Patent Document

CROSS REFERENCES TO RELATED APPLICATIONS 
       [0001]    The present application claims priority to Japanese Priority Patent Application JP 2009-200890 filed in the Japan Patent Office on Aug. 31, 2009, the entire content of which is hereby incorporated by reference. 
       BACKGROUND 
       [0002]    The present application relates to a fluorescence image producing method, a fluorescence image producing apparatus, and a fluorescence image producing program that are suitable for a field of observing a tissue section, for example. 
         [0003]    In general, a biological sample is fixed to a supporting base such as a glass slide and stained in a predetermined manner before being used in the field of pathology. 
         [0004]    In a pathological diagnosis, the presence of a malignant tumor is primarily determined in a morphological view using a sample made by performing a Hematoxylin-Eosin (HE) staining on the tissue section or performing a Papanicolaou staining on the secretion cell. If a malignant tumor or a suspected site with the malignant tumor is found, the presence, the type, and the stage of the malignant tumor are secondarily determined in a molecular biological view using a sample made by performing a fluorescence staining on the tissue section or the secretion cell. 
         [0005]    After a long storage period of such a sample, degradation of quality and discoloration of the biological sample occur and microscopic visibility of the biological sample also lowers, in general. Because the sample is sometimes diagnosed in a laboratory other than a facility where the sample is made such as a hospital, the biological sample is generally sent by mail, which takes a certain time. 
         [0006]    In view of such situations, there is proposed an apparatus for storing the biological sample in the form of an image data (see, for example, Japanese Unexamined Patent Application No. 2003-222801). 
         [0007]    By the way, in a case of obtaining an image of the entire biological sample enlarged to a predetermined scale, it is difficult to form the entire image on an imaging surface, and therefore a technique is generally used in which the biological sample is sectioned and enlargements of sectioned parts are connected together. Because this technique includes a step of moving a stage to focus on the sample with respect to each part of the sample, it takes a longer time to connect the enlargements of the parts. 
       SUMMARY 
       [0008]    However, in a case of connecting the enlarged images of the sample parts where the fluorescence staining is performed, due to the longer time taken to connect the enlargements of the parts, not only the discoloration of fluorescent material to be labeled to a target is accelerated but also the biological sample is overloaded. 
         [0009]    On the other hand, the sample performed with the fluorescence staining is not taken as an image because it is clear and colorless in an unexcited state, and therefore it is difficult to focus on the image based on the contrast of the image. 
         [0010]    It is therefore desirable to provide the fluorescence image obtaining method, the fluorescence image obtaining apparatus, and the fluorescence image obtaining program that can focus on an image without accelerating the discoloration of the fluorescent material to be labeled to the target or overloading the biological sample. 
         [0011]    In an embodiment, a method of producing an image associated with a biological sample is provided. The method includes focusing on the biological sample based on fluorescence of a first fluorescent material, wherein the biological sample is stained with the first fluorescent material and a second fluorescent material; and capturing the image based on fluorescence of the second fluorescent material, wherein the first and second fluorescent materials have different excitation wavelengths. 
         [0012]    In an embodiment, the image is produced in a dark field imaging mode. 
         [0013]    In an embodiment, the image is produced in a dark field imaging mode in combination with a bright field imaging mode. 
         [0014]    In an embodiment, the image is captured as an entire image. 
         [0015]    In an embodiment, the image is captured as a partial image. 
         [0016]    In an embodiment, the image is captured as a plurality of partial images that are connected to form the image. 
         [0017]    In an embodiment, image production is controlled by a data processing unit including control of focusing and capturing. 
         [0018]    In an embodiment, after focusing, an excitation light source is driven by the data processing unit to initiate image capturing. 
         [0019]    In an embodiment, a part of the biological sample is focused at a predetermined location. 
         [0020]    In an embodiment, a part of the biological sample is focused at a variable location. 
         [0021]    In another embodiment, an apparatus for producing an image associated with a biological sample is provided. The apparatus includes a base unit configured to receive the biological sample that is stained with a first fluorescent material and a second fluorescent material having different excitation wavelengths; a first light source configured to irradiate the biological sample thereby allowing focusing of the biological sample based on fluorescence of the first fluorescent material; and a second light source configured to irradiate the biological sample thereby allowing capturing of the image based on fluorescence of the second fluorescent material. 
         [0022]    In an embodiment, the apparatus further includes a data processing unit configured to control image production including in communication with the base unit, the first light source and the second light source. 
         [0023]    In an embodiment, the data processing unit stops driving the first light source and starts driving the second light source to initiate image capturing. 
         [0024]    In a further embodiment, a computer readable memory device is provided storing instructions to cause a data processing unit to focus on a biological sample based on fluorescence of a first fluorescent material, wherein the biological sample is stained with the first fluorescent material and a second fluorescent material; and capture an image associated with the biological sample based on fluorescence of the second fluorescent material, wherein the first and second fluorescent materials have different excitation wavelengths. 
         [0025]    In yet another embodiment, a method of producing an image associated with a biological sample is provided. The method includes irradiating the biological sample at a first excitation wavelength; focusing on the biological sample; irradiating the biological sample at a second excitation wavelength; and capturing the image associated with the biological sample. 
         [0026]    In yet a further embodiment, an apparatus for producing an image associated with a biological sample is provided. The apparatus includes a first light source configured to irradiate the biological sample at a first excitation wavelength thereby allowing focusing of the biological sample; and a second light source configured to irradiate the biological sample at a second excitation wavelength thereby allowing capturing of the image. 
         [0027]    According to an embodiment, the sample part is irradiated with the excitation light for the fluorescent material to be labeled to the control for counterstain, which is contained more than the target, when a dark field image of a focusing object is obtained, and the sample part is irradiated with the excitation light for the fluorescent material to be labeled to the target when a dark field image of a recording object is obtained. 
         [0028]    Therefore, the focus can be adjusted using the image of the sample part of the fluorescent material to be labeled to the control for counterstain, which is contained more than the target, without making the fluorescent material to be labeled to the target emit a light. As a result, the focus can be adjusted without accelerating the discoloration of the fluorescent material to be labeled to the target or overloading the biological sample. 
         [0029]    Additional features and advantages are described herein, and will be apparent from the following Detailed Description and the figures. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0030]      FIG. 1  schematically illustrates a configuration of a biological sample image obtaining apparatus; 
           [0031]      FIG. 2  schematically illustrates a fluorescence image; 
           [0032]      FIG. 3  is a block diagram showing a configuration of a data processing unit; 
           [0033]      FIG. 4  is a block diagram showing a functional configuration of a central processing unit (CPU) that executes a bright field imaging mode; 
           [0034]      FIG. 5  is a photograph showing a bright field entire image; 
           [0035]      FIG. 6  is a schematic diagram for explaining an allocation of an imaging area of a biological sample; 
           [0036]      FIG. 7  is a block diagram showing a functional configuration of the CPU that executes a dark field imaging mode; 
           [0037]      FIG. 8  is a photograph showing a dark field partial image of a control marker; 
           [0038]      FIGS. 9A and 9B  are schematic diagrams showing examples of an image to be used as a focusing object; 
           [0039]      FIG. 10  is a photograph showing a dark field partial image of a control marker and the fluorescence marker; 
           [0040]      FIG. 11  is a flowchart showing a dark field image obtaining process; 
           [0041]      FIG. 12  schematically illustrates an arrangement of a control-marker excitation light source according to another embodiment; and 
           [0042]      FIG. 13  schematically illustrates another arrangement of the control-marker excitation light source according to still another embodiment. 
       
    
    
     DETAILED DESCRIPTION 
       [0043]    The present application will be explained below in greater detail with reference to the drawings according to an embodiment. 
         [0044]    &lt;1. Embodiment&gt; 
         [0045]    [1-1. Configuration of biological sample image obtaining apparatus] 
         [0046]    [1-2. Configuration of microscope] 
         [0047]    [1-3. Configuration of data processing unit] 
         [0048]    [1-4. Specific processing of bright field imaging mode] 
         [0049]    [1-5. Specific processing of dark field imaging mode] 
         [0050]    [1-6. Image obtaining process in dark field imaging mode] 
         [0051]    [1-7. Effects] 
         [0052]    &lt;2. Other embodiments&gt; 
       1. Embodiment 
     1-1. Configuration of Biological Sample Image Obtaining Apparatus 
       [0053]      FIG. 1  illustrates a biological sample image obtaining apparatus  1  according to an embodiment. The biological sample image obtaining apparatus  1  includes a microscope  10  and a data processing unit  20 . 
       1-2. Configuration of Microscope 
       [0054]    The microscope  10  includes a stage (also referred to below as a movable stage)  31  movable in all directions (x-axis, y-axis, and z-axis directions) parallel to and perpendicular to a plane on which a slide SG such as a glass plate is placed (also referred to below as a slide placement plane). A slide holder  32  is provided on the slide placement plane. 
         [0055]    When the slide SG is set, the slide holder  32  is moved to a position specified as a setting place (also referred to below as a slide setting position). At the slide setting position, the slide SG contained in a slide container (not shown) is removed and set in the slide holder  32  by a slide setting mechanism (not shown). 
         [0056]    A tissue section or a secretion cell including a connective tissue such as blood, an epithelial tissue, or both of them is fixed to the slide SG contained in the slide container (not shown) as a biological sample SPL by a predetermined fixation technique, and stained as necessary. 
         [0057]    The staining includes not only typical staining techniques represented by a Hematoxylin-Eosin (HE) staining, a Giemsa staining, and a Papanicolaou staining but also fluorescence staining techniques represented by a fluorescence in situ hybridization (FISH) and an enzyme antibody technique. 
         [0058]    In addition to a fluorescent label applied to a probe (also referred to below as a fluorescence marker), the fluorescence staining technique generally uses another fluorescent label to be contrasted with the fluorescence marker on the probe (also referred to below as a control marker). 
         [0059]    The control marker has an excitation wavelength different from the excitation wavelength of the fluorescence marker. For example, the excitation wavelength may be approximately 365 nm, and 4′,6-diamidino-2-pheylindole (DAPI) is commonly used. With the DAPI, a target to be contrasted with the target of the fluorescence marker (also referred to below as a control target) is a cell nucleus. 
         [0060]    When an image of the biological sample SPL is taken, the slide holder  32  is moved to a position specified as a location for a microscopic examination (also referred to below as a microscopic position). In this case, either a bright field imaging mode or a dark field imaging mode is executed. 
         [0061]    In the case of the bright field imaging mode, an illuminating light is emitted from a bright field light source  41  to the biological sample SPL. The illuminating light is reflected by a reflection mirror  42 , irradiated onto the biological sample SPL in the microscopic position as a visible light through a bright field filter  43 , and then reaches an objective lens  44 . 
         [0062]    The magnification power of the objective lens  44  is either as low as an image of the entire biological sample SPL (also referred to below as a bright field entire image) can be formed or as high as an image of only a part of the biological sample SPL (also referred to below as a bright field partial image) can be formed. 
         [0063]    The microscope  10  forms an image of the biological sample SPL obtained through the illuminating light on an imaging surface of an imaging device  46  as the bright field entire image or the bright field partial image after enlarging the image using the objective lens  44  and an imaging lens  45 . 
         [0064]    As described above, the microscope  10  is configured to obtain an entire or partial bright field image (the bright field entire image or the bright field partial image) of the biological sample SPL in the bright field imaging mode. 
         [0065]    In  FIG. 1 , there are a dichroic mirror  54  and an emission filter  55  on a light path between the objective lens  44  and the imaging lens  45 . However, in the case of the bright field imaging mode, the dichroic mirror  54  and the emission filter  55  are retired from the light path so that the visible light entering through the bright field filter  43  is not absorbed or reflected by the mirror and the filter. 
         [0066]    On the other hand, in the case of the dark field imaging mode, a light that excites both the fluorescence marker on the probe and the control marker (also referred to below as an excitation light) is emitted from an excitation light source  51 . The magnification power of the objective lens  44  when the excitation light is emitted is as high as a part of a fluorescence image of the biological sample SPL (also referred to below as a dark field partial image) can be formed. 
         [0067]    A collimator lens  52  makes the excitation light emitted from the excitation light source  51  into a parallel beam, and an excitation filter  53  eliminates lights other than the excitation light. The excitation light transmitted through the excitation filter  53  is reflected by the dichroic mirror  54 , and then focused on the microscopic position by the objective lens  44 . 
         [0068]    When the probe is coupled to the target and the control target of the biological sample SPL in the microscopic position, the fluorescence marker applied to the probe and the control marker produce a luminescence by the excitation light. The luminescence is transmitted through the dichroic mirror  54  via the objective lens  44  and reaches the imaging lens  45  after the lights other than the luminescence of the fluorescent material are absorbed by the emission filter  55 . 
         [0069]    The microscope  10  enlarges an image obtained through the luminescence of the fluorescence marker and the control marker using the objective lens  44  and the imaging lens  45 , and forms the enlarged image on the imaging surface of the imaging device  46  as the dark field partial image. 
         [0070]    As described above, the microscope  10  is configured to obtain a fluorescence image of the sample part (dark field partial image) in the dark field imaging mode. 
         [0071]    Although  FIG. 1  shows a dichroic mirror  63  on the light path between the excitation filter  53  and the dichroic mirror  54 , the dichroic mirror  63  transmits the excitation light that is transmitted through the excitation filter  53 . 
         [0072]      FIG. 2  shows an example of the fluorescence image of the biological sample SPL (dark field partial image). The image shown in  FIG. 2  is taken by staining a mammary tissue by the FISH technique using a reagent called PathVysion HER-2DNA probe kit produced by Abbott Laboratories. 
         [0073]    Points indicated by arrows in  FIG. 2  represent the fluorescence marker, specifically the fluorescence marker applied to the probes for the HER2/neu genes encoding the HER2 protein. Concentrated areas with shades in  FIG. 2  represent the control marker forming an outer shape substantially matching the outer shape of the tissue with the HE stain. 
         [0074]    Although the reagent includes the fluorescence marker of the probes for the alpha satellite DNA sequence in the centromere region of the seventeenth chromosome as well as the probes for the HER2/neu genes, the fluorescence marker is omitted in  FIG. 2  for convenience. 
         [0075]    In addition to the configuration described above, the microscope  10  includes a light source (also referred to below as a control-marker excitation light source)  61  configured to emit an excitation light that excites the control marker while leaving the fluorescence marker unexcited (also referred to below as a control-marker exclusive excitation light). 
         [0076]    The control-marker exclusive excitation light is emitted from the control-marker excitation light source  61  in a focusing process when the dark field partial image of the biological sample SPL is obtained. 
         [0077]    The control-marker exclusive excitation light emitted from the control-marker excitation light source  61  is made into a parallel beam by a collimator lens  62 , reflected by the dichroic mirror  63  and the dichroic mirror  54 , and then focused on the microscopic position by the objective lens  44 . 
         [0078]    When the probe is coupled to the control target of the biological sample SPL in the microscopic position, the control marker applied to the probe produces a luminescence by the control-marker exclusive excitation light. The luminescence is transmitted through the dichroic mirror  54  via the objective lens  44  and reaches the imaging lens  45  after the lights other than the luminescence of the fluorescent material are absorbed by the emission filter  55 . 
         [0079]    The microscope  10  enlarges an image obtained through the luminescence of the control marker using the objective lens  44  and the imaging lens  45 , and forms the enlarged image on the imaging surface of the imaging device  46  as the dark field partial image. 
         [0080]    The data processing unit  20  controls the movable stage  31  based on the dark field partial image so that the corresponding sample part is focused on. Furthermore, when the sample part is focused on, the data processing unit  20  makes the excitation light source  51  emit the excitation light instead of the control-marker excitation light source  61 , and stores the dark field partial image obtained with the excitation light. 
         [0081]    As described above, the biological sample image obtaining apparatus  1  is configured to obtain the dark field partial image obtained with the control-marker exclusive excitation light as the dark field partial image of the focusing object, and obtain the dark field partial image obtained with the excitation light as the dark field partial image of the recording object. 
       1-3. Configuration of Data Processing Unit 
       [0082]    A configuration of the data processing unit  20  is explained below. As shown in  FIG. 3 , the data processing unit  20  includes a central processing unit (CPU)  71  that performs various controls and various types of hardware connected to the CPU  71 . 
         [0083]    Specifically, a read only memory (ROM)  72 , a random access memory (RAM)  73  that serves as a working memory of the CPU  71 , an operation input unit  74  that inputs a command corresponding to a user operation, an interface  75 , a display unit  76 , and a storage unit  77  are connected to the CPU  71  via a bus  78 . 
         [0084]    The ROM  72  stores computer programs for performing various processings. The interface  75  is connected to the microscope  10  (drive systems of the movable stage  31 , the light sources  41 ,  51 ,  61 , the imaging device  46 , the objective lens  44 , and the emission filter  55 ). 
         [0085]    As the display unit  76 , a liquid crystal display, an electroluminescence (EL) display, or a plasma display may be used. As the storage unit  77 , a magnetic disk such as a hard disk (HD), a semiconductor memory, or an optical disk may be used. Alternatively, a portable memory such as a universal serial bus (USB) memory and a compact flash (CF) memory may be used. 
         [0086]    The CPU  71  loads a computer program corresponding to the command provided by the operation input unit  74  from among a plurality of the computer programs stored in the ROM  72  to the RAM  73 , and controls the display unit  76  and the storage unit  77  according to the loaded program. 
         [0087]    The CPU  71  is also configured to control each unit of the microscope  10  via the interface  75  according to the loaded program. 
       1-4. Specific Processing of Bright Field Imaging Mode 
       [0088]    When the CPU  71  receives an execution command of the bright field imaging mode from the operation input unit  24 , the CPU  71  loads a computer program corresponding to the imaging mode to the RAM  23 . 
         [0089]    In this case, according to the computer program corresponding to the bright field imaging mode, the CPU  71  functions as a slide setting control unit  81 , a bright field image obtaining unit  82 , and a data recording unit  83 , as shown in  FIG. 4 . 
         [0090]    The slide setting control unit  81  drives the movable stage  31  and positions the slide holder  32  in the slide setting position when the slide setting control unit  81  receives the execution command of the imaging mode or a notice that the bright field image obtaining unit  82  or the slide should be replaced. 
         [0091]    In a case where the slide holder  32  is positioned in the slide setting position, the slide setting control unit  81  drives the movable stage  31  again after a predetermined waiting period to move the slide holder  32  from the slide setting position to the microscopic position. During the waiting period, as described with reference to  FIG. 1 , the slide SG contained in the slide container is set in the slide holder  32  by the slide setting mechanism. 
         [0092]    For example, at the time point of the slide holder  32  being positioned in the microscopic position, the bright field image obtaining unit  82  arranges the objective lens  44  with low power on an optical axis between the dichroic mirror  54  and the imaging lens  45 , and drives the bright field light source  41 . 
         [0093]    The bright field image obtaining unit  82  then focuses on the biological sample SPL based on the contrast of the bright field entire image formed by the imaging device  46 , thereby obtaining, for example, the bright field entire image as shown in  FIG. 5 . 
         [0094]    When the bright field entire image is obtained, bright field image obtaining unit  82  arranges a predetermined objective lens  44  with high power in a predetermined position on the light path instead of the objective lens  44  with low power arranged in the predetermined position on the light path. 
         [0095]    The bright field image obtaining unit  82  then detects a contour of the biological sample SPL from the bright field entire image. Applied to the detection of the contour is, for example, a technique of performing a binarization process for distinguishing the biological sample SPL from other areas and then performing an extraction process of extracting the outer shape of the biological sample SPL. 
         [0096]    Upon detecting the contour of the biological sample SPL from the bright field entire image, the bright field image obtaining unit  82  determines the size of an area in which the partial image should be taken (also referred to below as an imaging area), and allocates imaging areas AR to the biological sample SPL as shown in  FIG. 6 . 
         [0097]    The imaging area AR is determined at least based on the magnification power of the objective lens  44  and the size of the imaging surface of the imaging device  46 , and it is allocated under the condition of either including a part of the contour of the biological sample SPL or being included within the contour. Although the imaging areas AR do not overlap one another in  FIG. 6 , adjacent areas may partially overlap each other. 
         [0098]    After allocating the imaging areas AR to the biological sample SPL in the bright field entire image, the bright field image obtaining unit  82  sequentially moves the movable stage  31  so that each part of the biological sample SPL becomes the imaging area AR, and obtains the bright field partial image of each part corresponding to the imaging area AR. 
         [0099]    The bright field image obtaining unit  82  then connects the bright field partial images, thereby forming a connected image (also referred to below as a bright field part connected image). The bright field image obtaining unit  82  is configured to issue a notice to the slide setting control unit  81  at this time that the slide should be replaced. 
         [0100]    When the bright field part connected image is formed by the bright field image obtaining unit  82 , the data recording unit  83  generates data about the bright field partial images and the bright field entire image obtained in the process of generating the bright field part connected image as well as the bright field part connected image (also referred to below as bright field image data), and stores the data in the storage unit  77 . 
         [0101]    At this time, the data recording unit  83  generates data about the imaging areas AR allocated to the biological sample SPL in the bright field entire image by the bright field image obtaining unit  82  (also referred to below as imaging area allocation data), and associates the data with the bright field image data. 
         [0102]    The data recording unit  83  is also configured to associate data about the biological sample SPL (also referred to below as sample data) with the bright field image data. 
         [0103]    The sample data includes, for example, information such as the name, sexuality, and age of a subject of the examination, date of collecting the biological sample SPL, and the like. Also additional information can be obtained through an input from the operation input unit  74  or by a read unit that scans a bar-code on the glass slide SG. 
       1-5. Specific Processing of Dark Field Imaging Mode 
       [0104]    When the CPU  71  receives an execution command of the dark field imaging mode from the operation input unit  24 , the CPU  71  loads a computer program corresponding to the imaging mode to the RAM  23 . 
         [0105]    In this case, according to the computer program corresponding to the dark field imaging mode, the CPU  71  functions as the slide setting control unit  81 , a dark field image obtaining unit  92 , and a data recording unit  93 , as shown in  FIG. 7  in which the same reference numeral is applied to the same constituent shown in  FIG. 4 . 
         [0106]    For example, at the time point of the slide holder  32  being positioned in the microscopic position, the dark field image obtaining unit  92  arranges the objective lens  44  with high power on the optical axis between the dichroic mirror  54  and the imaging lens  45 . 
         [0107]    At this time, the dark field image obtaining unit  92  also obtains the sample data about the biological sample SPL provided to the slide holder  32 , and searches in the storage unit  77  for the sample data including, for example, the name of the subject and the date of collection matching those in the obtained sample data. 
         [0108]    In a case where the sample data is not found in the storage unit  77 , the dark field image obtaining unit  92  issues a notice of the fact through, for example, the display unit  76 . 
         [0109]    On the other hand, when the sample data is found in the storage unit  77 , the dark field image obtaining unit  92  reads the imaging area allocation data associated with the sample data from the storage unit  77 . The dark field image obtaining unit  92  then allocates the imaging areas AR ( FIG. 6 ) to the biological sample SPL provided to the slide holder  32  at this time point in the same state as the allocation to the biological sample SPL in the bright field imaging mode. 
         [0110]    Thus, only the part corresponding to the biological sample SPL on the glass slide SG fixed to the slide holder  32  is precisely allocated as an allocation object even when the fluorescent material in the biological sample SPL is not excited. 
         [0111]    After allocating the imaging areas AR to the biological sample SPL, the dark field image obtaining unit  92  selects each sample part allocated with the imaging area AR as an imaging object in a predetermined order, and obtains an image (dark field partial image) of the sample part selected as the imaging object. 
         [0112]    The way of obtaining the image (dark field partial image) of a single sample part selected as the imaging object is specifically explained below. That is, after moving the movable stage  31  in the x-axis direction or the y-axis direction so that the sample part of the biological sample SPL to be imaged is included in the imaging area, the dark field image obtaining unit  92  drives the control-marker excitation light source  61  to irradiate the biological sample SPL with the control-marker exclusive excitation light. 
         [0113]    As a result, the imaging device  46  forms a dark field image (dark field partial image) of the control marker in the sample part to be imaged from among the control marker excited by the control-marker exclusive excitation light.  FIG. 8  shows an example of the dark field partial image of the control marker. 
         [0114]    The dark field image obtaining unit  92  obtains a part of the dark field partial image from the imaging device  46 , and focuses on the sample part of the imaging object based on the contrast of the part. Accordingly, the focusing time is reduced compared with the case of focusing based on the contrast of the entire dark field partial image. 
         [0115]    The part of the dark field partial image used as the focusing object may be, for example, defined by lines at a predetermined interval as shown in  FIG. 9A , or defined as a group of a center pixel and pixels surrounding the center pixel as shown in  FIG. 9B . 
         [0116]    Upon completion of focusing on the sample part of the imaging object, the dark field image obtaining unit  92  stops driving the control-marker excitation light source  61  and starts driving the excitation light source  51 . As a result, the imaging device  46  forms a dark field image (dark field partial image) of the control marker and the fluorescence marker in the sample part of the imaging object as shown in  FIG. 10 , for example, among the whole control marker excited by the control-marker excitation light and the whole fluorescence marker. 
         [0117]    The dark field image obtaining unit  92  obtains the entire dark field partial image from the imaging device  46  as the dark field partial image of the recording object. 
         [0118]    As described above, the dark field image obtaining unit  92  obtains the dark field partial image of each sample part of the recording object in a predetermined order using the above-described obtaining technique. 
         [0119]    When the dark field partial images of all the sample parts of the recording object are obtained, the dark field image obtaining unit  92  generates a connected image (also referred to below as a dark field part connected image) by connecting the dark field partial images to one another. At this time, the dark field image obtaining unit  92  notifies the slide setting control unit  81  that the slide should be replaced. 
         [0120]    When the dark field partial images of all the sample parts of the recording object are obtained by the dark field image obtaining unit  92 , the data recording unit  93  generates data about the dark field partial images and the dark field part connected image obtained in the process of generating the images (also referred to below as the dark field image data). 
         [0121]    The data recording unit  93  then stores the dark field image data in the storage unit  77  in association with the bright field image data associated with the imaging area allocation data read by the dark field image obtaining unit  92  in the process of generating the dark field image data. 
       1-6. Image Obtaining Process in Dark Field Imaging Mode 
       [0122]    A dark field image obtaining process is explained below with reference to a flowchart shown in  FIG. 11 . 
         [0123]    Upon receipt of the execution command of the imaging mode, the CPU  71  starts the dark field image obtaining process, moves to Step SP 1 , positions the slide holder  32  in the slide setting position, and then moves to Step SP 2 . 
         [0124]    At Step SP 2 , after the predetermined waiting period, the CPU  71  positions the slide holder  32  in the microscopic position from the slide setting position, and moves to Step SP 3 . At Step SP 3 , the CPU  71  arranges the objective lens  44  with high power on the optical axis between the dichroic mirror  54  and the imaging lens  45 , and moves to Step SP 4 . 
         [0125]    At Step SP 4 , the CPU  71  allocates the imaging areas AR ( FIG. 6 ) to the biological sample SPL provided to the slide holder  32  in the same state as the allocation to the biological sample SPL in the bright field imaging mode, and moves to Step SP 5 . 
         [0126]    At Step SP 5 , the CPU  71  selects one of the sample parts, as the imaging object, to which the imaging areas AR are allocated, and moves to Step SP 6 . At Step SP 6 , the CPU  71  drives the control-marker excitation light source  61 , focuses on the sample part of the imaging object based on the contrast of a part of the dark field image (dark field partial image) of the control marker in the sample part of the imaging object, and moves to Step SP 7 . 
         [0127]    At Step SP 7 , the CPU  71  stops driving the control-marker excitation light source  61  and starts driving the excitation light source  51  at the same time, and then moves to Step SP 8 . At Step SP 8 , the CPU  71  obtains the dark field image of the control marker and the fluorescence marker in the sample part of the imaging object as the dark field partial image of the recording object, and moves to Step SP 9 . 
         [0128]    At Step SP 9 , the CPU  71  stops driving the excitation light source  51 , and moves to Step SP 10 . At Step SP 10 , the CPU  71  determines whether the dark field partial images of all the sample parts allocated with the imaging areas AR are obtained, and returns to Step SP 5  when there remains any sample part of which the dark field partial image is not obtained. 
         [0129]    On the other hand, when the dark field partial images of all the sample parts are obtained, the CPU  71  moves to Step SP 11 , generates the dark field part connected image by connecting the dark field partial images, and moves to Step SP 12 . 
         [0130]    At Step SP 12 , the CPU  71  generates the dark field image data about the dark field part connected image obtained at Step SP 11  and the dark field partial images before being connected, stores the dark field image data in the storage unit  77  in association with the corresponding bright field image data, and moves to Step SP 13 . 
         [0131]    At Step SP 13 , the CPU  71  determines whether there is any glass slide SG left in the slide container, and if there is any left, the CPU  71  returns to Step SP 1  to repeat the process described above. On the other hand, if there is no glass slide SG left in the slide container, the CPU  71  terminates the dark field image obtaining process. 
       1-7. Effects 
       [0132]    With the configuration described above, in a case of obtaining the dark field partial image, the biological sample image obtaining apparatus  1  drives the control-marker excitation light source  61  to irradiate the sample part of the imaging object with the control-marker exclusive excitation light. The biological sample image obtaining apparatus  1  then focuses on the part of the biological sample based on the contrast in the dark field image of the control marker in the sample part excited by the control-marker exclusive excitation light. 
         [0133]    Upon completion of focusing on the sample part of the imaging object, the biological sample image obtaining apparatus  1  drives the excitation light source  51  instead of the control-marker excitation light source  61  to irradiate the sample part of the imaging object with the excitation light. The biological sample image obtaining apparatus  1  then obtains the dark field image of the fluorescence marker and the control marker in the sample part excited by the excitation light as the dark field partial image to be recorded. 
         [0134]    Because the control marker targets a cell nucleus, its amount included in the biological sample SPL is much larger than that of the fluorescence marker that targets a certain gene, and the control marker produces a light vaguely presenting the biological sample as a whole. 
         [0135]    Therefore, the biological sample image obtaining apparatus  1  can focus on each sample part of the biological sample SPL with a high accuracy without making the fluorescence marker emit a light and, as a result, obtain the dark field partial image of each sample part to be recorded. 
         [0136]    By the way, as the excitation light source  51 , a mercury lamp, a xenon lamp, or a metal halide lamp is used in general. Partially because the wavelength spectra of these lamps are stable, the transmittances of the excitation filer  53  and the emission filter  55  are adjusted so that the fluorescence marker appears brighter than the control marker. 
         [0137]    Therefore, to observe the control marker using the excitation light source  51 , the irradiation intensity of the excitation light source  51  is made higher compared with the case of observing the fluorescence marker. As a result, in this case, discoloration of both the fluorescence marker and the control marker is accelerated and also the biological sample SPL is overloaded. 
         [0138]    On the other hand, to extract only a light that excites the control marker alone from the excitation light emitted from the excitation light source  51  using a filter, the filter is replaced when the dark field partial image is focused on and when dark field partial image is obtained. As a result, in this case, the size of the apparatus increases due to the increased number of the filters. Furthermore, it takes a longer time to obtain the dark field partial image in this case, and therefore this approach is not realistic. 
         [0139]    On the contrary, the biological sample image obtaining apparatus  1  emits the control-marker exclusive excitation light from the control-marker excitation light source  61 . Therefore, the biological sample image obtaining apparatus  1  can obtain the dark field partial image faster than the case of observing the control marker using the excitation light source  51  without accelerating the discoloration of both the fluorescence marker and the control marker. 
         [0140]    With the configuration described above, the biological sample image obtaining apparatus  1  can adjust the focus without accelerating the discoloration of the fluorescent material to be labeled to the target or overloading the biological sample by using the dark field image of the control marker excited by the control-marker exclusive excitation light. 
       2. Other Embodiments 
       [0141]    In the embodiment described above, a sample surface of the biological sample SPL facing the objective lens  44  is irradiated with the control-marker exclusive excitation light. However, a sample surface on the opposite side of the sample surface facing the objective  44  may be irradiated with the control-marker exclusive excitation light. 
         [0142]      FIG. 12  illustrates an example with the same reference numerals and symbols applied to the constituents corresponding to those in  FIG. 1 . In the example shown in  FIG. 12 , in the case of the dark field imaging mode, the control-marker excitation light source  61  is arranged in a predetermined light source position, where the bright field light source  41  is generally arranged, instead of the bright field light source  41 . Furthermore, the collimator lens  62  is arranged on the light path between the light source and the reflection mirror  42 , and the bright field filter  43  is removed from the light path. The control-marker exclusive excitation light emitted from the control-marker excitation light source  61  is made into a parallel beam by the collimator lens  62 , and conducted to the sample surface on the opposite side of the sample surface facing the objective lens  44  via the reflection mirror  42 . In the example shown in  FIG. 12 , the dichroic mirror  63  shown in  FIG. 1  is omitted. 
         [0143]      FIG. 13  illustrates another example with the same reference numerals and symbols applied to the constituents corresponding to those in  FIG. 1 . In the example shown in  FIG. 13 , the control-marker excitation light source  61  and the collimator lens  62  are arranged so that the control-marker exclusive excitation light directly falls on the sample surface on the opposite side of the sample surface facing the objective lens  44  at a slant. Like the example shown in  FIG. 12 , the dichroic mirror  63  shown in  FIG. 1  is also omitted in the example shown in  FIG. 13 . Furthermore, the example shown in  FIG. 13  is advantageous because the biological sample SPL can be directly irradiated with the control-marker exclusive excitation light without moving any filter or lens as in the example shown in  FIG. 12 . 
         [0144]    In the embodiments described above, a part of the dark field partial image of the control marker ( FIG. 8 ) is used as the focusing object corresponding to the sample part. While the part is fixed to each sample part allocated with the imaging area AR, it may be variable. 
         [0145]    In general, in the biological sample SPL (especially in a tissue section), cells are not evenly present but there are regions where the cells are concentrated and regions where the cells are sparse. In the concentrated region, the fluorescence marker that is truly targeted is more likely to exist compared with the sparse region, and the light intensity of the dark field partial image of the control marker corresponding to the concentrated region is also higher. That is, the concentrated region of the dark field partial image of the control marker is a notable region (a region to be used for focusing). 
         [0146]    When the dark field partial image of the control marker corresponding to the sample part to be obtained is read from the imaging device  46  for the first time, all the dark field partial images are read out, and a part of the read dark field partial images is determined to be used for focusing. 
         [0147]    This determination technique determines a predetermined number of the parts to be used for focusing in the descending order of the density in a search area smaller than the dark field partial image such as 16×16 pixels or 8×8 pixels. The density can be scaled by, for example, the number of pixels exhibiting the intensity no lower than a threshold in the search area, or the kurtosis (linearity) of intensity histogram of the search area. 
         [0148]    While the intensity histogram indicates the distribution of the intensity values in the search area based on the pixels, it is preferable to normalize the distribution assuming the variance indicative of the expanse of the distribution as one. This is because the dark field partial images of sample parts allocated with the imaging areas AR can be scaled in the same units. 
         [0149]    In this manner, in each sample part allocated with the imaging area AR, a part in which the fluorescence marker is very likely to exist can be focused on. 
         [0150]    In the embodiments described above, the DAPI, which employs the cell nucleus for contrast, is used as the control marker. However, the control marker is not limited to the DAPI. For example, a control marker that targets a cell tissue (or a molecule unique to the cell tissue) may be used, or a control marker that targets a cell cytoplasm (or a molecule unique to the cell cytoplasm) may be used. Naturally, another control marker that targets something else may be used. 
         [0151]    Although one type of the control marker is used in the embodiments described above, two or more types of the control marker may be used. In such a case, an exclusive excitation light source is provided with respect to each control marker, or a single excitation light source that excites each control marker but does not excite the fluorescent material in the target is provided, thereby the same effect as in the embodiments described above can be obtained. 
         [0152]    In the embodiments described above, a gene is used as the target in the biological sample SPL. However, the target in the biological sample SPL is not limited to the gene. For example, various molecules including a protein molecule such as a cell membrane channel, a glycoprotein molecule, and a sugar chain molecule can be used as the target. 
         [0153]    To adjust the focus in the embodiments described above, the movable stage  31  is moved in the z-axis direction (optical axis direction) in the direction of the optical axis of the objective lens  44  that is fixed. Alternatively, the movable stage  31  may be fixed and the objective lens  44  may be moved in the z-axis direction (optical axis direction) against the movable stage  31 . 
         [0154]    It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Technology Category: 3