JP2008051773A - Fluorescence image acquisition device and fluorescence image acquisition method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence image acquisition device and a fluorescence image acquisition method capable of acquiring suitably a fluorescence observation image of a sample with high resolution. <P>SOLUTION: This fluorescence image acquisition device has a configuration equipped with a sample stage 15, an image acquisition part 30 including an imaging device 31 for acquiring an image using a prescribed direction as a longitudinal direction, a fluorescence optical system unit 44 including a dichroic mirror 45 for vertical illumination by excitation light from an excitation light source 40, a scanning part for acquiring a partial image by scanning the sample S by the imaging device 31 repeatedly in a plurality of times, an image synthesis part for generating the fluorescence observation image of the sample S by synthesizing a plurality of partial images, a correction data storage part 76 for storing shading correction data in correspondence with the optical system unit 44, and an image correction processing part 75 for performing shading correction to each of the plurality of partial images on reference to the shading correction data. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a fluorescence image acquisition device and a fluorescence image acquisition method for acquiring a fluorescence observation image of a sample.

  In recent years, in the field of pathology and the like, a virtual microscope that can be operated as if a sample is being viewed with an actual microscope in a virtual space such as a personal computer is known. The sample data handled by this virtual microscope is based on image data of a sample acquired in advance with high resolution using an actual microscope.

As described above, an image acquisition apparatus that acquires image data of a sample used in a virtual microscope is required to acquire an image of the sample with a sufficiently high resolution in order to realize an image operation with the virtual microscope. For such high-resolution image acquisition, for example, a method of acquiring an observation image for the entire sample by scanning the sample two-dimensionally with an imaging device is used. In addition to the observation image of the sample stained with the light-absorbing dye, the sample that is the target of image acquisition with such an image acquisition device is a fluorescence that targets the sample stained with the fluorescent dye. Acquisition of observation images has been studied (for example, see Patent Document 1: Japanese Patent Laid-Open No. 2005-164815, Patent Document 2: Japanese Patent Laid-Open No. 2004-514920).
JP 2005-164815 A JP-T-2004-514920 JP 2004-347454 A

  As the above-described high-resolution image acquisition method, specifically, for example, an imaging device that acquires an image having a predetermined direction as a longitudinal direction is used, and a sample is scanned in a direction orthogonal to the longitudinal direction by the imaging device. There is a method for acquiring a plurality of partial images by acquiring a partial image and repeating the acquisition of the partial image a plurality of times while shifting the imaging position. In this case, it is possible to obtain a fluorescence observation image of a high-resolution sample by arranging the obtained partial images side by side.

  On the other hand, in a fluorescence microscope for acquiring a fluorescence observation image of a sample, the sample is irradiated with excitation light using a dichroic mirror provided between a sample stage on which the sample is placed and an imaging device for acquiring the image. An epi-illumination configuration is used. In such a configuration, shading occurs in an obtained image due to an excitation light irradiation optical system including a dichroic mirror, an imaging device used for image acquisition, or the like (Patent Document 3: Japanese Patent Application Laid-Open No. 2004-347454). reference). The effect of such shading is noticeable when, for example, a fluorescence observation image is generated by combining a plurality of partial images as described above, such as an unnatural image change at the boundary between adjacent partial images. appear.

  The present invention has been made to solve the above problems, and provides a fluorescence image acquisition apparatus and a fluorescence image acquisition method capable of suitably acquiring a fluorescence observation image of a sample at high resolution. For the purpose.

  In order to achieve such an object, the fluorescence image acquisition apparatus according to the present invention includes (1) a sample stage on which a sample to be subjected to fluorescence measurement is placed, and (2) a first fluorescence observation image of the sample. Image acquisition means including an imaging device for acquiring a one-dimensional image or a two-dimensional image whose direction is the longitudinal direction, (3) excitation light supply means for supplying excitation light for fluorescence measurement to the sample, and (4) A fluorescent optical system unit including a dichroic mirror that reflects the excitation light from the excitation light supply means to irradiate the sample and passes the fluorescence from the sample to the image acquisition means; The sample stage and the image are acquired so as to acquire a partial image by scanning in the direction of 2 and repeatedly acquiring the partial image a plurality of times while shifting the imaging position along the first direction. Scanning means for adjusting the positional relationship with the acquisition means, (6) image synthesizing means for generating a fluorescence observation image of the sample by arranging and synthesizing a plurality of partial images in the first direction, and (7) an imaging device. Correction data storage means for storing the shading correction data for the image acquired in accordance with the fluorescence optical system unit, and (8) a plurality of partial images with reference to the shading correction data stored in the correction data storage means And an image correction processing means for performing shading correction for each of the above.

  In the fluorescence image acquisition method according to the present invention, (a) a sample placed on a sample stage is a target for fluorescence measurement, and a fluorescence observation image of the sample is obtained in the first direction by an imaging device included in the image acquisition unit. And (b) supplying excitation light for fluorescence measurement to the sample, reflecting the excitation light and irradiating the sample, An excitation light irradiation step of irradiating the sample with excitation light via a fluorescence optical system unit including a dichroic mirror that passes fluorescence from the sample to the image acquisition means; and (c) the sample is moved in the second direction by the imaging device. A sample stage that scans and acquires partial images, and acquires the partial images by repeating the acquisition several times while shifting the imaging position along the first direction. A scanning step for adjusting the positional relationship with the image acquisition means; (d) an image combining step for generating a fluorescence observation image of the sample by combining a plurality of partial images in a first direction; and (e) imaging. A correction data storage step for storing shading correction data for an image acquired by the apparatus in association with the fluorescence optical system unit; and (f) a plurality of portions with reference to the shading correction data stored in the correction data storage step. And an image correction processing step for performing shading correction for each of the images.

  In the fluorescent image acquisition device and the fluorescent image acquisition method described above, an image acquisition unit that acquires an image of a sample uses an imaging device that acquires an image having a first direction as a longitudinal direction. The imaging apparatus scans the sample a plurality of times while shifting the imaging position with the second direction as the scanning direction, and obtains a fluorescence observation image of the sample by synthesizing the obtained partial images. Thereby, it is possible to efficiently acquire a fluorescence observation image of the sample with sufficiently high resolution.

  Furthermore, the excitation light irradiation optical system for irradiating the sample with excitation light is configured as an epi-illumination optical system using a fluorescence optical system unit (fluorescence cube) including a dichroic mirror, and is adapted to the fluorescence optical system unit. Shading correction data is prepared, and shading correction is performed for each of a plurality of partial images. Here, in the fluorescence measurement of the sample, the fluorescence optical system unit to be used may be exchanged depending on the type of the target sample and the type of fluorescence measurement. On the other hand, according to the above configuration, it is possible to perform the optimum shading correction for each fluorescence optical system unit. This makes it possible to acquire a fluorescence observation image of the sample with high resolution in a suitable state.

  Regarding the scanning of the sample by the imaging apparatus, the fluorescence image acquisition apparatus has a second direction in which the scanning unit is set to a direction orthogonal to the first direction that is the longitudinal direction of the image acquired by the imaging apparatus. It is preferable that Similarly, in the fluorescence image acquisition method, in the scanning step, a direction orthogonal to the first direction that is the longitudinal direction of the image acquired by the imaging device is preferably the second direction that is the scanning direction of the sample.

  In the fluorescence image acquisition device, the correction data storage unit stores dark correction data for the image acquired by the imaging device in correspondence with the exposure time in the imaging device, and the image correction processing unit performs shading correction. In addition, it is preferable to perform dark correction on each of the plurality of partial images with reference to the dark correction data stored in the correction data storage means.

  Similarly, in the fluorescence image acquisition method, dark correction data for an image acquired by the imaging device is stored in correspondence with the exposure time in the imaging device in the correction data storage step, and shading correction is performed in the image correction processing step. In addition, it is preferable to perform dark correction on each of the plurality of partial images with reference to the dark correction data stored in the correction data storage step.

  In this way, by performing dark correction in addition to shading correction on an image acquired by the imaging apparatus, it is possible to obtain a fluorescence observation image of the obtained sample as a more accurate image. Also, by preparing dark correction data corresponding to the exposure time in image acquisition, optimal dark correction can be performed according to the set exposure time.

  Further, the image acquisition means used for acquiring the fluorescence observation image of the sample has a wavelength resolving optical system that decomposes fluorescence from the sample into three different wavelength components, and is decomposed by the wavelength resolving optical system as an imaging device. It is preferable to include three imaging devices that acquire fluorescence observation images based on the three wavelength components. In such a configuration, the three imaging devices acquire optical images of samples formed by different wavelength components (color components), respectively. Thereby, it becomes possible to acquire the fluorescence observation image in the color of the sample based on the optical image acquired by each of the three imaging devices.

  In this case, in the fluorescence image acquisition device, it is preferable that the correction data storage means store three types of shading correction data for images acquired by each of the three imaging devices. Similarly, in the fluorescence image acquisition method, it is preferable to store three types of shading correction data for images acquired by each of the three imaging devices in the correction data storage step. Thereby, in each of the three imaging devices included in the image acquisition unit, it is possible to acquire the fluorescence observation image of the sample at a high resolution in a suitable state.

  As for the imaging device in the image acquisition means, a one-dimensional sensor capable of acquiring a one-dimensional image having the first direction as the longitudinal direction, or acquiring a two-dimensional image having the first direction as the longitudinal direction and TDI driving. It is preferable to use a configuration which is a two-dimensional sensor capable of According to such a configuration, it is possible to efficiently obtain a fluorescence observation image of a sample with sufficiently high resolution by scanning the sample with a one-dimensional sensor or a TDI-driven two-dimensional sensor.

  In addition, the imaging device in the image acquisition unit includes a plurality of pixels arranged in an array, and a plurality of light detection units that output charges generated according to the amount of light incident on the pixels, and a plurality of light detection units. Charge transfer means having a plurality of partial charge transfer sections divided along the arrangement direction, each of the plurality of partial charge transfer sections being included in the light detection means. Alternatively, a configuration may be used in which charges from pixels in the predetermined light detection region are transferred in the output direction and output from the output end.

  In the imaging apparatus having the above-described configuration, the plurality of pixels each having an output end are provided with a plurality of pixels of the light detection means or charge transfer means to which charges from a plurality of vertical shift registers constituting the light detection means are input in parallel. A multi-tap structure divided into charge transfer units is employed. With such a configuration, reading of the charges generated in each pixel of the light detection means is speeded up. Further, by performing the shading correction on the obtained image as described above, an unnatural image change between adjacent taps can be prevented.

  According to the fluorescence image acquisition device and the fluorescence image acquisition method of the present invention, an image pickup device that acquires an image having the first direction as the longitudinal direction is used for image acquisition of the sample, and the sample is moved in the second direction by the image pickup device. Fluorescence optical system unit including a dichroic mirror is used as the optical system for irradiating the sample with excitation light. Fluorescent observation image of the sample at high resolution by configuring shading correction data corresponding to the fluorescence optical system unit and performing shading correction for each of the partial images Can be acquired in a suitable state.

  Hereinafter, preferred embodiments of a fluorescence image acquisition device and a fluorescence image acquisition method according to the present invention will be described in detail with reference to the drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the dimensional ratios in the drawings do not necessarily match those described.

  First, the overall configuration of the fluorescence image acquisition device will be described. FIG. 1 is a block diagram showing a configuration of an embodiment of a fluorescence image acquisition apparatus according to the present invention. The fluorescence image acquisition apparatus according to the present embodiment is a fluorescence microscope system used for acquiring a fluorescence observation image of a sample S with high resolution. The microscope apparatus 10 that acquires the fluorescence observation image of the sample S, and the microscope apparatus 10 And a control device 60 that performs control of image acquisition and the like. As the sample S to be subjected to fluorescence measurement, for example, a slide in which a biological sample such as a tissue section stained with a fluorescent dye is sealed on a slide glass when acquiring image data used in a virtual microscope ( An example is a preparation).

  The microscope apparatus 10 includes a sample storage unit 11, a macro image acquisition unit 20, and a micro image acquisition unit 30. The sample storage unit 11 is storage means configured to be capable of storing a plurality of samples (for example, a plurality of slides each sealed with a biological sample) S that are objects of image acquisition. The sample storage unit 11 is provided with a door 12 used for storing and taking out the sample S by an operator. In the present embodiment, an interlock mechanism 13 is provided to prevent the door 12 from being accidentally opened during image acquisition.

  The macro image acquisition unit 20 is a first image acquisition unit for acquiring a macro image that is a low-magnification image of the sample S. In the image acquisition unit 20, a macro image with a low resolution corresponding to the entire image of the sample S is acquired prior to acquisition of a micro image that is a fluorescence observation image. For example, the macro image is used as necessary in order to set an imaging condition when acquiring a fluorescence observation image of the sample S. Further, a macro light source 25 that irradiates light for generating a light image of the sample S at the time of macro image acquisition is installed in the macro image acquisition unit 20.

  On the other hand, the micro image acquisition unit 30 is a second image acquisition unit for acquiring a micro image that is a high-magnification image of the sample S. In the image acquisition unit 30, a micro image of the target sample S at a high resolution is acquired. In the present embodiment, as will be described later, the micro image acquisition unit 30 functions as an image acquisition unit including an imaging device for acquiring the fluorescence observation image of the sample S at a high resolution. Further, a micro light source 35 that irradiates light for generating a light image of the sample S at the time of micro image acquisition is installed in the micro image acquisition unit 30.

  In addition, the microscope apparatus 10 is provided with a sample transport unit 14 and a sample stage 15 as sample moving means for moving the sample S between positions in the microscope apparatus 10. The sample transport unit 14 transports the sample S between the storage position of the sample S in the sample storage unit 11 and the image acquisition positions in each of the macro image acquisition unit 20 and the micro image acquisition unit 30 as necessary. It is the conveyance means to do. The sample stage 15 is used for setting and adjusting the image acquisition position of the sample S while the sample S is placed when acquiring a macro image or a micro image.

  The control device 60 is a control unit that performs control of an image acquisition operation in the microscope apparatus 10, setting of image acquisition conditions, processing of acquired image data of the sample S, and the like. The control device 60 is configured by a computer including a CPU and a storage device such as a necessary memory and a hard disk. A display device 71 and an input device 72 are connected to the control device 60. The display device 71 is, for example, a CRT display or a liquid crystal display, and is used for displaying an operation screen necessary for the operation of the image acquisition device, displaying an image of the acquired sample S, or the like. The input device 72 is, for example, a keyboard or a mouse, and is used for inputting information necessary for image acquisition, inputting instructions for an image acquisition operation, and the like.

  The configuration of the image acquisition apparatus shown in FIG. 1 will be further described. FIG. 2 is a block diagram showing an example of the configuration of the microscope apparatus 10 and the control apparatus 60 in the fluorescence image acquisition apparatus. FIG. 3 is a diagram schematically illustrating an example of the configuration of an optical system for obtaining a macro image in the microscope apparatus 10. FIG. 4 is a diagram schematically showing an example of the configuration of an optical system for micro image acquisition (for high-resolution fluorescence observation image acquisition) in the microscope apparatus 10.

  Here, as shown in FIGS. 3 and 4, in the configuration of the microscope apparatus 10, two directions orthogonal to each other in the horizontal direction are defined as an X-axis direction and a Y-axis direction, and a vertical direction orthogonal to the horizontal direction is defined as a Z-axis direction. To do. Of these, the Z-axis direction, which is the vertical direction, is the direction of the optical axis for image acquisition in the microscope system. In FIG. 2, the sample storage unit 11, the sample transport unit 14, and the like are not shown. In addition, as described above, the sample S that is an object of image acquisition in the microscope apparatus 10 is a slide in which a biological sample stained with, for example, a fluorescent pigment is sealed.

  The sample S to be subjected to fluorescence measurement is placed on the sample stage 15 when the image acquisition units 20 and 30 acquire images. The sample stage 15 is configured as an XY stage that can move in the X-axis direction and the Y-axis direction (horizontal direction) using a stepping motor, a DC motor, a servo motor, or the like. In such a configuration, the image acquisition position with respect to the sample S in the image acquisition units 20 and 30 is set and adjusted by driving the sample stage 15 in the XY plane. In the present embodiment, the sample stage 15 is movable between an image acquisition position in the macro image acquisition unit 20 and an image acquisition position in the micro image acquisition unit 30.

  With respect to the macro image acquisition position for acquiring the macro image of the sample S, as shown in FIG. 3, the macro image acquisition unit 20 and the macro light source 25 are respectively installed at predetermined positions with respect to the optical axis 20a. . The macro light source 25 is illumination means for irradiating the sample S with light for generating a macro image acquisition light image.

  The macro image acquisition unit 20 is configured using a macro imaging device 21 such as a two-dimensional CCD sensor capable of acquiring a two-dimensional image from an optical image of the sample S. An imaging optical system 22 is provided between the macro image acquisition position where the sample S is arranged and the imaging device 21 as an optical system for guiding the optical image of the sample S.

  In the configuration example shown in FIG. 3, two light sources, a dark field light source 26 and a bright field light source 27, are provided as the macro light source 25. Among these, the macro dark field light source 26 is dark field illumination means used when acquiring a dark field macro image by detecting scattered light from the sample S as a macro image of the sample S. It arrange | positions in the position which irradiates light diagonally from the opposite side to the macro image acquisition part 20 with respect to the surface orthogonal to the optical axis at the time of acquisition.

  Specifically, in the configuration shown in FIG. 3, the dark field light source 26 is installed as oblique illumination means at a position where light is irradiated from an obliquely lower side of the sample S with respect to the optical axis 20a. According to such a dark field light source 26, a macro image of the sample S can be suitably acquired with sufficient contrast even when the sample S stained with a fluorescent dye is an object of image acquisition. . Such a high-contrast macro image is effective for setting imaging conditions such as the image acquisition range when acquiring a micro image that is a fluorescence observation image, and reliably sets the imaging conditions for the fluorescence observation image. It becomes possible to do.

  In this configuration example, a bright field light source 27 is installed as the macro light source 25 in addition to the dark field light source 26. The macro bright field light source 27 is a bright field illumination unit used when acquiring a bright field macro image as a macro image of the sample S, and is installed below the sample stage 15 as a transmission illumination unit. The dark field light source 26 and the bright field light source 27 are selectively used as needed depending on the specific state of the sample S and the like.

  On the other hand, as shown in FIG. 4, with respect to the micro image acquisition position for acquiring the micro image of the sample S, the micro image acquisition unit 30 and the micro light source 35 are installed at predetermined positions with respect to the optical axis 30a. ing. The micro light source 35 is an illuminating unit that irradiates the sample S with light for generating a micro image acquisition light image.

  The micro image acquisition unit 30 includes a micro imaging device 31 that can acquire a high-resolution image based on an optical image of the sample S. Specifically, the micro imaging device 31 is an imaging device capable of acquiring a one-dimensional image or a two-dimensional image in which a predetermined direction (first direction) is a longitudinal direction (major axis direction). It is used to acquire a fluorescence observation image or other observation image at a high resolution.

  For the imaging device 31 of such a micro image acquisition unit 30, the sample stage 15 scans the sample S in the second direction with the imaging device 31 to acquire a partial image when acquiring a fluorescence observation image. Furthermore, it functions as a scanning unit that adjusts the positional relationship between the sample stage 15 and the image acquisition unit 30. Furthermore, the sample stage 15 repeats the acquisition of the partial image described above a plurality of times while shifting the imaging position along the first direction so that the sample stage 15 and the image acquisition unit 30 acquire a plurality of partial images. Adjust the positional relationship.

  Here, the first direction which is the longitudinal direction of the imaging region (field of view) with respect to the sample S by the imaging device 31 and the second direction which is the scanning direction of the sample S are different directions in the XY plane (horizontal plane). , Preferably set as directions orthogonal to each other. The scanning method of the sample S when acquiring the fluorescence observation image of the sample S with high resolution will be described in detail later.

  In addition, between the micro image acquisition position where the sample S is arranged on the sample stage 15 and the imaging device 31, an objective lens 32 and a guide are provided as an optical system that guides the optical image of the sample S to the imaging device 31. An optical optical system 34 is provided. The objective lens 32 receives light (for example, fluorescence) from the sample S and generates a light image (for example, fluorescence image) of the sample S. The light guide optical system 34 is constituted by, for example, a tube lens, and guides a light image such as a fluorescent image of the sample S that has passed through the objective lens 32 to the micro imaging device 31.

  In the configuration shown in FIG. 4, a Z stage 33 using a stepping motor or a piezo actuator is provided for the objective lens 32, and the objective lens 32 is driven in the Z-axis direction by the Z stage 33. Thus, focusing on the sample S and the like are possible.

  In the configuration example shown in FIG. 4, two light sources, a transmissive light source 36 and an excitation light source 40, are provided as the micro light source 35. Among these, the transmission light source 36 is a transmission illumination means used when acquiring a transmission observation image as a micro image of the sample S, and is installed so as to irradiate light from below the sample stage 15 together with the transmission illumination optical system 37. Has been. The transmission illumination optical system 37 is provided with a shutter 38 for switching ON / OFF of the transmission illumination.

  The excitation light source 40 is excitation light supply means used when acquiring a fluorescence observation image by fluorescence generated in the sample S as a micro image of the sample S, and supplies excitation light for fluorescence measurement to the sample S. It is configured as an epi-illumination means. Such an excitation light source 40 is configured using, for example, a discharge tube. An excitation light irradiation optical system 41 including a light guide optical system 42 and a fluorescence optical system unit 44 is installed as an optical system for irradiating the excitation light to the sample S with respect to the excitation light source 40.

  Of the excitation light irradiation optical system 41, a light guide optical system 42 that constitutes an optical system portion on the excitation light source 40 side is provided with a shutter 43 for switching ON / OFF of epi-illumination by excitation light. Further, the fluorescence optical system unit (fluorescence cube) 44 reflects the excitation light from the excitation light source 40 and irradiates the sample S on the sample stage 15, and the fluorescence from the sample S is captured by the image acquisition unit 30. It includes a dichroic mirror 45 that passes through to 31.

  Further, as shown in FIGS. 4 and 5, the fluorescence optical system unit 44 includes an insertion position including the optical axis 30a and a standby position off the optical axis 30a with respect to the optical axis 30a for micro image acquisition. It is configured to be movable between. When a transmission observation image of the sample S is acquired by transmission illumination from the transmission light source 36, as shown in FIG. 5A, the optical system unit 44 is in a standby position off the optical axis 30a (broken line in FIG. 4). (Position indicated by). On the other hand, when acquiring a fluorescence observation image of the sample S by epi-illumination from the excitation light source 40, the optical unit 44 is inserted at the insertion position including the optical axis 30a (shown by a solid line in FIG. 4), as shown in FIG. (Position indicated by).

  For the optical system portion other than the fluorescence optical system unit 44 in the excitation light irradiation optical system 41, specifically, various configurations may be used. As an example of such an excitation light irradiation optical system 41, a configuration including a cylindrical lens system for shaping the irradiation region of the excitation light irradiated from the excitation light source 40 to the sample S can be used. In the configuration shown in FIG. 4, this cylindrical lens system is provided, for example, in the light guide optical system 42. By using such a cylindrical lens system in the optical system 41, the irradiation area of the excitation light with respect to the sample S can be set to an area having a predetermined direction as a longitudinal direction (long axis direction).

  In addition, the excitation light irradiation optical system 41 including this cylindrical lens system has a major axis direction that is different between the irradiation region of the excitation light on the sample S and the imaging region (imaging field of view) on the sample S by the imaging device 31. It is preferable that the sample S be configured to irradiate the excitation light so that the irradiation area is wider than the imaging area. In this case, the excitation light irradiation optical system 41 irradiates the sample S with the excitation light under the irradiation conditions in which the imaging region of the sample S is included in the excitation light irradiation region.

  An optical filter for selecting excitation light or fluorescence is installed on the optical path between the sample S and the imaging device 31 or on the optical path between the excitation light source 40 and the dichroic mirror 45 as necessary. May be. Such an optical filter such as an excitation filter or a fluorescence filter may be provided integrally with the dichroic mirror 45 in the fluorescence optical system unit 44. In FIG. 4, as an example of such a configuration, a configuration in which an excitation filter 45a is provided on the excitation light source 40 side of the dichroic mirror 45 and a fluorescence filter 45b is provided on the sample stage 15 side in the fluorescence optical system unit 44 is shown. ing.

  FIG. 6 is a diagram showing a specific example of the configuration of the optical system for obtaining a micro image shown in FIG. In this configuration example, a mercury lamp 40a, which is a discharge tube, is used as the excitation light source 40 that supplies excitation light. The light guide optical system 42 between the excitation light source 40 and the fluorescence optical system unit 44 is composed of three lens systems, a cylindrical lens system 46, a focus lens system 47, and a collimating lens system 48.

  These lens systems 46, 47, and 48 that constitute the excitation light irradiation optical system 41 together with the dichroic mirror 45 and the like of the fluorescence optical system unit 44 are each constituted by one or a plurality of lenses. The transmission illumination optical system 37 between the transmission light source 36 and the sample S is constituted by a light guide 37a, a collimating lens 37b, a mirror 37c, and a condenser lens 37d.

  As described above, the imaging device 31 in the micro image acquisition unit 30 used for acquiring a micro image such as a fluorescence observation image of the sample S acquires a one-dimensional image or a two-dimensional image having the first direction as the longitudinal direction as described above. Possible imaging devices are used. Specifically, a one-dimensional sensor capable of acquiring a one-dimensional image with the first direction as the longitudinal direction can be used as the imaging device 31. Alternatively, as the imaging device 31, a two-dimensional sensor capable of acquiring a two-dimensional image having the first direction as a longitudinal direction and TDI driving can be used.

  Control for driving these sample stage 15, macro image acquisition unit 20, micro image acquisition unit 30, light sources 26 and 27 that are macro light sources 25, and light sources 36 and 40 that are micro light sources 35. As means, a stage control unit 16, a macro light source control unit 17, and a micro light source control unit 18 are provided. The macro image acquisition unit 20 and the micro image acquisition unit 30 are configured to include the functions of the imaging control unit.

  The stage control unit 16 drives and controls the sample stage 15 and the Z stage 33 that are XY stages, thereby setting and adjusting the imaging conditions for the sample S, scanning the sample S by the imaging device 31 when acquiring the fluorescence observation image, and the like. To control. Further, the macro light source control unit 17 controls the irradiation of light when acquiring the dark field macro image and the bright field macro image of the sample S by driving and controlling the dark field light source 26 and the bright field light source 27. In addition, the micro light source control unit 18 controls the irradiation of light when acquiring the transmission observation image and the fluorescence observation image of the sample S by drivingly controlling the transmission light source 36, the excitation light source 40, and the shutters 38 and 43. To do.

  The control device 60 includes an image acquisition control unit including a macro image acquisition control unit 61 and a micro image acquisition control unit 62, an image data processing unit including a macro image processing unit 68 and a micro image processing unit 69, and an imaging condition setting unit 65. And have. The image acquisition control unit controls the image acquisition operation of the sample S in the microscope apparatus 10 through the above-described control units 16 to 18 and the like.

  In addition, the image data processing unit includes the image data of the macro image acquired by the image acquisition unit 20 and the micro image acquired by the image acquisition unit 30 via the image data communication I / F (interfaces) 66 and 67. Image data is input, and necessary data processing is performed on these image data. In addition, image data input to the image data processing unit, various data and information obtained by processing the image data, or control information used in the image acquisition control unit are stored in the data storage unit as necessary. , Retained.

  Specifically, the macro image acquisition control unit 61 of the image acquisition control unit is configured to set the macro image acquisition position of the sample S via the stage control unit 16, the macro image acquisition unit 20, and the macro light source control unit 17. Macro-image acquisition operation by the imaging device 21 of the macro-image acquisition unit 20, dark-field macro image acquisition light irradiation operation by the dark-field light source 26, and bright-field macro image acquisition light irradiation operation by the bright-field light source 27 To control.

  The micro image acquisition control unit 62 is configured to set the micro image acquisition position of the sample S via the stage control unit 16, the micro image acquisition unit 30, and the micro light source control unit 18, and the imaging device 31 of the micro image acquisition unit 30. The micro image acquisition operation, the light irradiation operation for transmitting observation image acquisition by the transmission light source 36, the excitation light irradiation operation for acquiring the fluorescence observation image by the excitation light source 40, and the movement operation of the optical system unit 44 are controlled. The micro image acquisition control unit 62 controls acquisition of the micro image of the sample S with reference to the imaging conditions set by the imaging condition setting unit 65.

  The macro image processing unit 68 receives image data of the macro image of the sample S acquired by the imaging device 21 of the macro image acquisition unit 20 via the macro image data communication I / F 66. The image processing unit 68 performs necessary data processing such as correction, processing, and storage on the image data of the input macro image.

  The micro image processing unit 69 receives image data of the micro image of the sample S acquired by the imaging device 31 of the micro image acquisition unit 30 via the micro image data communication I / F 67. Similar to the image processing unit 68, the image processing unit 69 performs necessary data processing such as correction, processing, and storage on the input image data of the micro image. In the present embodiment, the micro image processing unit 69 uses the acquired micro image image data to create sample data that is high-resolution fluorescence observation image data of the target sample S.

  The imaging condition setting unit 65 is a setting unit that sets the imaging condition of the micro image with reference to the macro image of the sample S acquired by the macro image acquisition unit 20 of the microscope apparatus 10. The imaging condition setting unit 65 refers to the macro image on which the necessary processing is performed in the macro image processing unit 68, and sets the imaging condition of the micro image such as the fluorescence observation image of the sample S in a range including the target of image acquisition. Set the corresponding image acquisition range. Alternatively, the imaging condition setting unit 65 may include other imaging conditions as necessary, for example, focus-related information such as a focus measurement position for performing focusing and focus information for acquiring an image of an object in an image acquisition range. Set.

  Moreover, in the fluorescence image acquisition apparatus of this embodiment, as shown in FIG. 4, with respect to the micro image acquisition unit 30 for acquiring a fluorescence observation image, an image correction processing unit 75 and a correction data storage unit 76 are provided. Is provided. The correction data storage unit 76 is a storage unit that stores shading correction data for an image acquired by the imaging device 31. The shading correction data is stored in the correction data storage unit 76 in correspondence with the fluorescence optical system unit 44 applied to the fluorescence measurement for the sample S.

  The image correction processing unit 75 refers to the shading correction data stored in the correction data storage unit 76 and performs shading correction on the image acquired by the imaging device 31. Specifically, shading correction is performed for each of a plurality of partial images obtained by scanning the sample S two-dimensionally with the imaging device 31.

  Further, the correction data storage unit 76 may store dark correction data for an image acquired by the imaging device 31 in correspondence with the exposure time in the imaging device 31 as necessary. In this case, the image correction processing unit 75 preferably performs dark correction on each of the plurality of partial images with reference to the dark correction data stored in the correction data storage unit 76 in addition to the shading correction.

  Here, acquisition of the macro image and the micro image of the sample S in the image acquisition units 20 and 30 will be described. The macro image acquisition unit 20 acquires a macro image that is an overall image of the sample S used for setting the imaging conditions of a micro image such as a fluorescence observation image. For example, when a slide in which a biological sample such as a tissue section is sealed on the above-described slide glass is used as the sample S, the macro image includes an image of a predetermined range including the whole slide or a biological sample stained with a fluorescent dye. Is acquired.

  Further, in the optical system having the configuration shown in FIG. 3, in acquiring a macro image, the macro image is obliquely below the sample S depending on the type of the sample S to be acquired or the type of the macro image to be acquired. The oblique illumination by the arranged dark field light source 26 or the transmitted illumination by the bright field light source 27 arranged below the sample S is appropriately selected and used.

  The micro image acquisition unit 30 acquires a micro image of the sample S at a target resolution with reference to the set imaging conditions. The acquisition of the micro image is preferably performed by two-dimensionally scanning the sample S at a predetermined resolution higher than that of the macro image, as schematically shown in FIG. Here, regarding acquisition of a micro image using the imaging device 31 such as a one-dimensional CCD camera or a TDI-driven two-dimensional CCD camera, for example, in the XY plane parallel to the sample S, the length of the imaging region by the imaging device 31 The axial direction (first direction) is the X-axis direction, and the direction orthogonal to the long-axis direction is the Y-axis direction. At this time, in the micro image acquisition, the direction orthogonal to the major axis direction of the imaging region by the imaging device 31, and the negative direction of the Y axis in FIG. 7A is the scanning direction (second direction) with respect to the sample S. )

  In acquisition of a fluorescence observation image using the imaging device 31 of the image acquisition unit 30 (image acquisition step), first, excitation light is applied to the sample S via the excitation light irradiation optical system 41 including the fluorescence optical system unit 44. (Excitation light irradiation step), the sample S on the sample stage 15 is scanned in the scanning direction by the imaging device 31 to obtain a strip-shaped partial image A having a desired resolution.

  Furthermore, as shown in FIG. 7A, such partial image acquisition is repeated a plurality of times while shifting the imaging position along the long axis direction (the positive direction of the X axis) of the imaging surface. Images A, B, ..., I are acquired (scanning step). Such scanning of the sample S is performed, for example, by driving the sample stage 15 that functions as a scanning unit and adjusting the positional relationship between the sample stage 15 and the imaging device 31 of the image acquisition unit 30.

  By combining the partial images A to I obtained in this way in the X-axis direction, a micro image such as an entire fluorescence observation image of the sample S can be generated. According to such a micro image acquisition method, the image data of the sample S can be preferably acquired with sufficiently high resolution.

  In FIG. 7A, the region having the X-axis direction indicated by the oblique lines in the partial image A as the longitudinal direction corresponds to the imaging surface of the imaging device 31, and the X-axis direction is the long-axis direction. An imaging region on the sample S is shown. In addition, when the excitation light irradiation optical system 41 including a cylindrical lens system is used for such an imaging region, the irradiation region of the excitation light by the optical system 41 is the above-described region in FIG. As indicated by a broken line surrounding the imaging region, it is preferable that the major axis direction of the irradiation region and the imaging region coincide with each other and that the irradiation region is wider than the imaging region. Such excitation light irradiation regions may be set in various regions depending on the specific configuration of the excitation light irradiation optical system 41.

  In addition, regarding the setting of the imaging conditions of the micro image such as the fluorescence observation image, the imaging condition setting unit 65 refers to the macro image acquired by the macro image acquisition unit 20 and the image acquisition range as the micro image imaging condition, It is preferable to set the focus measurement position. Thereby, it is possible to suitably set parameters used for micro image acquisition, and to acquire a fluorescence observation image of a sample in a good state with high resolution.

  Specifically, when the slide is the sample S as described above, as shown in FIG. 7B, the image acquisition range for the sample S is a rectangle including the biological sample L in the slide that is the target of image acquisition. It can be set by the shape range R. Two-dimensional scanning (see FIG. 7A) of the sample S in the micro image acquisition unit 30 is performed within the image acquisition range R set in this way. The focus measurement position is used when the micro image acquisition unit 30 acquires focus information for the sample S prior to acquisition of the micro image of the sample S. The micro image acquisition unit 30 performs focus measurement on the set one or a plurality of focus measurement positions to obtain a focus position, a focal plane, a focus map, and the like as focus information when acquiring a micro image of the sample S. decide.

  FIG. 7B shows an example in which nine focus measurement positions are automatically set for setting a focus measurement position using a macro image. Here, the image acquisition range R previously set for the sample S is divided into 9 equal parts by 3 × 3, and nine focus measurement positions P are set by the center points of the respective areas.

  In addition, here, among the nine focus measurement positions, eight of the initial positions are included in the range of the biological sample L that is the object of image acquisition, and thus are set as the focus measurement positions as they are. The On the other hand, since the lower left point is outside the range of the biological sample L, the focus measurement position at the lower left is focused on the position Q obtained by, for example, moving toward the center within the image acquisition range R. It may be set as a measurement position. Alternatively, such a position may be excluded from the focus measurement position. Further, such a focus measurement position may be set manually by an operator instead of automatically.

  As in the example described above, when the sample S is a slide, with respect to the imaging conditions for acquiring the micro image, preferably, referring to the macro image acquired by the macro image acquisition unit 20 first, the micro image is obtained. As an image capturing condition, an image acquisition range R including the biological sample L and a focus measurement position P with a predetermined number of points are set. Then, the micro image acquisition unit 30 acquires focus information about a focus position or a focal plane with respect to the sample S based on the focus measurement position P, and captures the obtained focus information and the set image acquisition range R and the like. Based on the conditions, acquisition of a micro image such as a fluorescence observation image of the sample S is performed.

  Further, the creation of the image data of the fluorescence observation image of the sample S in the micro image processing unit 69 is shown in FIG. 8 based on the plurality of partial images A, B,..., I shown in FIG. To be done. Here, as the image data of the micro image acquired by the imaging device 31 of the micro image acquisition unit 30, the image data group of the strip-shaped partial images A, B, C,... Is transmitted via the image data communication I / F 67. It is input to the control device 60.

  The micro image processing unit 69 generates image data that becomes a fluorescence observation image for the entire sample S by combining the plurality of partial images in the longitudinal direction of the imaging region, and uses the image data as sample data (image combining step). ). Thereby, the micro image processing unit 69 functions as an image combining unit that generates a fluorescence observation image of the sample S by combining a plurality of partial images. The sample data generated by the image processing unit 69 can be used as fluorescence observation image data in a virtual microscope, for example.

  In the present embodiment, shading correction or dark correction is performed as necessary for each of the plurality of partial images A, B,... The correction data storage unit 76 stores and prepares shading correction data corresponding to the fluorescence optical system unit 44 (correction data storage step). When dark correction is performed in addition to shading correction, dark correction data corresponding to the set exposure time is stored and prepared in the correction data storage unit 76.

  The image correction processing unit 75 refers to these shading correction data and dark correction data, and performs necessary shading correction or further dark correction on the image acquired by the imaging device 31 (image correction processing step). As the correction data used here, correction data prepared in advance or correction data obtained by performing a preliminary measurement prior to the main measurement can be used.

  The effects of the fluorescence image acquisition device and the fluorescence image acquisition method according to the present embodiment will be described.

  In the fluorescent image acquisition device and the fluorescent image acquisition method described above, the micro image acquisition unit 30 that is an image acquisition unit that acquires a high-resolution image of the sample S acquires an image having the first direction as the longitudinal direction. An imaging device 31 is used. Then, the imaging device 31 scans the sample S a plurality of times while shifting the imaging position in the first direction with the second direction as the scanning direction, and the micro image processing unit 69 synthesizes the obtained partial images. Thus, the fluorescence observation image of the sample S is acquired. Thereby, the fluorescence observation image of the sample S can be efficiently acquired with sufficiently high resolution.

  Further, the excitation light irradiation optical system 41 for the sample S is configured as an epi-illumination optical system using the fluorescence optical system unit 44 including the dichroic mirror 45, and the correction data storage unit 76 corresponds to the fluorescence optical system unit 44. Shading correction data is prepared, and using this correction data, the image correction processing unit 75 performs shading correction for each of a plurality of partial images.

  Here, in the fluorescence measurement of the sample S, the illumination unevenness with respect to the sample S by the excitation light irradiation optical system 41 including the excitation light source 40 and the dichroic mirror 45, or the sensitivity unevenness in the imaging device 31 used for acquiring the fluorescence observation image. For example, shading occurs in the longitudinal direction of an image acquired by the imaging device 31 due to signal unevenness or the like. In the above configuration, the fluorescence optical system unit 44 to be used may be exchanged depending on the type of the sample S to be subjected to fluorescence measurement, a specific method of fluorescence measurement, or the like.

  For example, when a fluorescence filter is provided in the fluorescence optical system unit 44 in addition to the dichroic mirror 45, an optical system unit having a fluorescence filter having characteristics suitable for the fluorescence to be measured is selected from a plurality of types of fluorescence optical system units. Selected and used. As described above, when the fluorescence optical system unit 44 applied to the excitation light irradiation optical system 41 is replaced, the shading characteristics generated in the acquisition of the fluorescence observation image also change. The same applies when an excitation filter is provided in the fluorescence optical system unit 44.

  On the other hand, according to the above configuration, by preparing shading correction data corresponding to the fluorescence optical system unit 44, the shading correction for the image is performed using the optimum correction data for each fluorescence optical system unit to be used. It can be performed. This makes it possible to acquire a fluorescence observation image of the sample with high resolution in a suitable state. In addition, by performing shading correction on each of a plurality of partial images acquired by scanning the sample S two-dimensionally, in the fluorescence observation image generated by combining the partial images, adjacent partial images Degradation of image quality such as an unnatural image change (for example, discontinuous luminance change) at the boundary is prevented.

  Such shading correction is correction for data variation caused by optical systems such as the excitation light irradiation optical system 41 and the image acquisition unit 30 as described above. Therefore, the shading correction data can be acquired by performing fluorescence measurement using a uniform fluorescent sample as a calibration sample. In addition, as such shading correction data, for example, correction data input from the outside to the control device 60 can be used. Or you may use the correction data produced in the micro image processing part 69 of the control apparatus 60 with reference to the image acquired by the fluorescence measurement performed for calibration. In this case, the micro image processing unit 69 functions as correction data creation means.

  As for the correction performed by the image correction processing unit 75, as described above, dark correction data is prepared in the correction data storage unit 76 in correspondence with the exposure time in the imaging device 31, and the dark correction data. It is good also as performing dark correction about each of several partial images using. In this way, by performing dark correction in addition to shading correction on the image acquired by the imaging device 31, the fluorescence observation image of the sample S obtained can be made a more accurate image.

  Here, in the fluorescence measurement of the sample S, the exposure time of imaging by the imaging device 31 may be changed according to the amount of fluorescence generated in the sample S or the like. Further, the correction amount by dark correction varies depending on the exposure time. Therefore, by preparing the dark correction data corresponding to the exposure time as described above, the dark correction can be performed on the image using the optimal correction data for each exposure time set for the sample S. it can. The accuracy of the shading correction data described above can be further improved by acquiring it according to the exposure time.

  Such dark correction is correction for dark output that the imaging device 31 used for acquiring the fluorescence observation image has in the dark. Therefore, the dark correction data can be acquired by performing imaging using the imaging device 31 under dark conditions. The creation of dark correction data and the like is as described above with respect to shading correction data.

  Specifically, the image correction processing unit 75 and the correction data storage unit 76 illustrated in FIG. 4 can be provided in the micro imaging device 31 or the control device 60. Alternatively, the image correction processing device may be provided separately from the imaging device 31 and the control device 60.

  FIG. 9 is a block diagram illustrating an example of the configuration of an imaging device and a control device used for acquiring a fluorescence observation image. In this configuration example, the image correction processing unit and the correction data storage unit are provided in the imaging device 31. Specifically, the imaging device 31 performs A / D on the light detection unit 31a having a plurality of pixels arranged in a one-dimensional array or a two-dimensional array, and charges output from the pixels of the light detection unit 31a. An A / D conversion unit 31b that performs conversion, an image correction processing unit 75a, a correction data storage unit 76a, and an image data communication I / F 31c are included.

  The control device 60 includes an image data communication I / F 67, a storage device 60a such as a hard disk, and a memory 60b. In such a configuration, the correction data input from the outside to the control device 60 or the correction data created by the micro image processing unit 69 is stored in the storage device 60a. The correction data storage unit 76a of the imaging device 31 acquires necessary correction data stored in the storage device 60a via the communication I / Fs 31c and 67. Then, using the correction data, the image correction processing unit 75a corrects the image. For example, a CameraLink I / F can be used as the image data communication I / F.

  FIG. 10 is a block diagram illustrating another example of the configuration of the imaging device and the control device. In this configuration example, the image correction processing unit and the correction data storage unit are provided in the control device 60. Specifically, the imaging device 31 includes a light detection unit 31a, an A / D conversion unit 31b, and an image data communication I / F 31c.

  The control device 60 includes an image data communication I / F 67, a memory 60b, an image correction processing unit 75b, and a correction data storage unit 76b. In such a configuration, the correction data is directly stored in the correction data storage unit 76 b provided in the control device 60. Then, using the correction data, the image correction processing unit 75b corrects the image. The correction data storage unit 76b is configured by a storage device such as a hard disk.

  In the fluorescence image acquisition apparatus of the above embodiment, the excitation light source is configured such that the irradiation region with respect to the sample S becomes a region having the predetermined direction as the major axis direction by the cylindrical lens system 46 installed in the excitation light irradiation optical system 41. The excitation light beam from 40 is shaped, and the major axis direction of the irradiation region of the excitation light on the sample S and the major axis direction of the imaging region serving as a field of view by the imaging device 31 are made to coincide with each other. Thereby, the light quantity of the extra excitation light irradiated out of the visual field of the imaging device 31 among the light quantity of the excitation light supplied to the sample S is reduced.

  In the above configuration, the irradiation region of the excitation light to the sample S is set so that the irradiation region is wider than the imaging region. As a result, even when the irradiation region changes according to the wavelength due to chromatic aberration, the imaging region by the imaging device 31 can be reliably positioned within the irradiation region with each wavelength component of the excitation light. It becomes.

  In the fluorescence image acquisition apparatus described above, as shown in FIG. 4, the excitation light irradiation optical system 41 reflects the excitation light from the excitation light source 40 and irradiates the sample S. A fluorescence optical system unit (epi-illumination optical system unit) 44 including a dichroic mirror 45 that allows fluorescence to pass to the imaging device 31 of the image acquisition unit 30 is provided. According to such a configuration, the incident illumination optical system for the excitation light with respect to the sample S and the light guide optical system for the fluorescence image acquisition unit 30 generated in the sample S can be preferably made compatible.

  For such a fluorescence optical system unit 44, as shown in FIG. 5, the insertion position including the optical axis 30a and the standby position deviating from the optical axis 30a with respect to the optical axis 30a for micro image acquisition. It is preferable that it is configured to be movable between. In such a configuration, the acquisition of the transmission observation image of the sample S by the transmission light source 36 and the acquisition of the fluorescence observation image by the excitation light source 40 can be preferably made compatible.

  FIGS. 11 and 12 are perspective views showing an example of the configuration of an epi-illumination optical system for excitation light including a moving mechanism of the fluorescence optical system unit (fluorescence cube) 44. FIG. In this configuration example, a cylindrical lens system 46, an epi-illumination shutter 43, a focus lens system 47, and a collimating lens system 48 are sequentially arranged on the optical system support plate 41 a arranged in a predetermined direction with respect to the excitation light source 40 from the light source 40 side. , And a fluorescence optical system unit 44 are installed. Further, a moving mechanism 44a is provided for the fluorescence optical system unit 44, and the fluorescence optical system unit 44 is placed in a standby position (FIGS. 11 and 5A) and an insertion position (FIGS. 12 and 5B). ) Between each other. In FIGS. 11 and 12, the specific structure of the moving mechanism 44a is not shown.

  In addition, as described above, in such a fluorescence optical system unit 44, an excitation filter for selecting a wavelength component of excitation light in addition to the dichroic mirror 45, or a fluorescence filter for the fluorescence component, and the like are further installed as necessary. An optical system unit may be configured. Moreover, it is preferable that the moving mechanism 44a has a configuration in which the fluorescence optical system unit 44 can be attached and detached.

  There are various types of samples S to be subjected to fluorescence measurement. For this reason, in the excitation light irradiation optical system 41, it is preferable to provide an optical element installation part for installing an ND filter or the like for adjusting the amount of excitation light as required. In addition, it is preferable to set the front focal position and the rear focal position of each lens system in consideration of such an installation position of the optical element.

  When a discharge tube is used as the excitation light source 40 in the fluorescence image acquisition device described above, the discharge tube is preferably a white light source that supplies white light as excitation light. Specific examples of such a discharge tube include a mercury lamp and a halogen lamp. Further, other discharge tubes or light source devices other than the discharge tubes may be used as the excitation light source. Examples of such an excitation light source include a light emitting element such as an LED.

  Further, as described above, a one-dimensional CCD sensor can be used as the imaging device for acquiring the fluorescence observation image of the sample S. In addition to the one-dimensional sensor, for example, a two-dimensional CCD sensor capable of TDI driving can be used. Use of such a TDI drive two-dimensional sensor is effective in increasing the speed and sensitivity of obtaining a high-resolution fluorescence observation image by scanning the sample S with an imaging device.

  FIG. 13 is a block diagram illustrating an example of a configuration of an imaging apparatus including a two-dimensional CCD sensor capable of TDI driving. The imaging device 90 according to this configuration example includes a light detection unit 91, a charge transfer unit 92, and an A / D conversion unit 93. The imaging device 90 has a multi-tap configuration having a plurality of charge output ends. According to such a multi-tap structure, reading of charges generated in each pixel of the light detection unit 91 is speeded up. Further, by performing the shading correction on the obtained image as described above, an unnatural image change between adjacent taps can be prevented.

  The light detection unit 91 is arranged in a two-dimensional array, has a plurality of pixels each having a photoelectric conversion function, and is configured to output charges generated according to the amount of light incident on the pixels. In addition, with respect to the light detection unit 91, a charge transfer unit 92 is provided along the horizontal direction (left-right direction in the drawing) that is one of the arrangement directions of the plurality of pixels below the drawing. The charge transfer unit 92 is a horizontal shift register that transfers charges output from a plurality of vertical shift registers constituting the light detection unit 10 and input in parallel to each other in a horizontal direction and output from an output terminal.

  In the present configuration example, the charge transfer unit 92 includes 16 partial charge transfer units T01 to T16 divided into a plurality in the horizontal direction. Each of the partial charge transfer units T01 to T16 includes a plurality of and the same number of cells, and is arranged in order from the left side to the right side in the drawing. In addition, the charge transfer direction in each of the partial charge transfer units T01 to T16 is a direction from the right side to the left side in the drawing in the horizontal direction, and the left end portion is an output end of charges.

  Further, with respect to the partial charge transfer units T01 to T16 in the charge transfer unit 92, the photodetection unit 91 can be divided into 16 corresponding photodetection regions R01 to R16. In such a configuration, the first partial charge transfer unit T01 of the charge transfer unit 92 transfers the charges output from the pixels in the first photodetection region R01 of the photodetection unit 91 by the vertical shift register in the output direction. And output from the output terminal. At this time, a signal output from the output terminal of the partial charge transfer unit T01 is sequentially a signal due to charges input in parallel to a plurality of cells of the partial charge transfer unit T01 from a plurality of vertical shift registers in the photodetection region R01. This is the output signal sequence. The configurations of the second to sixteenth partial charge transfer units T02 to T16 of the charge transfer unit 92 and the second to sixteenth photodetection regions R02 to R16 of the photodetection unit 91 also include the first partial charge transfer unit T01 and This is the same as the first light detection region R01.

  Corresponding to these partial charge transfer units T01 to T16, the A / D conversion unit 93 is provided with 16 A / D converters C01 to C16. The analog signal due to the charge from the photodetection unit 91 output from the output terminals of the partial charge transfer units T01 to T16 is converted into a digital data signal in the corresponding A / D converter among the A / D converters C01 to C16. And output to a subsequent processing circuit or the like.

  FIG. 14 is a plan view illustrating an example of the configuration of the light detection unit and the charge transfer unit in the imaging apparatus illustrated in FIG. 13. In this configuration example, the light detection unit 91 includes 4096 × 70 pixels in a two-dimensional array with 4096 cells in the horizontal direction and 70 lines in the vertical direction. In the vertical direction, the upper and lower three lines are each a dummy area 91b, and the inner 64 lines are an area 91a used for light detection.

  Corresponding to the photodetection unit 91, the charge transfer unit 92 is composed of 16 partial charge transfer units T01 to T16 each having 256 cells. A dummy cell is provided on the output end side of each partial charge transfer unit as necessary. Further, in this configuration example, a storage unit 92 a having 4096 cells and 70 lines is provided between the light detection unit 91 and the charge transfer unit 92, similarly to the light detection unit 91.

  Further, in the configuration of FIG. 14, a charge transfer unit 93 including a storage unit 93 a having the same configuration as the lower storage unit 92 a and the charge transfer unit 92, and partial charge transfer units T <b> 17 to T <b> 32 above the light detection unit 91. Is provided. With this configuration, in the present configuration example, the charge transfer direction in the vertical shift register of the light detection unit 91 can be set to any of the two upper and lower directions. The configuration of FIG. 14 shows an example of the configuration of a multi-tap two-dimensional CCD sensor capable of TDI driving. Specifically, for example, a multi-tap one-dimensional CCD sensor is also available. Alternatively, various configurations such as a single tap structure CCD sensor can be used.

  When a one-dimensional sensor or a two-dimensional sensor having a multi-tap structure as shown in FIG. 13 is used as the imaging device 31, an A / D converter or the like provided for each of 16-channel outputs in the multi-tap structure In the readout circuit, characteristic variations may occur between channels. Such variation in characteristics causes shading such as an unnatural image change at the boundary between images between adjacent channels. On the other hand, in the configuration in which the shading correction is performed on the image acquired by the imaging device 31 as described above, it is possible to prevent the deterioration of the image quality due to such a multi-tap structure.

  In addition, the image acquisition unit used for acquiring the fluorescence observation image of the sample uses the micro image acquisition unit 30 having the single imaging device 31 in the above embodiment, but the configuration of the image acquisition unit is specifically described. In particular, various configurations can be used. As one of such configurations, a wavelength-resolving optical system that decomposes fluorescence from the sample S into three different wavelength components and an imaging device for acquiring a fluorescence observation image of the sample are used by the wavelength-resolving optical system. A configuration including three imaging devices that acquire fluorescence observation images based on the three decomposed wavelength components can be used. In such a configuration, the three imaging devices acquire optical images of samples formed by different wavelength components (color components), respectively. Thereby, it becomes possible to acquire the fluorescence observation image in the color of the sample based on the optical image acquired by each of the three imaging devices.

  FIG. 15 is a configuration diagram illustrating a modification of the image acquisition unit (micro image acquisition unit 30 in the above embodiment) used for acquiring the fluorescence observation image of the sample. The image acquisition unit 80 of this configuration example includes a low-pass filter 81 for suppressing moire fringes, and an IR filter 82 that further cuts infrared light out of the light transmitted through the low-pass filter 81. The image acquisition unit 80 also includes a color separation optical system 83 that is a wavelength separation optical system that separates the incident light L10 transmitted through the IR filter 82 into green light L11, red light L12, and blue light L13 having three wavelength components. .

  The color separation optical system 83 includes three prisms (dichroic prisms) 84, 85, 86 arranged in order from the IR filter 82 side. A dichroic film 84 a that reflects a light component having a wavelength in the red region is formed on a surface facing the prism 85 among the prisms 84 constituting a part of the color separation optical system 83. In addition, a dichroic film 85 a that reflects light components having a wavelength in the blue region is formed between the prism 85 and the prism 86. These dichroic films 84a and 85a both transmit light components having a wavelength in the green region.

  Therefore, among the incident light L10 incident from the light receiving surface F10 that the prism 84 has and faces the IR filter 82, the green light L11 travels straight in the color separation optical system 83 and is output from the light emitting surface F11 that the prism 86 has. . Further, the red light L12 is reflected by the dichroic film 84a and then further reflected within the prism 84, and then output from the light exit surface F12 of the prism 84. Further, the blue light L13 is transmitted through the dichroic film 84a, then reflected by the dichroic film 85a, further reflected in the prism 85, and then output from the light exit surface F13 of the prism 85.

  A CCD sensor, which is an imaging device 88 for acquiring a fluorescence observation image of the sample S, is provided on each of the light emission surfaces F11, F12, and F13 via a trimming filter 87 for adjusting the spectral characteristics of the light of each color component. Is provided. Further, each of these three imaging devices 88 is connected to an imaging control unit 89. Thus, according to the configuration using the color separation optical system 83 and the three imaging devices 88, it is possible to acquire a fluorescence observation image in the color of the sample S as described above.

  In such a case, the correction data storage unit 76 stores three types of shading correction data for images acquired by the three imaging devices 88, and the image correction processing unit 75 stores the three imaging devices 88. It is preferable to perform shading correction using the corresponding shading correction data for each of the above. Thereby, in each of the three imaging devices included in the image acquisition unit, it is possible to acquire the fluorescence observation image of the sample at a high resolution in a suitable state. This is the same when dark correction is performed on an image. In such a configuration, a triple band pass filter may be provided as the fluorescent filter.

  Here, shading correction of an image when the 16-tap CCD sensor shown in FIG. 13 is used as each of the three imaging devices 88 in the configuration shown in FIG. 15 will be described together with a specific example. FIG. 16 is a graph illustrating an example of image data acquired by each of the three imaging devices. Here, one-dimensional image data is shown along the longitudinal direction (4096 pixels) of the imaging surface of the imaging apparatus. The number of pixels per tap in the 16 tap structure is 256.

  In the graph of FIG. 16, the horizontal axis indicates the pixels in the longitudinal direction, and the vertical axis indicates the luminance value (8 bits, 0 to 255) in each channel. Graph R1 shows image data obtained by the red light imaging device, graph G1 shows image data obtained by the green light imaging device, and graph B1 obtained by the blue light imaging device. Image data is shown. In these graphs, the analog gain of the camera is set to 1 time for all the taps and all the imaging devices. The analog offset is adjusted so that the dark level is uniform.

  In each of the graphs R1, G1, and B1 shown in FIG. 16, shading occurs due to variations in characteristics between taps in the optical system and the imaging device. In addition, the luminance value levels differ between the graphs due to differences in spectral characteristics of RGB. In addition, graphs R2, G2, and B2 in FIG. 17 indicate image data after the analog gain is adjusted for the image data in the graphs R1, G1, and B1 illustrated in FIG. Here, the RGB levels are approximately the same, but the influence of shading due to characteristic variations between taps remains.

  On the other hand, graphs R3, G3, and B3 in FIG. 18 show image data after the shading correction is further performed on the graphs R2, G2, and B2 shown in FIG. 17 by the method described above. In these graphs, it is understood that uniform image data is obtained by removing the influence of all the shading elements described above. In the graphs shown in FIGS. 17 and 18, a part of the vertical axis of the luminance value is enlarged.

  An example of an image acquisition method for the sample S using the fluorescence image acquisition apparatus having the above configuration will be described. FIG. 19 is a diagram schematically illustrating a method for acquiring a fluorescent image of a sample and a method for driving each unit of the fluorescent image acquiring apparatus. First, the slide S, which is a sample for executing image acquisition, is taken out from the sample storage unit 11, transported by the sample transport unit 14, and loaded at a predetermined position on the sample stage 15 (step 1). Then, the macro image acquisition unit 20 acquires a macro image of the slide S.

  In acquiring the macro image of the slide S, the dark field light source 26 is used when acquiring the image of the biological sample L in the slide S (step 2). Further, when acquiring an image of another part such as a label of the slide S, the bright field light source 27 is used (step 3). In the macro image acquisition, in the micro image acquisition optical system, the transmission illumination shutter 38 and the excitation light epi-illumination shutter 43 are both closed. In FIG. 19, the state where the shutter is closed is indicated by “x”, and the state where the shutter is open is indicated by “◯”.

  The image data of the macro image acquired by the macro image acquisition unit 20 is input to the macro image processing unit 68 of the control device 60, and necessary processing is performed on the macro image. Subsequently, the imaging condition setting unit 65 refers to the macro image of the slide S, and acquires an image according to a range including the biological sample L that is an object of image acquisition as an imaging condition for the micro image that is the fluorescence observation image. A range R is set, and a focus measurement position P is set.

  On the other hand, the slide S for which the acquisition of the macro image has been completed is moved from the image acquisition position in the macro image acquisition unit 20 by the sample transport unit 14 or the sample stage 15 and set to the image acquisition position in the micro image acquisition unit 30. Is done. Then, automatic focusing is performed by performing focus measurement on each of the set focus measurement positions P, and an image acquisition of the biological sample L that is the object in the image acquisition range R is performed as an imaging condition for the micro image. Focus information about is acquired (step 4).

  In acquiring the focus information, the transmission illumination shutter 38 is opened, and the focus information is acquired by the transmission illumination from the transmission light source 36 (see FIG. 5A). At this time, the fluorescence optical system unit 44 is disposed at the standby position (OUT) or the insertion position (IN) as necessary. Further, an exposure time is set for the slide S to be subjected to fluorescence measurement (step 5). Here, the epi-illumination shutter 43 is opened, the fluorescence optical system unit 44 is disposed at the insertion position (IN), and the exposure time is set by the epi-illumination of the excitation light from the excitation light source 40. (See FIG. 5B).

  Subsequently, a micro image that is a fluorescence observation image of the biological sample L in the slide S is acquired based on the acquired focus information and the set exposure time (step 6). At this time, when the image pickup device 31 scans the slide S to acquire a partial image, the epi-illumination shutter 43 is opened and the fluorescence optical system unit 44 is disposed at the insertion position. In addition, when the imaging position is moved during the acquisition of the partial image, the fluorescence optical system unit 44 remains in the insertion position, but the epi-illumination shutter 43 is closed. When the acquisition of the fluorescence observation image for the slide S is completed by the above steps, the slide S is unloaded and returned to the sample storage unit 11 (step 7).

  Next, a method for acquiring correction data held in the correction data storage unit 76 and a method for acquiring a fluorescence observation image using the correction data will be described. The following operations such as image acquisition shown in FIGS. 20 and 21 are basically performed using the micro image acquisition unit 30 for acquiring a high-resolution fluorescence observation image.

  FIG. 20 is a flowchart illustrating an example of a method for acquiring shading correction data. First, an appropriate fluorescence optical system unit 44 is selected for the sample S to be subjected to fluorescence measurement, and is set at a predetermined position of the optical system (step S01). Further, a calibration slide for acquiring shading correction data is set at a predetermined storage position of the sample storage unit 11 of the microscope apparatus 10 (S02). As the calibration slide, a slide in which a fluorescent substance is uniformly placed as described above can be used (for example, see Japanese Patent Application Laid-Open No. 11-352299).

  Next, the calibration slide is loaded on the sample stage 15 (S03). Then, the image data of the calibration slide serving as calibration data for shading correction is acquired by the imaging device 31 of the micro image acquisition unit 30 (S04). Here, a calibration partial image is obtained by scanning the calibration slide once in the Y-axis direction by a one-dimensional sensor or a TDI-driven two-dimensional sensor capable of obtaining a one-dimensional image having the X-axis direction as a longitudinal direction. .

  The acquired image data for calibration is subjected to necessary image processing in the micro image processing unit 69 of the control device 60 (S05). Examples of the image processing here include dust removal processing. That is, when acquiring the calibration partial image, there may be dust on the calibration slide in the scanning range. In contrast, first, the mode value in the Y-axis direction is obtained for each pixel in the X-axis direction in the obtained partial image, and pixels outside the range of ± several% from this mode value are determined as dust. , Excluding the pixel line containing the pixel. Then, for the remaining lines, processing such as obtaining an average value in the Y-axis direction for each pixel is performed to create final shading correction data D1 (S06).

  The created shading correction data D1 is stored in the correction data storage unit 76 in the imaging device 31 or the control device 60 in association with the set fluorescence optical system unit 44. When the creation and storage of the shading correction data D1 is finished, the calibration slide is unloaded (S07), and the shading correction data acquisition operation is finished. In addition, when three imaging devices are used as the imaging device and it is necessary to acquire three types of corresponding shading correction data (for example, three types of RGB correction data), images are acquired by the respective imaging devices. It is preferable to prepare three types of calibration slides corresponding to the wavelength components to be obtained, and to obtain calibration data and create correction data for each of them.

  FIG. 21 is a flowchart illustrating an example of a method for acquiring dark correction data and a fluorescence observation image of a sample. First, a slide S of a sample that is actually subject to fluorescence measurement is set in the sample storage unit 11 (S11). Here, as for the fluorescence optical system unit 44, an appropriate unit is selected and set when the shading correction data is acquired previously.

  Next, the slide S is loaded on the sample stage 15 (S12). Then, in the control device 60, an exposure time suitable for acquiring the fluorescence observation image of the slide S is set (S13). Further, under the dark condition with the shutter closed, the calibration device for dark correction corresponding to the set exposure time is acquired by the imaging device 31 of the micro image acquisition unit 30 (S14), and the micro image processing of the control device 60 is performed. In the part 69, dark correction data D2 is created (S15). The created dark correction data D2 is stored in the correction data storage unit 76 in the imaging device 31 or the control device 60 in association with the set exposure time.

  Subsequently, the main measurement of the fluorescence measurement is performed on the slide S, and image data of the fluorescence observation image is acquired (S16). At this time, the image correction processing unit 75 performs necessary data correction with reference to the shading correction data and dark correction data stored in the correction data storage unit 76. When the acquisition of the fluorescence observation image data is completed, the slide S is unloaded (S17), and the operation for acquiring the fluorescence observation image of the slide S is ended. In addition, when there are a plurality of slides S to be subjected to fluorescence measurement, necessary steps are repeatedly executed.

  The fluorescence image acquisition device and the fluorescence image acquisition method according to the present invention are not limited to the above-described embodiments and configuration examples, and various modifications are possible. For example, in the above-described embodiment, the macro image acquisition unit 20 is provided in addition to the micro image acquisition unit 30 for acquiring a high-resolution fluorescence observation image. May be omitted if unnecessary. Further, FIG. 4 shows an example of a specific configuration of an optical system including an image acquisition unit for acquiring a fluorescence observation image. Specifically, various configurations may be used.

  In the above description, the three-plate CCD is exemplified for the micro image acquisition unit 30. However, the present invention is not limited to this, and various modifications such as a single-plate CCD are possible. When a single-plate CCD is used in this way, it is possible to use a configuration in which the wavelength selection filter is exchangeable in front of the light source or the single-plate CCD sensor, images are acquired three times, and they are combined. Alternatively, an image may be acquired in a single color without selecting a wavelength.

  INDUSTRIAL APPLICABILITY The present invention can be used as a fluorescence image acquisition apparatus and a fluorescence image acquisition method that can suitably acquire a fluorescence observation image of a sample with high resolution.

It is a block diagram which shows the structure of one Embodiment of a fluorescence image acquisition apparatus. It is a block diagram which shows an example of a structure of a microscope apparatus and a control apparatus. It is a figure which shows schematically an example of a structure of the optical system for macro image acquisition. It is a figure which shows roughly an example of a structure of the optical system for the micro image acquisition which is a fluorescence observation image. It is a figure shown about the standby position and insertion position of a fluorescence optical system unit. It is a figure which shows the specific example of a structure of the optical system for micro image acquisition shown in FIG. It is a figure which shows typically the acquisition method of the observation image of a sample. It is a figure showing typically about creation of sample data using a micro picture. It is a block diagram which shows an example of a structure of an imaging device and a control apparatus. It is a block diagram which shows the other example of a structure of an imaging device and a control apparatus. It is a perspective view which shows an example of a structure of the epi-illumination optical system of excitation light. It is a perspective view which shows an example of a structure of the epi-illumination optical system of excitation light. It is a block diagram which shows an example of a structure of an imaging device. It is a top view which shows an example of a structure of the photon detection part and electric charge transfer part in the imaging device shown in FIG. It is a block diagram which shows the modification of an image acquisition part. It is a graph which shows an example of image data. It is a graph which shows an example of the image data after gain adjustment. It is a graph which shows an example of the image data after shading correction. It is a figure shown about the fluorescence image acquisition method of a sample, and the drive method of each part of a fluorescence image acquisition apparatus. It is a flowchart which shows the acquisition method of shading correction data. It is a flowchart which shows the acquisition method of the dark correction data and the fluorescence observation image of a sample.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Microscope apparatus, 11 ... Sample storage part, 12 ... Door, 13 ... Interlock mechanism, 14 ... Sample conveyance part, 15 ... Sample stage, 16 ... Stage control part, 17 ... Macro light source control part, 18 ... Micro Light source control unit, 20 ... macro image acquisition unit, 21 ... macro imaging device, 22 ... imaging optical system, 25 ... macro light source, 26 ... dark field light source, 27 ... bright field light source, 30 ... micro image acquisition unit, 31 Image capturing device for micro, 32 ... objective lens, 33 ... Z stage, 34 ... light guide optical system, 35 ... light source for micro, 36 ... transmission light source, 37 ... transmission illumination optical system, 38 ... shutter for transmission illumination, 40 ... Excitation light source, 41 ... excitation light irradiation optical system, 42 ... light guide optical system, 43 ... epi-illumination shutter, 44 ... fluorescence optical system unit, 45 ... dichroic mirror, 46 ... cylindrical lens system, 47 ... Okasurenzu system, 48 ... the collimating lens system,
DESCRIPTION OF SYMBOLS 60 ... Control apparatus, 61 ... Macro image acquisition control part, 62 ... Micro image acquisition control part, 65 ... Imaging condition setting part, 66, 67 ... Image data communication I / F, 68 ... Macro image processing part, 69 ... Micro image Processing unit 71... Display device 72. Input device 75. Image correction processing unit 76.

Claims (12)

A sample stage on which a sample to be subjected to fluorescence measurement is placed;
An image acquisition means including an imaging device for acquiring a one-dimensional image or a two-dimensional image having a first direction as a longitudinal direction as a fluorescence observation image of the sample;
Excitation light supply means for supplying excitation light for fluorescence measurement to the sample;
A fluorescence optical system unit including a dichroic mirror that reflects the excitation light from the excitation light supply means and irradiates the sample with fluorescence, and allows the fluorescence from the sample to pass to the image acquisition means;
The sample is scanned in the second direction by the imaging device to acquire a partial image, and the acquisition of the partial image is repeated a plurality of times while shifting the imaging position along the first direction. Scanning means for adjusting the positional relationship between the sample stage and the image acquisition means so as to acquire;
Image combining means for generating a fluorescence observation image of the sample by combining the plurality of partial images in the first direction; and
Correction data storage means for storing shading correction data for an image acquired by the imaging device in association with the fluorescence optical system unit;
An fluorescence image acquisition apparatus comprising: an image correction processing unit that performs shading correction for each of the plurality of partial images with reference to the shading correction data stored in the correction data storage unit. The correction data storage means stores dark correction data for an image acquired by the imaging device in correspondence with an exposure time in the imaging device;
The image correction processing unit performs dark correction on each of the plurality of partial images with reference to the dark correction data stored in the correction data storage unit in addition to the shading correction. Item 2. The fluorescent image acquisition device according to Item 1. The image acquisition means has a wavelength resolving optical system for decomposing fluorescence from the sample into three different wavelength components,
3. The fluorescent image acquisition device according to claim 1, wherein the imaging device includes three imaging devices that acquire the fluorescence observation image based on each of the three wavelength components resolved by the wavelength resolving optical system. .   4. The fluorescent image acquisition apparatus according to claim 3, wherein the correction data storage means stores three types of shading correction data for images acquired by each of the three imaging devices.   The imaging device in the image acquisition means may be a one-dimensional sensor capable of acquiring a one-dimensional image having the first direction as a longitudinal direction, or acquiring a two-dimensional image having the first direction as a longitudinal direction and TDI. The fluorescence image acquisition apparatus according to any one of claims 1 to 4, wherein the fluorescence image acquisition apparatus is a two-dimensional sensor that can be driven. The imaging device in the image acquisition means is
A light detection means having a plurality of pixels arranged in an array, and outputting charges generated according to the amount of light incident on the pixels;
Charge transfer means having a plurality of partial charge transfer portions provided along one arrangement direction of the plurality of pixels with respect to the light detection means and divided in the arrangement direction;
2. Each of the plurality of partial charge transfer units transfers charges from pixels in a predetermined light detection region of the light detection means in an output direction and outputs the charges from an output end. The fluorescence image acquisition apparatus as described in any one of -5. A sample placed on the sample stage is a target for fluorescence measurement. As a fluorescence observation image of the sample, a one-dimensional image or a two-dimensional image having a first direction as a longitudinal direction by an imaging device included in an image acquisition unit is used. An image acquisition step to acquire;
Fluorescence optics including a dichroic mirror that supplies excitation light for fluorescence measurement to the sample, reflects the excitation light to irradiate the sample, and passes fluorescence from the sample to the image acquisition means An excitation light irradiation step of irradiating the sample with the excitation light via a system unit;
The sample is scanned in the second direction by the imaging device to acquire a partial image, and the acquisition of the partial image is repeated a plurality of times while shifting the imaging position along the first direction. A scanning step of adjusting a positional relationship between the sample stage and the image acquiring means so as to acquire;
An image synthesis step for generating a fluorescence observation image of the sample by combining the plurality of partial images in the first direction,
A correction data storage step for storing shading correction data for an image acquired by the imaging device in association with the fluorescence optical system unit;
A fluorescence image acquisition method comprising: an image correction processing step of performing shading correction on each of the plurality of partial images with reference to the shading correction data stored in the correction data storage step. In the correction data storing step, dark correction data for an image acquired by the imaging device is stored in correspondence with an exposure time in the imaging device, and
The dark correction is performed on each of the plurality of partial images with reference to the dark correction data stored in the correction data storage step in addition to the shading correction in the image correction processing step. Item 8. The fluorescent image acquiring method according to Item 7. The image acquisition means has a wavelength resolving optical system for decomposing fluorescence from the sample into three different wavelength components,
9. The fluorescent image acquisition method according to claim 7, wherein the imaging device includes three imaging devices that acquire the fluorescence observation image based on each of the three wavelength components resolved by the wavelength resolving optical system. .   10. The fluorescent image acquisition method according to claim 9, wherein in the correction data storage step, three types of shading correction data for images acquired by each of the three imaging devices are stored.   The imaging device in the image acquisition means may be a one-dimensional sensor capable of acquiring a one-dimensional image having the first direction as a longitudinal direction, or acquiring a two-dimensional image having the first direction as a longitudinal direction and TDI. The fluorescence image acquisition method according to claim 7, wherein the fluorescence image acquisition method is a two-dimensional sensor that can be driven. The imaging device in the image acquisition means is
A light detection means having a plurality of pixels arranged in an array and outputting charges generated according to the amount of light incident on the pixels;
Charge transfer means provided along one arrangement direction of the plurality of pixels with respect to the light detection means, and having a plurality of partial charge transfer units divided in the arrangement direction;
8. Each of the plurality of partial charge transfer units transfers charges from pixels in a predetermined light detection region of the light detection means in an output direction and outputs the charges from an output end. The fluorescence image acquisition method as described in any one of -11.