Of these, a range to be used for the focus determination in the third step is determined, a focus determination target region is specified for a plurality of color images within the range, and focus determination is performed for these focus determination target regions. 2. The method for generating a focused color image according to claim 1, wherein:   In the focus determination in the third step, a focus evaluation value is obtained for each of the plurality of focus determination target areas, and the target pixel of the focus determination target area that has the maximum value among the focus evaluation values The in-focus color image generating method according to claim 3, wherein the in-focus position is determined. A confocal image acquisition means for acquiring a confocal image by changing the relative position of the objective lens that collects light from the light source on the sample and the sample in the optical axis direction of the objective lens ;
Height information acquisition means for obtaining height information of a focus position for each pixel position of each point of the sample based on the plurality of confocal images;
Color image acquisition means for capturing a color image by changing the relative position between the focusing position of the objective lens and the sample in the optical axis direction of the objective lens ;
A focus determination is performed on the pixel position of each point of the sample of the color image captured by the color image acquisition unit based on the height information corresponding to the pixel position. In-focus color image acquisition means for acquiring
Characterized by comprising a three-dimensional color image generation Te扱that by combining the height information and focus color image acquired by the in-focus color image acquisition means to generate a three-dimensional color image of the sample Confocal microscope.   The in-focus color image acquisition means is based on the height information corresponding to the pixel position obtained by the height information acquisition means and the height information of the pixel position for the pixel position of each point of the sample. The confocal microscope according to claim 5, further comprising: a focus determination start determination unit that determines start of the focus determination.   The focused color image acquisition unit captures the pixel position of each point of the sample with the color image acquisition unit based on height information corresponding to the pixel position obtained by the height information acquisition unit. Of the color images, a range to be used for the focus determination is determined, a focus determination target region is specified for a plurality of color images within the range, and focus determination is performed on these focus determination target regions. The confocal microscope according to claim 5, further comprising a focus determination unit.   The focus determination unit obtains a focus evaluation value for each of the plurality of focus determination target areas, and focuses a target pixel in the focus determination target area whose value is large among the focus evaluation values. The confocal microscope according to claim 7, wherein the confocal microscope is determined as a position.

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