JP2008051772A - 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 the first direction as a longitudinal direction as the fluorescence observation image of the sample S, an excitation light source 40 for supplying excitation light, and an excitation light irradiation optical system 41 including a cylindrical lens system for shaping an irradiation domain of the excitation light to the sample S. The device also has a configuration wherein a position relation between the stage 15 and the image acquisition part 30 is adjusted by the stage 15 which is an XY-stage so that the sample S is scanned in the second direction by the imaging device 31. The optical system 41 is constituted so that each long-axis direction agrees with each other in the irradiation domain to the sample S and in an imaging domain by the imaging device 31, and that the irradiation domain is wider than the imaging domain. <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, an observation image for the entire sample is acquired by scanning the sample with an imaging device capable of acquiring a one-dimensional image or a two-dimensional image having a predetermined direction as a longitudinal direction. The method 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

  In a fluorescence microscope for acquiring a fluorescence observation image of a sample, epi-illumination that irradiates the sample with excitation light using a dichroic mirror provided between the sample stage on which the sample is placed and the imaging device that acquires the image Is used. In such a fluorescence microscope, generally, a configuration is used in which excitation light supplied from an excitation light source is collimated, and the microscope objective lens functions as a condenser lens to irradiate the sample with excitation light.

  On the other hand, when acquiring a one-dimensional image having a predetermined direction as a longitudinal direction as described above, in the irradiation region of excitation light corresponding to a circular field of view with a normal microscope, excitation supplied to the sample There is a problem that a large part of the amount of light is wasted. In addition, when extra excitation light is irradiated outside the field of view corresponding to the imaging region by the imaging device in this way, it causes discoloration in the sample.

  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. An image acquisition means including an imaging device for acquiring a one-dimensional image or a two-dimensional image having a direction as a longitudinal direction, (3) an excitation light source for supplying excitation light for fluorescence measurement to the sample, and (4) to the sample And an excitation light irradiation optical system including a cylindrical lens system for shaping the irradiation region of the excitation light irradiated, and (5) a sample stage and image acquisition so that the sample is scanned in the second direction by the imaging device And (6) an excitation light irradiation optical system in which the major axis direction of the irradiation area on the sample and the imaging area by the imaging device coincide with each other and the irradiation area is imaged. Be wider than the area The sea urchin excitation light and irradiating to the specimen.

  The fluorescent image acquisition method according to the present invention includes: (a) a sample placed on a sample stage as a target of fluorescence measurement, and a fluorescence observation image of the sample as a longitudinal direction in a first direction by an imaging device 1 An image acquisition step for acquiring a two-dimensional image or a two-dimensional image, and (b) supplying excitation light for fluorescence measurement from the excitation light source to the sample, and shaping the irradiation region of the excitation light irradiated to the sample An excitation light irradiation step of irradiating the sample with excitation light via an excitation light irradiation optical system including a cylindrical lens system; and (c) a sample stage and an image so that the sample is scanned in the second direction by the imaging device. A scanning step that adjusts the positional relationship with the acquisition means, and (d) in the excitation light irradiation step, the major axis direction of the irradiation region on the sample and the imaging region by the imaging device coincide with each other; Morphism region and then irradiating the excitation light to be wider than the imaging area to the sample.

  In the fluorescence image acquisition device and the fluorescence image acquisition method described above, a cylindrical lens system is installed in the excitation light irradiation optical system that guides the excitation light for fluorescence measurement supplied from the excitation light source to the sample, and the cylindrical lens system The excitation light beam is shaped so that the irradiation region on the sample is a region having a predetermined direction as a major axis direction. Further, with respect to the irradiation region thus shaped, the major axis direction of the irradiation region of the excitation light on the sample and the major axis direction of the imaging region serving as a field of view by the imaging device are made to coincide with each other. As a result, of the amount of excitation light supplied to the sample, the amount of excess excitation light irradiated outside the field of view of the imaging device is reduced.

  Here, in the configuration in which the excitation light irradiation region is shaped as described above, the position, range, etc. of the irradiation region changes depending on the wavelength due to chromatic aberration for each wavelength component included in the excitation light irradiated on the sample. There is. In contrast, in the above configuration, the irradiation region of the sample with the excitation light beam shaped by the cylindrical lens system 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 can be reliably positioned within the irradiation region at each wavelength of the excitation light. . As the excitation light source, for example, a discharge tube can be used. Moreover, you may use light emitting elements, such as LED, as an excitation light source.

  In the fluorescence image acquisition apparatus described above, the excitation light irradiation optical system reflects the excitation light from the excitation light source and irradiates the sample, and also includes fluorescence optics including a dichroic mirror that passes the fluorescence from the sample to the image acquisition means. It is preferable to have a system unit (epi-illumination optical system unit). According to such a configuration, the incident illumination optical system for the excitation light with respect to the sample and the light guide optical system to the image acquisition means for the fluorescence generated in the sample can be preferably made compatible.

  Further, in the fluorescence image acquisition device, 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 as an imaging device, wavelength decomposition It is preferable to include three imaging devices that acquire fluorescence observation images based on each of the three wavelength components resolved by the optical system. 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 addition, in the fluorescence image acquisition device, for the imaging device in the image acquisition means, a one-dimensional sensor capable of acquiring a one-dimensional image with the first direction as the longitudinal direction, or a two-dimensional with the first direction as the longitudinal direction. It is preferable to use a configuration that is a two-dimensional sensor capable of acquiring images and TDI driving.

  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. For example, in such a configuration, a strip-shaped partial image obtained by scanning a sample in one direction with a one-dimensional sensor or a TDI-driven two-dimensional sensor is acquired with high resolution, and a plurality of partial images are synthesized in the other direction. By using a fluorescence observation image for the whole, image data of the sample can be suitably acquired with sufficiently high resolution.

  The excitation light source used in the fluorescence image acquisition device is preferably a white light source that supplies white light as excitation light. Examples of such excitation light sources include discharge tubes such as mercury lamps and halogen lamps, and LEDs.

  With respect to a specific method for acquiring the fluorescence observation image, the fluorescence image acquisition apparatus is configured such that the scanning unit sets the second scanning direction to the direction perpendicular to the first direction that is the longitudinal direction of the image acquired by the imaging device. The partial image is obtained by scanning the sample in the second direction by the imaging device and acquiring the partial image, and repeating the acquisition of the partial image a plurality of times while shifting the imaging position along the first direction. It is preferable to adjust the positional relationship between the sample stage and the image acquisition means so as to acquire.

  Similarly, in the fluorescence image acquisition method, in the scanning step, a direction orthogonal to the first direction, which is the longitudinal direction of the image acquired by the imaging device, is the second direction, which is the sample scanning direction, and the sample is captured by the imaging device. The sample stage is scanned so as to acquire a partial image by scanning in the second direction, and the partial image acquisition is repeated a plurality of times while shifting the imaging position along the first direction. It is preferable to adjust the positional relationship with the image acquisition means.

  Further, in this case, the fluorescence image acquisition device includes an image synthesis unit that generates a fluorescence observation image of the sample by arranging a plurality of partial images scanned in the second direction side by side and synthesizing the partial images. It is preferable. Similarly, the fluorescence image acquisition method may include an image synthesis step of generating a fluorescence observation image of the sample by arranging a plurality of partial images scanned in the second direction side by side and synthesizing the partial images. preferable.

  Regarding the irradiation condition of the excitation light to the sample, the fluorescence image acquisition apparatus has an excitation light irradiation optical system in which the irradiation area is 1.05 to 3 times in the major axis direction and 2 in the minor axis direction with respect to the imaging area. It is preferable to irradiate the sample with excitation light so as to be double to 20 times. Similarly, in the fluorescence image acquisition method, in the excitation light irradiation step, excitation is performed so that the irradiation area is 1.05 to 3 times in the long axis direction and 2 to 20 times in the short axis direction with respect to the imaging area. It is preferable to irradiate the sample with light. As a result, both the reduction of the amount of excess excitation light irradiated outside the field of view of the imaging device and the configuration in which the imaging region by the imaging device is reliably positioned within the irradiation region at each wavelength of the excitation light are preferably compatible. It becomes possible to do.

  According to the fluorescence image acquisition device and the fluorescence image acquisition method of the present invention, a cylindrical lens system for shaping the irradiation region of the excitation light irradiated from the excitation light source to the sample is installed in the excitation light irradiation optical system, and the sample Excitation supplied to the sample by irradiating the sample with excitation light under irradiation conditions in which the major axis directions of the irradiation region at the imaging device and the imaging region by the imaging device coincide with each other and the irradiation region is wider than the imaging region Of the amount of light, the amount of excess excitation light irradiated outside the field of view of the imaging device is reduced. Further, even when the irradiation region changes according to the wavelength due to the chromatic aberration, the imaging region by the imaging device can be reliably positioned within the irradiation region at each wavelength of the excitation light.

  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 the micro image acquisition unit 30, the sample stage 15 is configured so that the sample S is scanned in the second direction by the imaging device 31 when acquiring the fluorescence observation image. Functions as scanning means for adjusting the positional relationship between the image acquisition unit 30 and the image acquisition unit 30. 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.

  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. An epi-illumination means is constituted by a light source such as 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).

  The excitation light irradiation optical system 41 includes a cylindrical lens system for shaping the irradiation region of the excitation light irradiated from the excitation light source 40 to the sample S. 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, the irradiation region of the excitation light with respect to the sample S is set to a region whose predetermined direction is the 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 configured to irradiate the sample S with excitation light so that the irradiation area is wider than the imaging area. That is, 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.

  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.

  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, the sample S is irradiated with excitation light via the excitation light irradiation optical system 41 (excitation light). (Irradiation step), the sample S on the sample stage 15 is scanned in the scanning direction by the imaging device 31 (scanning step), and a strip-shaped partial image A having a desired resolution is obtained. 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. 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. Further, with respect to such an imaging region, the irradiation region of the excitation light by the excitation light irradiation optical system 41 including the cylindrical lens system is irradiated as shown by a broken line surrounding the imaging region in FIG. The major axis direction of the region and the imaging region are set to be equal to each other, and the irradiation region is set to be wider than the imaging region.

  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.

  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, an imaging device 31 that can acquire a one-dimensional image or a two-dimensional image with the first direction as the longitudinal direction is used as an imaging device for image acquisition. A fluorescence observation image of the sample S is acquired by scanning the sample S in the second direction by the imaging device 31. According to such a configuration, the fluorescence observation image of the sample S can be efficiently acquired with sufficiently high resolution.

  Further, for such a configuration of the image acquisition system, a cylindrical lens system 46 is installed in the excitation light irradiation optical system 41 that guides the excitation light for fluorescence measurement supplied from the discharge tube of the excitation light source 40 to the sample S, By this cylindrical lens system 46, the excitation light beam from the excitation light source 40 is shaped so that the irradiation region of the sample S is a region having a predetermined direction as the major axis direction. Further, with respect to the irradiation region shaped in this way, the major axis direction of the irradiation region of the excitation light on the sample S and the major axis direction (the above-described first direction) of the imaging region serving as the field of view by the imaging device 31. Match 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.

  Here, in the configuration in which the irradiation region of the excitation light to the sample S is shaped by the cylindrical lens system 46 of the excitation light irradiation optical system 41 as described above, irradiation with the sample S is performed for each wavelength component included in the excitation light by chromatic aberration. The range, area, etc. of the region may change depending on the wavelength. For example, FIGS. 9A to 9C schematically show irradiation regions R1, R2, and R3 that change at three wavelengths λ1, λ2, and λ3.

  In contrast, in the above configuration, the irradiation area of the excitation light beam shaped by the cylindrical lens system 46 on the sample S is set so that the irradiation area is wider than the imaging area. As a result, even when the irradiation region changes according to the wavelength due to chromatic aberration, as shown in FIG. 9D, the imaging region R0 by the imaging device 31 is changed at each wavelength component of the excitation light. It becomes possible to position in the irradiation area | region R1-R3 reliably. Further, in the example shown in FIG. 9, as shown in the setting range R5 in FIG. 9E, the setting range of the imaging region R0 may be within the range of the irradiation region R3 in the minor axis direction. In this case, for example, a method of setting the imaging region R0 substantially at the center of the irradiation region R3 can be used.

  A configuration of such an excitation light irradiation optical system 41 and a specific example of an irradiation region of the excitation light with respect to the sample S will be described. FIG. 10 is a diagram showing an example of the configuration of the excitation light irradiation optical system. FIG. 10A is a configuration diagram of the optical system in the major axis direction (first direction) of the irradiation region with respect to the sample S. FIG. (B) is a block diagram of the optical system about the short-axis direction (second direction) of the irradiation region. Here, in the present configuration example, the cylindrical lens system 46 is arranged in the direction where f = ∞ in the configuration of FIG. 10B.

  In the configuration example shown in FIG. 10, the excitation light emitted from the light emission point at the lamp house of the excitation light source 40 is built in front thereof and is paralleled by a collector lens adjusted so that the front focal position matches the light emission point. After being converted into a light beam, the light enters the cylindrical lens system 46. Here, by making the collector lens movable along the optical axis, it is possible to adjust the convergence and divergence state of the excitation light beam emitted from the excitation light source 40.

  The parallel luminous flux emitted from the collector lens is compressed in the first direction as shown in FIG. 10A to form a first linear excitation light image having the second direction as the major axis direction. At this time, the excitation light beam remains a parallel light beam in the second direction.

  Next, the focus lens system 47 is installed so that the front focal position matches the first linear excitation light image. Thereby, in the first direction, the compressed excitation light beam is returned to the parallel light beam again. On the other hand, in the second direction, which has remained a parallel light beam, the light beam is compressed to the rear focal position of the focus lens system 47. As a result, at the rear focal position of the focus lens system 47, a second linear excitation light image having a first axis orthogonal to the first linear excitation light image by the cylindrical lens system 46 as a major axis direction is obtained. It is formed.

  Further, a collimating lens system 48 is installed so that the front focal position matches the second linear excitation light image. Thereby, in the second direction, the compressed excitation light beam is returned to the parallel light beam again. On the other hand, in the first direction, which is a parallel light beam, the light beam is compressed at the rear focal position of the collimating lens system 48, and a third linear excitation light image is formed again with the second direction as the major axis direction. Is done.

  Here, the microscope objective lens 32 is arranged so that its entrance pupil matches the third linear excitation light image. As a result, the second linear excitation light image having the first direction as the major axis direction is projected onto the sample surface by the collimating lens system 48 and the objective lens 32, thereby forming an excitation light irradiation region for the sample S. It will be. In such a configuration, the optical image width on the sample surface in the first direction which is the major axis direction is, for example, about 1 mm. Further, the optical image width on the sample surface in the second direction which is the minor axis direction is, for example, about 15 μm.

  In such an excitation light irradiation optical system 41, the focal length of each lens and the lens system, the arrangement, and the combination thereof are the shape of the excitation light image formed on the sample surface that becomes the irradiation region of the excitation light to the sample S, It is preferable to set according to the size or the like or the incident condition of the excitation light beam with respect to the entrance pupil of the objective lens 32.

  In such a configuration, the major axis direction of the excitation light beam is a Kohler illumination configuration, and uniform illumination by the excitation light can be realized. On the other hand, the minor axis direction in which the excitation light beam is compressed has a critical illumination configuration in which the luminance distribution at the light emission point of the excitation light source 40 is reflected. However, since the minor axis direction of the excitation light irradiation region is the scanning direction of the sample S by the imaging device 31, even if the illumination by the excitation light becomes uneven in this direction, the fluorescence observation image There will be no problems with the acquisition.

  11 and 12 are diagrams illustrating examples of irradiation regions of excitation light irradiated onto the sample S by the excitation light irradiation optical system 41. FIG. Here, FIG. 11 is a diagram illustrating an irradiation region (irradiation light amount distribution of excitation light) when the sample S is irradiated with excitation light having a wavelength of 365 nm under the condition of no slit. FIG. 12 is a diagram showing an irradiation region when the sample S is irradiated with excitation light having a wavelength of 576 nm under the same conditions without slits.

  In the excitation light irradiation region shown in FIGS. 11 and 12, it can be seen that the shape and range of the irradiation region change depending on the wavelength. Further, when there is an axial misalignment or the like of an optical element such as a lens in the excitation light irradiation optical system 41, the positional deviation of the irradiation region also occurs as schematically shown in FIG. On the other hand, as described above, by setting the irradiation area of the excitation light on the sample surface wider than the imaging area, the irradiation area changes according to the wavelength due to chromatic aberration. In addition, the imaging region by the imaging device 31 can be surely positioned within the excitation light irradiation region.

  Here, regarding the specific irradiation conditions of the excitation light to the sample S, the excitation light irradiation optical system 41 has an irradiation area of the excitation light that is 1.05 times longer than the imaging area of the imaging device 31 in the major axis direction. It is preferable to irradiate the sample with excitation light so as to be 3 times and 2 times to 20 times in the minor axis direction. Thereby, it is preferable that the amount of excess excitation light emitted outside the field of view of the imaging device 31 is reduced and the imaging region by the imaging device 31 is reliably positioned within the irradiation region at each wavelength of excitation light. It is possible to achieve both. Specifically, it is preferable to set the irradiation area in consideration of conditions such as aberration of a lens used in the optical system.

  FIG. 13 is a graph showing the wavelength dependence of the light amount efficiency of excitation light on the sample surface when a slit is placed on the light guide optical path of excitation light. Here, with respect to the configuration of the lens or the like in the excitation light irradiation optical system, an optimized arrangement is assumed with the center wavelength of the excitation light being 470 nm, and the light amount emitted from the excitation light source is 1 at each wavelength. The result of having simulated the light quantity efficiency is shown. In FIG. 13, graph (a) shows the wavelength dependence of the light efficiency when the slit width is set to a slightly narrow 181 μm, and graph (b) shows the wavelength dependence of the light efficiency when the slit width is expanded to 800 μm. Showing sex.

  As can be seen from these graphs, in the graph (a) in which the slit width is narrowed, the light quantity efficiency of the excitation light on the sample surface varies with the wavelength. This is because the optical path of the excitation light changes according to the wavelength due to the influence of chromatic aberration, as described above for the irradiation region of the excitation light. On the other hand, in the graph (b) in which the slit width is widened, the light quantity efficiency of the excitation light is not affected by the wavelength.

  Here, in the configuration in which the irradiation area of the excitation light to the sample S is shaped using the cylindrical lens system 46 as described above, the irradiation area can be shaped without using a slit. Such a configuration is also effective in improving the light quantity efficiency of the excitation light and suppressing the wavelength dependence of the light quantity efficiency.

  Further, when a slit is used for shaping the irradiation region of the excitation light, the light amount of the excitation light rapidly changes at the edge of the irradiation region. Such an abrupt change in the amount of excitation light irradiation may cause the influence of fluorescence from outside the field of view, or the influence of chromatic aberration or fading when acquiring a fluorescence observation image. On the other hand, in the configuration in which the irradiation area is shaped by the optical system 41 including the cylindrical lens system 46, the change in the amount of excitation light at the edge of the irradiation area can be moderated as compared with the case where the slit is used. . However, even in such a case, a configuration may be adopted in which a slit is additionally provided in the excitation light irradiation optical system 41 as necessary.

  The effects of the fluorescent image acquisition device and the fluorescent image acquisition method according to the above embodiment and the specific configuration thereof will be further described.

  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, and also emits fluorescence from the sample S. A fluorescence optical system unit (an epi-illumination optical system unit) 44 including a dichroic mirror 45 that passes through 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.

  14 and 15 are perspective views showing an example of a 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 disposed in a predetermined direction with respect to the excitation light source 40. , 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. 14 and 5A) and an insertion position (FIGS. 15 and 5B). ) Between each other. 14 and 15, the specific structure of the moving mechanism 44a is not shown.

  Further, in such a fluorescence optical system unit 44, if necessary, an excitation filter for selecting the wavelength component of the excitation light in addition to the dichroic mirror 45, or a fluorescence filter for the fluorescence component, is further installed to install the optical system unit. It may be configured. In addition, since there are various types of samples S to be subjected to fluorescence measurement, the excitation light irradiation optical system 41 is provided with an ND filter or the like for adjusting the amount of excitation light as necessary. It is preferable to provide an optical element installation portion. 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.

  The discharge tube used as the excitation light source 40 in the fluorescent image acquisition device described above 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. Moreover, as an excitation light source, you may use light emitting elements, such as LED, for example.

  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. 16 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 addition to the one-dimensional CCD sensor, for example, a two-dimensional CCD sensor capable of TDI drive can be used as the imaging device for acquiring the fluorescence observation image of the sample S as described above. 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. 17 is a block diagram illustrating an example of a configuration of an imaging device 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. Such a multi-tap structure is effective in terms of speeding up the reading of charges from the imaging device 90.

  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. 18 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. 17. 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. 18, a charge transfer unit 93 having a storage unit 93a having a configuration similar to that of the lower storage unit 92a and the charge transfer unit 92, and partial charge transfer units T17 to T32 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.

  Then, an example of the image acquisition method of the sample S using the fluorescence image acquisition apparatus of the said structure is demonstrated. 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).

  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. In addition, as for the configuration of the excitation light irradiation optical system 41, the above-described configuration including the light guide optical system 42 and the fluorescence optical system unit 44 shows an example, and various other 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 schematic diagram shown about the irradiation area | region of the excitation light with respect to a sample, and the imaging area | region by an imaging device. It is a figure which shows an example of a structure of an excitation light irradiation optical system. It is a figure which shows an example of the irradiation area | region of the excitation light irradiated to a sample. It is a figure which shows an example of the irradiation area | region of the excitation light irradiated to a sample. It is a graph which shows the wavelength dependence of the light quantity efficiency of the excitation light on a sample surface. 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 the modification of an image acquisition part. 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 figure shown about the fluorescence image acquisition method of a sample, and the drive method of each part of a fluorescence image acquisition apparatus.

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.

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;
An excitation light source for supplying excitation light for fluorescence measurement to the sample;
An excitation light irradiation optical system including a cylindrical lens system for shaping the irradiation region of the excitation light irradiated to the sample;
Scanning means for adjusting the positional relationship between the sample stage and the image acquisition means so that the sample is scanned in the second direction by the imaging device;
The excitation light irradiation optical system emits the excitation light so that the major axis direction of the irradiation region on the sample and the imaging region by the imaging device coincide with each other and the irradiation region is wider than the imaging region. A fluorescent image acquisition apparatus irradiating the sample.   The excitation light irradiating optical system includes a dichroic mirror that reflects the excitation light from the excitation light source to irradiate the sample and allows the fluorescence from the sample to pass to the image acquisition unit. The fluorescence image acquisition apparatus according to claim 1, wherein 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. .   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 device according to any one of claims 1 to 3, wherein the fluorescence image acquisition device is a two-dimensional sensor that can be driven.   The fluorescence image acquisition apparatus according to any one of claims 1 to 4, wherein the excitation light source is a white light source that supplies white light as the excitation light.   The scanning means sets the direction orthogonal to the first direction, which is the longitudinal direction of the image acquired by the imaging device, as the second direction, which is the scanning direction of the sample, and causes the sample to be captured by the imaging device. The sample stage is configured to acquire a partial image by scanning in the direction of 2, and to acquire the partial image by repeating the acquisition of the partial image a plurality of times while shifting the imaging position along the first direction. The fluorescent image acquisition apparatus according to claim 1, wherein a positional relationship between the image acquisition unit and the image acquisition unit is adjusted.   The image combining means for generating a fluorescence observation image of the sample by combining the plurality of partial images scanned in the second direction in a row in the first direction. 6. The fluorescence image acquisition device according to 6.   The excitation light irradiation optical system applies the excitation light to the sample so that the irradiation region is 1.05 to 3 times in the long axis direction and 2 to 20 times in the short axis direction with respect to the imaging region. The fluorescence image acquisition apparatus according to claim 1, wherein the fluorescence image acquisition apparatus is irradiated with a fluorescent light. An image acquisition step of acquiring a one-dimensional image or a two-dimensional image having a first direction as a longitudinal direction by an imaging device as a fluorescence observation image of the sample placed on the sample stage, as a fluorescence observation image of the sample; ,
The excitation light for fluorescence measurement is supplied from the excitation light source to the sample, and the excitation light irradiation optical system includes a cylindrical lens system for shaping the irradiation region of the excitation light irradiated to the sample. An excitation light irradiation step for irradiating the sample with excitation light;
A scanning step of adjusting a positional relationship between the sample stage and the image acquisition unit so that the sample is scanned in the second direction by the imaging device;
In the excitation light irradiation step, the excitation light is applied so that the major axis direction of the irradiation region on the sample and the imaging region by the imaging device coincide with each other and the irradiation region is wider than the imaging region. A fluorescent image acquisition method characterized by irradiating a sample.   In the scanning step, a direction orthogonal to the first direction, which is a longitudinal direction of an image acquired by the imaging device, is defined as the second direction, which is a scanning direction of the sample, and the sample is removed by the imaging device. The sample stage is configured to acquire a partial image by scanning in the direction of 2, and to acquire the partial image by repeating the acquisition of the partial image a plurality of times while shifting the imaging position along the first direction. The fluorescence image acquisition method according to claim 9, wherein a positional relationship between the image acquisition unit and the image acquisition unit is adjusted.   The image synthesizing step of generating a fluorescence observation image of the sample by combining the plurality of partial images respectively scanned in the second direction in the first direction. The fluorescent image acquisition method according to 10.   In the excitation light irradiation step, the excitation light is applied to the sample such that the irradiation region is 1.05 to 3 times in the long axis direction and 2 to 20 times in the short axis direction with respect to the imaging region. The fluorescence image acquisition method according to any one of claims 9 to 11, wherein irradiation is performed.

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