Abstract:
An image capturing apparatus is configured to store the control result of a focus position during scanning of segmented regions and determine an initial focus position in scanning of the (n+1)th segmented region, based on the control result stored during scanning of the nth (n is an integer of 1 or more) or earlier segmented region. The foregoing technique allows this image capturing apparatus to roughly determine the initial focus position in the next-scanned segmented region by making use of the control result of the segmented region the scanning of which has been already completed. This can suppress increase in processing time necessary for imaging, by simplification of pre-focus.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to an image capturing apparatus used for capturing of images of a sample or the like, and an image capturing method thereby. 
       BACKGROUND ART 
       [0002]    As an image capturing apparatus there is a virtual microscope device, for example, configured to preliminarily divide an imaging region of a sample into a plurality of regions, capture images of the respective segmented regions at a high magnification, and thereafter synthesize these images. The conventional image capturing with such a virtual microscope is carried out as follows: a focus map for an entire region of the sample as an object is set as an imaging condition in capturing images of the sample such as a biological sample, and the image capturing of the sample is carried out while performing focus control based on the focus map. 
         [0003]    For creation of the focus map, a macro image of the entire sample is first captured with use of an image capturing apparatus having a macro optical system. Next, an imaging range of the sample is set using the captured macro image, the imaging range is divided into a plurality of segmented regions, and focus acquisition positions are set for the respective segmented regions. After the focus acquisition positions are set, the sample is transferred to an image capturing apparatus having a micro optical system, focus positions are captured at the set focus acquisition positions, and the focus map is created from these focus positions. 
         [0004]    However, there was a problem that the creation of the focus map as described above needed some time for processing. The time necessary for processing can be reduced by decreasing the interval and number of focuses to be acquired, but in that case there arose another problem of reduction in focus accuracy. For this reason, development of dynamic focus has been advanced to capture high-magnification images of the sample while acquiring the focus positions. This method is a method of detecting a deviation direction of a focus position with respect to a current height of an objective lens, based on a light intensity difference or contrast difference between an optical image which is focused at the front of an optical image made incident into an imaging device for capturing an image (front focus) and an optical image which is focused at the rear thereof (rear focus), moving the objective lens in a direction to cancel the deviation, and then capturing an image (e.g., cf. Patent Literature 1). 
         [0005]    On the other hand, there is a method using the conventional virtual microscope not using the dynamic focus, to capture a set of Z-plane images at different focus positions for a sample with some thickness (images on planes in focus at positions indicated by broken lines in  FIG. 16 ) (Z-stack images). For example, Patent Literature 2 below discloses a method to capture the Z-stack images by moving the objective lens relatively to a sample stage (e.g., by advancing the Z-plane level downward). 
       CITATION LIST 
     Patent Literatures 
       [0006]    Patent Literature 1: Japanese Patent Application Laid-open Publication No. 2011-081211 
         [0007]    Patent Literature 2: Japanese Patent Application Laid-open Publication No. 2008-500643 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0008]    For capturing the Z-stack by moving the objective lens relatively to the sample stage as in the above-cited Patent Literature 2, it is necessary to use information of a reference position of the objective lens such as the focus map. However, the focus map is not acquired in the dynamic focus and thus the foregoing information of the reference position of the objective lens is not available. Therefore, in the case of the dynamic focus being used, it is difficult to capture the Z-stack images by adopting the method of simply moving the objective lens relatively to the sample stage. 
         [0009]    The present invention has been accomplished in order to solve the above problem and it is an object of the present invention to provide an image capturing apparatus and an image capturing method thereby capable of readily capturing the Z-stack images in capturing the images by the dynamic focus. 
       Solution to Problem 
       [0010]    In order to solve the above problem, an image capturing apparatus according to the present invention comprises: a stage on which a sample is placed; a stage control unit which scans the stage at a predetermined speed; a light source which radiates light to the sample; a light guiding optical system including a light dividing unit which divides an optical image of the sample into a first optical path for capturing an image and a second optical path for focus control; a first imaging unit which captures a first image by a first optical image divided into the first optical path; a second imaging unit which captures a second image by a second optical image divided into the second optical path; a focus control unit which analyzes the second image so as to control a focus position of the image pickup by the first imaging unit based on the analysis result; and an optical-path-difference changing unit which changes an optical path difference between the first optical path and the second optical path based on a predetermined target focus interval, wherein every time the optical-path-difference changing unit changes the optical path difference, while the focus control unit controls the focus position, the stage control unit scans the stage and the first imaging unit captures the first image, thereby capturing Z-stack images consisting of a plurality of the first images in a depth direction of the sample. 
         [0011]    This image capturing apparatus comprises the optical-path-difference changing unit which changes the optical path difference between the first optical path and the second optical path. The focus position of the image pickup by the first imaging unit varies (or moves) in the depth direction of the sample according to the foregoing optical path difference changed by the optical-path-difference changing unit. Therefore, every time the optical-path-difference changing unit changes the optical path difference based on the predetermined target focus interval, while the focus control unit controls the focus position of the image pickup by the first imaging unit, the stage control unit scans the stage and the first imaging unit captures the first image, whereby the apparatus can readily capture the Z-stack images consisting of the first images in the depth direction of the sample according to the change of the optical path difference. Since this technique comprises performing the dynamic focus by the focus control unit after the change of the optical path difference, the apparatus captures the Z-stack images consisting of images of sections along curved surfaces approximately similar to the surface shape (relief) of the sample, different from the images of sections of the sample by XY planes parallel to the stage plane. 
         [0012]    The optical-path-difference changing unit may move the second imaging unit along an optical-axis direction of the second optical path based on a moving distance calculated by Formula (1) below. In this manner, the second imaging unit may be moved based on the below Formula (1) so as to change the optical path difference between the first optical path and the second optical path, whereby the apparatus can capture the first images at a plurality of focus positions with the predetermined target focus interval in between. 
         [0013]    The optical-path-difference changing unit may move the first imaging unit along an optical-axis direction of the first optical path based on a moving distance calculated by Formula (2) below. In this manner, the first imaging unit may be moved based on the below Formula (2) so as to change the optical path difference between the first optical path and the second optical path as well, whereby the apparatus can also capture the first images at a plurality of focus positions with the predetermined target focus interval in between. 
         [0000]      Moving distance=the predetermined target focus interval×(a square of an optical magnification in the second optical path)  (1)
 
         [0000]      Moving distance=the predetermined target focus interval×(a square of an optical magnification in the first optical path)  (2)
 
         [0014]    The image capturing apparatus may further comprise: region control unit which sets at an imaging area of the second imaging unit a first imaging region and a second imaging region for capturing a partial image of the second optical image; and an optical-path-difference producing member which is disposed on the second optical path, having a portion whose thickness continuously varies along an in-plane direction of the imaging area, and giving an optical path difference to the second optical image along the in-plane direction of the imaging area, wherein the optical-path-difference changing unit may make the region control unit change set positions of the first imaging region and the second imaging region based on a rate of variation of the thickness in the in-plane direction of the imaging area and the target focus interval. In this manner, the set positions of the first imaging region and the second imaging region may be changed so as to change the optical path difference between the first optical path and the second optical path as well, whereby the apparatus can also capture the first images at a plurality of focus positions with the predetermined target focus interval in between. 
         [0015]    An image capturing method according to the present invention is an image capturing method by an image capturing apparatus comprising: a stage on which a sample is placed; a stage control unit which scans the stage at a predetermined speed; a light source which radiates light to the sample; a light guiding optical system including a light dividing unit which divides an optical image of the sample into a first optical path for capturing an image and a second optical path for focus control; a first imaging unit which captures a first image by a first optical image divided into the first optical path; a second imaging unit which captures a second image by a second optical image divided into the second optical path; a focus control unit which analyzes the second image so as to control a focus position of the image pickup by the first imaging unit based on the analysis result; and an optical-path-difference changing unit which changes an optical path difference between the first optical path and the second optical path based on a predetermined target focus interval, wherein the method comprises, every time changing the optical path difference by the optical-path-difference changing unit, while controlling the focus position by the focus control unit, scanning the stage by the stage control unit and capturing the first image by the first imaging unit, thereby capturing Z-stack images consisting of a plurality of the first images in a depth direction of the sample. 
         [0016]    Making use of the fact that the focus position of the image pickup by the first imaging unit varies (or moves) in the depth direction of the sample according to the optical path difference between the first optical path and the second optical path, this image capturing method comprises every time changing the optical path difference between the first optical path and the second optical path, while controlling the focus position of the image pickup by the first imaging unit, scanning the stage and capturing the first image by the first imaging unit. This allows the method to readily capture the Z-stack images consisting of a plurality of first images in the depth direction of the sample according to the change of the optical path difference. In this technique, the dynamic focus is carried out every change of the optical path difference, whereby the method can capture the Z-stack images consisting of the images of sections along curved surfaces approximately similar to the surface shape (relief) of the sample, different from the images of sections of the sample by XY planes parallel to the stage plane. 
         [0017]    The method may comprise moving the second imaging unit along an optical-axis direction of the second optical path by the optical-path-difference changing unit based on a moving distance calculated by Formula (1) below. In this manner, the second imaging unit may be moved based on the below Formula (1) so as to change the optical path difference between the first optical path and the second optical path, whereby the method can capture the first images at a plurality of focus positions with the predetermined target focus interval in between. 
         [0018]    The method may comprise moving the first imaging unit along an optical-axis direction of the first optical path by the optical-path-difference changing unit based on a moving distance calculated by Formula (2) below. In this manner, the first imaging unit may be moved based on the below Formula (2) so as to change the optical path difference between the first optical path and the second optical path as well, whereby the method can also capture the first images at a plurality of focus positions with the predetermined target focus interval in between. 
         [0000]      Moving distance=the predetermined target focus interval×(a square of an optical magnification in the second optical path)  (1)
 
         [0000]      Moving distance=the predetermined target focus interval×(a square of an optical magnification in the first optical path)  (2)
 
         [0019]    The image capturing apparatus may further comprise: region control unit which sets at an imaging area of the second imaging unit a first imaging region and a second imaging region for capturing a partial image of the second optical image; and an optical-path-difference producing member which is disposed on the second optical path, having a portion whose thickness continuously varies along an in-plane direction of the imaging area, and giving an optical path difference to the second optical image along the in-plane direction of the imaging area, wherein the method may comprise changing set positions of the first imaging region and the second imaging region by the region control unit based on a rate of variation of the thickness in the in-plane direction of the imaging area and the target focus interval. In this manner, the set positions of the first imaging region and the second imaging region may be changed so as to change the optical path difference between the first optical path and the second optical path as well, whereby the method can also capture the first images at a plurality of focus positions with the predetermined target focus interval in between. 
       Advantageous Effect of Invention 
       [0020]    The present invention enables the Z-stack images to be readily captured in capturing the images by the dynamic focus. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0021]      FIG. 1  is a drawing showing one embodiment of a macro image capturing device which constitutes an image capturing apparatus according to the present invention. 
           [0022]      FIG. 2  is a drawing showing one embodiment of a micro image capturing device which constitutes the image capturing apparatus according to the present invention. 
           [0023]      FIG. 3  is a drawing showing a second imaging device. 
           [0024]      FIG. 4  is a drawing showing an example of a combination of an optical-path-difference producing member and the second imaging device. 
           [0025]      FIG. 5  is a drawing showing an example of the optical-path-difference producing member whose thickness continuously varies along an in-plane direction of an imaging area of the second imaging device. 
           [0026]      FIG. 6  is a block diagram showing functional components of the image capturing apparatus. 
           [0027]      FIG. 7  is a drawing showing an analysis result of contrast values in a situation where a distance to the surface of a sample is coincident with the focal length of an objective lens. 
           [0028]      FIG. 8  is a drawing showing an analysis result of contrast values in a situation where a distance to the surface of the sample is longer than the focal length of the objective lens. 
           [0029]      FIG. 9  is a drawing showing an analysis result of contrast values in a situation where a distance to the surface of the sample is shorter than the focal length of the objective lens. 
           [0030]      FIG. 10  is a drawing showing a relationship of the distance between the objective lens and the stage with respect to scanning time of the stage. 
           [0031]      FIG. 11  is a drawing showing control of a scanning direction of the stage by a stage control portion. 
           [0032]      FIG. 12  is a drawing showing control of a scanning speed of the stage by the stage control portion. 
           [0033]      FIG. 13  is a drawing used for explaining Z-stack images captured by the image capturing apparatus according to one embodiment of the present invention. 
           [0034]      FIG. 14  is a drawing used for explaining a method for changing an optical path difference between a first optical path and a second optical path by moving positions of a first imaging region and a second imaging region. 
           [0035]      FIG. 15  is a flowchart showing an operation of the image capturing apparatus according to one embodiment of the present invention. 
           [0036]      FIG. 16  is a drawing used for explaining the Z-stack images captured by a conventional image capturing apparatus. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0037]    Preferred embodiments of the image capturing apparatus and the image capturing method thereby according to the present invention will be described below in detail with reference to the drawings. 
         [0038]      FIG. 1  is a drawing which shows one embodiment of the macro image capturing device which constitutes the image capturing apparatus of the present invention.  FIG. 2  is a drawing which shows one embodiment of the micro image capturing device which constitutes the image capturing apparatus of the present invention. As shown in  FIG. 1  and  FIG. 2 , an image capturing apparatus M is constituted with a macro image capturing device M 1  for capturing a macro image of a sample S and a micro image capturing device M 2  for capturing a micro image of the sample S. The image capturing apparatus M is an apparatus which sets, for example, a plurality of line-shaped divided regions  40  with respect to the macro image captured by the macro image capturing device M 1  (refer to  FIG. 11 ) and produces a virtual micro image by capturing and synthesizing each of the divided regions  40  by the micro image capturing device M 2  at a high magnification. 
         [0039]    As shown in  FIG. 1 , the macro image capturing device M 1  is provided with a stage  1  which supports the sample S. The stage  1  is an XY stage which is actuated in a horizontal direction by a motor or an actuator such as a stepping motor (pulse motor) or a piezo actuator, for example. The sample S which is observed by using the image capturing apparatus M is, for example, a biological sample such as cells and placed on the stage  1  in a state of being sealed on a slide glass. The stage  1  is actuated inside the XY plane, by which an imaging position with respect to the sample S is allowed to move. 
         [0040]    The stage  1  is able to move back and forth between the macro image capturing device M 1  and the micro image capturing device M 2  and provided with functions to deliver the sample S between the devices. It is acceptable that when a macro image is captured, an entire image of the sample S is picked up at one time or the sample S is divided into a plurality of regions to pick up each of the images. It is also acceptable that the stage  1  is installed both on the macro image capturing device M 1  and on the micro image capturing device M 2 . 
         [0041]    A light source  2  which radiates light to the sample S and a condensing lens  3  which concentrates light from the light source  2  at the sample S are disposed on a bottom of the stage  1 . It is acceptable that the light source  2  is disposed so as to radiate light obliquely to the sample S. Further, a light guiding optical system  4  which guides an optical image from the sample S and an imaging device  5  which images the optical image of the sample S are disposed on an upper face of the stage  1 . The light guiding optical system  4  is provided with an image forming lens  6  which forms the optical image from the sample S at an imaging area of the imaging device  5 . Still further, the imaging device  5  is an area sensor which is capable of capturing, for example, a two-dimensional image. The imaging device  5  captures an entire image of the optical image of the sample S made incident into the imaging area via the light guiding optical system  4  and is housed at a virtual micro image storage  39  to be described later. 
         [0042]    As shown in  FIG. 2 , the micro image capturing device M 2  is provided on the bottom of the stage  1  with a light source  12  and a condensing lens  13 , as with the macro image capturing device M 1 . Further, a light guiding optical system  14  which guides an optical image from the sample S is disposed on the upper face of the stage  1 . The optical system which radiates light from the light source  12  to the samples may include an excitation light radiating optical system which radiates excitation light to the sample S and a dark-field illuminating optical system which captures a dark-field image of the sample S. 
         [0043]    The light guiding optical system  4  is provided with an objective lens  15  disposed so as to face to the sample S and a beam splitter (light dividing unit)  16  disposed at a rear stage of the objective lens  15 . The objective lens  15  is provided with a motor and an actuator such as a stepping motor (pulse motor) or a piezo actuator for actuating the objective lens  15  in a Z direction orthogonal to a face on which the stage  1  is placed. A position of the objective lens  15  in the Z direction is changed by these actuation units, thus making it possible to adjust a focus position of image pickup when an image of the sample S is captured. It is acceptable that the focus position is adjusted by changing a position of the stage  1  in the Z direction or by changing positions of both the objective lens  15  and the stage  1  in the Z direction. 
         [0044]    The beam splitter  16  is a portion which divides an optical image of the sample S into a first optical path L 1  for capturing an image and a second optical path L 2  for focus control. The beam splitter  16  is disposed at an angle of approximately 45 degrees with respect to an optical axis from the light source  12 . In  FIG. 2 , an optical path passing through the beam splitter  16  is given as the first optical path L 1 , while an optical path reflected at the beam splitter  16  is given as the second optical path. 
         [0045]    On the first optical path L 1 , there are disposed an image forming lens  17  which forms the optical image of the sample S (first optical image) which has passed through the beam splitter  16  and a first imaging device (first imaging unit)  18  in which an imaging area is disposed at an image forming position of the image forming lens  17 . The first imaging device  18  is a device which is capable of capturing a one-dimensional image (first image) by the first optical image of the sample S and is configured so as to be movable by an arbitrary distance in both directions along the optical-axis direction of the first optical path L 1 . The first imaging device  18  to be used is, for example, a two-dimension CCD sensor or a line sensor capable of realizing TDI (time delay integration) actuation. Further, in a method which captures images of the sample S sequentially, with the stage  1  controlled at a constant speed, the first imaging device  18  may be a device which is capable of capturing a two-dimensional image such as a CMOS sensor or a CCD sensor. First images picked up by the first imaging device  18  are sequentially stored in a temporary storage memory such as a lane buffer, thereafter, compressed and output at an image producing portion  38  to be described later. 
         [0046]    On the other hand, on the second optical path L 2 , there are disposed a view-field adjusting lens  19  which contracts an optical image of a sample reflected by the beam splitter  16  (second optical image) and a second imaging device (second imaging unit)  20 . Further, at a front stage of the second imaging device  20 , there is disposed an optical path difference producing member  21  which gives an optical path difference to the second optical image. It is preferable that the view-field adjusting lens  19  is constituted in such a manner that the second optical image is formed at the second imaging device  20  in a dimension similar to that of the first optical image. 
         [0047]    The second imaging device  20  is a device which is capable of capturing a two-dimensional image (second image) by the second optical image of the sample S and is configured so as to be movable by an arbitrary distance in both directions along the optical-axis direction of the second optical path L 2 . The second imaging device  20  to be used is, for example, a sensor such as a CMOS (complementary metal oxide semiconductor) or a CCD (charge coupled device). Furthermore, a line sensor may be used. 
         [0048]    An imaging area  20   a  of the second imaging device  20  is disposed so as to be substantially in alignment with an XZ plane orthogonal to the second optical path L 2 . As shown in  FIG. 3 , a first imaging region  22 A and a second imaging region  22 B which capture a partial image of the second optical image are set on the imaging area  20   a . The first imaging region  22 A and the second imaging region  22 B are set in a direction perpendicular to a direction (scanning direction: Z direction) at which the second optical image moves on the imaging area  20   a  in association with scanning of the sample S. The first imaging region  22 A and the second imaging region  22 B are set, with a predetermined interval kept, and both of them capture a part of the second optical image in a line shape. Thereby, an optical image at the same region as that of the first optical image of the sample S captured by the first imaging device  18  can be captured as the second optical image at the first imaging region  22 A and the second imaging region  22 B. It is acceptable that each of the first imaging region  22 A and the second imaging region  22 B is set by using a separate line sensor. In this case, each of the line sensors is controlled separately, thus making it possible to shorten the time necessary for setting the first imaging region  22 A and the second imaging region  22 B. 
         [0049]    The optical path difference producing member  21  is a glass member which gives an optical path difference to the second optical image along an in-plane direction of the imaging area  20   a . In an example shown in  FIG. 4 , the optical path difference producing member  21 A is formed in the shape of a prism having a triangular cross section and disposed in such a manner that an apex thereof is substantially in alignment with a central part of the imaging area  20   a  in the Z direction. Therefore, the second optical image which is made incident into the imaging area  20   a  is longest in optical path at the central part of the imaging area  20   a  in the Z direction and becomes shorter in optical path when moving toward both ends of the imaging area  20   a  in the Z direction. Further, it is preferable that the optical path difference producing member  21  is disposed in such a manner that a face which faces to the second imaging device  20  is parallel with the imaging area (light receiving face)  20   a  of the second imaging device. Thereby, it is possible to reduce deflection of light by the face which faces to the second imaging device  20  and also to secure the amount of light which is received by the second imaging device  20 . 
         [0050]    Accordingly, the second imaging device  20  is able to capture an optical image which is focused at the front of a first optical image made incident into the first imaging device  18  (front focus) and an optical image which is focused at the rear thereof (rear focus) based on a position of the first imaging region  22 A and that of the second imaging region  22 B. In the present embodiment, the position of the first imaging region  22 A and that of the second imaging region  22 B are set in such a manner that, for example, the first imaging region  22 A is given as the front focus and the second imaging region  22 B is given as the rear focus. A focus difference between the Front focus and the rear focus is dependent on a difference between a thickness t 1  and an index of refraction of the optical path difference producing member  21 A through which the second optical image made incident into the first imaging region  22 A passes, and a thickness t 2  and an index of refraction of the optical path difference producing member  21 A through which the second optical image made incident into the second imaging region  22 B passes. 
         [0051]    The optical-path-difference producing member  21 B of the prism shape of a right triangle cross section as shown in  FIG. 5  may be used as the optical-path-difference producing member  21  while it is arranged so that the thickness continuously increases along the in-plane direction (Z-direction) of the imaging area  20   a.    
         [0052]      FIG. 6  is a block diagram which shows functional components of the image capturing apparatus. As shown in the diagram, the image capturing apparatus M is provided with a computer system having a CPU, a memory, a communication interface, a storage such as a hard disk, an operation portion  31  such as a keyboard, a monitor  32  etc. The functional components of the control portion  33  include a focus control portion  34 , a region control portion  35 , an objective lens control portion  36 , a stage control portion  37 , an image producing portion  38 , a virtual micro image storage  39 , and an optical-path-difference changing portion  50 . 
         [0053]    The focus control portion  34  is a portion which analyzes a second image captured by the second imaging device  20  so as to control a focus position of an image picked up by the first imaging device  18  based on the analysis result. More specifically, the focus control portion  34  first determines a difference between a contrast value of the image obtained at the first imaging region  22 A and a contrast value obtained at the second imaging region  22 B in the second imaging device  20 . 
         [0054]    Here, as shown in  FIG. 7 , where a focus position of the objective lens  15  is in alignment with the surface of the sample S, an image contrast value of the front focus obtained at the first imaging region  22 A is substantially in agreement with an image contrast value of the rear focus obtained at the second imaging region  22 B. Thereby, a difference value between them is almost zero. 
         [0055]    On the other hand, as shown in  FIG. 8 , where a distance to the surface of the sample S is longer than a focal length of the objective lens  15 , an image contrast value of the rear focus obtained at the second imaging region  22 B is greater than an image contrast value of the front focus obtained at the first imaging region  22 A. Therefore, a difference value between them is a positive value. In this case, the focus control portion  34  outputs instruction information to the objective lens control portion  36  so as to be actuated in a direction at which the objective lens  15  is brought closer to the sample S. 
         [0056]    Further, as shown in  FIG. 9 , where a distance to the surface of the samples is shorter than a focal length of the objective lens  15 , an image contrast value of the rear focus obtained at the second imaging region  22 B is smaller than an image contrast value of the front focus obtained at the first imaging region  22 A. Therefore, a difference value between them is a negative value. In this case, the focus control portion  34  outputs instruction information to the objective lens control portion  36  so as to be actuated in a direction at which the objective lens  15  is brought away from the sample S. 
         [0057]    The region control portion  35  is a portion which controls a position of the first imaging region  22 A and a position of the second imaging region  22 B at the imaging area  20   a  of the second imaging device  20 . The region control portion  35  sets at first the first imaging region  22 A at a predetermined position based on operation from the operation portion  31  and releases the setting of the first imaging region  22 A after image pickup at the first imaging region  22 A. Then, the region control portion  35  sets the second imaging region  22 B, with a predetermined interval kept in the Z direction (scanning direction) from the first imaging region  22 A, and releases the setting of the second imaging region  22 B after image pickup at the second imaging region  22 B. 
         [0058]    At this time, waiting time W from image pickup at the first imaging region  22 A to image pickup at the second imaging region  22 B is set based on an interval d between the first imaging region  22 A and the second imaging region  22 B, and a scanning velocity v of the stage  1 . For example, where the waiting time W is given as time W 1  from the start of image pickup at the first imaging region  22 A to the start of image pickup at the second imaging region  22 B, it is possible to determine the waiting time with reference to a formula of W 1 =d/v−el−st, with consideration given to exposure time el of image pickup at the first imaging region  22 A and time st from release of the setting of the first imaging region  22 A to the setting of the second imaging region  22 B. 
         [0059]    Further, where the waiting time W is given as waiting time W 2  from the start of image pickup at the first imaging region  22 A to completion of image pickup at the second imaging region  22 B, it is possible to determine the waiting time with reference to a formula of W 2 =d/v−st, with consideration given to time st from release of the setting of the first imaging region  22 A to setting of the second imaging region  22 B. Still further, an interval d between the first imaging region  22 A and the second imaging region  22 B is set based on a difference in optical path length made by the optical path difference producing member  21 . However, the interval d actually corresponds to a distance of the sample S on a slide. Eventually, it is necessary to convert the interval d to the number of pixels at the second imaging region  22 B. Where a pixel size of the second imaging device  20  is expressed in terms of AFpsz and magnification is expressed in terms of AFmag, the number of pixels dpix corresponding to the interval d can be determined with reference to a formula of dpix=d÷(AFpsz/AFmag). 
         [0060]    Further, the region control portion  35  is able to change at least one of a position of the first imaging region  22 A and that of the second imaging region  22 B along an in-plane scanning direction (here, the Z direction) of the imaging area  20   a  based on operation from the operation portion  31 . In this case, it is acceptable to change only one of the position of the first imaging region  22 A and that of the second imaging region  22 B or both of the position of the first imaging region  22 A and that of the second imaging region  22 B. It is also acceptable to change both of the position of the first imaging region  22 A and that of the second imaging region  22 B, with the interval d between the first imaging region  22 A and the second imaging region  22 B being kept. 
         [0061]    The first imaging region  22 A and the second imaging region  22 B are changed in position, by which, for example, use of a prism-like optical path difference producing member  21  ( 21 A or  21 B) as shown in  FIG. 4  or  FIG. 5  makes it possible to change the thickness t 1  of the optical path difference producing member  21 A through which the second optical image made incident into the first imaging region  22 A passes and the thickness t 2  of the optical path difference producing member  21 A through which the second optical image made incident into the second imaging region  22 B passes. Thereby, an interval between the front focus and the rear focus is changed, thus making it possible to adjust resolution on determination of a difference in contrast value. 
         [0062]    The objective lens control portion  36  is a portion which controls actuation of the objective lens  15 . Upon receiving instruction information output from the focus control portion  34 , the objective lens control portion  36  actuates the objective lens  15  in the Z direction in accordance with contents of the instruction information. It is, thereby, possible to adjust a focus position of the objective lens  15  with respect to the sample S. 
         [0063]    The objective lens control portion  36  does not actuate the objective lens  15  during analysis of the focus position which is being performed by the focus control portion  34  and actuates the objective lens  15  only in one direction along the Z direction until the next analysis of focus position is initiated.  FIG. 10  is a drawing which shows a relationship of the distance between the objective lens and the stage with respect to scanning time of the stage. As shown in the drawing, during scanning of the sample S, an analysis period A of the focus position and an objective lens actuation period B based on an analysis result thereof are taken place alternately. By keeping the positional relationship between the objective lens  15  and the sample S unchanged during the analysis of focus position in this manner, analysis accuracy of focus position can be guaranteed. 
         [0064]    The stage control portion  37  is a portion which controls actuation of the stage  1 . More specifically, the stage control portion  37  allows the stage  1  on which the sample S is placed to scan at a predetermined speed based on operation from the operation portion  31 . By the scanning of the stage  1 , an imaging field of the sample S moves relatively and sequentially at the first imaging device  18  and the second imaging device  20 . The scanning direction of the stage  1  may be determined to be one-directional scanning, as shown in (a) of  FIG. 11 , which is carried out in such a manner that the position of the stage  1  is returned to a scan start position every completion of scanning of one segmented region  40  and the next segmented region  40  is then scanned in the same direction, or may be determined to be bidirectional scanning, as shown in (b) of  FIG. 11 , which is carried out in such a manner that, after completion of scanning of one segmented region  40 , the stage  1  is moved in a direction perpendicular to the scanning direction and the next segmented region  40  is then scanned in the opposite direction. 
         [0065]    Although the stage  1  is scanned at a constant speed while images are captured, actually, immediately after the start of scanning, there is a period during which the scanning speed is unstable due to influences of vibrations of the stage  1  etc. For this reason, it is preferable, as shown in  FIG. 12 , to set a scanning width longer than the segmented regions  40  and make each of an acceleration period C for the stage  1  to accelerate, a stabilization period D for the scanning speed of the stage  1  to stabilize, and a deceleration period F for the stage  1  to decelerate, occur during scanning outside the segmented regions  40 . This allows capturing of images to be carried out in accord with a constant speed period E where the scanning speed of the stage  1  is constant. It is also possible to adopt a technique of starting imaging in the stabilization period D and deleting data part obtained in the stabilization period D after the image has been captured. Such a technique can be suitably applied to cases using an imaging device which requires void reading of data. 
         [0066]    The image producing portion  38  is a portion at which an captured image is synthesized to produce a virtual micro image. The image producing portion  38  receives sequentially first images output from the first imaging device  18 , that is, images of individual divided regions  40 , synthesizing these images to produce an entire image of the sample S. Then, based on the synthesized image, prepared is an image, the resolution of which is lower than that of the synthesized image, and housed in a virtual micro image storage  39  by associating a high resolution image with a low resolution image. It is acceptable that an image captured by the macro image capturing device M 1  is also associated with them in the virtual micro image storage  39 . The virtual micro image may be stored as a single image or may be stored as a plurality of divided images. 
         [0067]    The optical-path-difference changing portion  50  is a portion which changes the optical path difference between the first optical path L 1  and the second optical path L 2 . The optical-path-difference changing portion  50  has a function to drive the first imaging device  18  by an arbitrary distance in both directions along the optical-axis direction of the first optical path L 1 . Furthermore, the optical-path-difference changing portion  50  has a function to drive the second imaging device  20  by an arbitrary distance in both directions along the optical-axis direction of the second optical path L 2 . In addition, the optical-path-difference changing portion  50  has a function to output instruction information to change the set positions of the first imaging region  22 A and the second imaging region  22 B in the imaging area  20   a  of the imaging device  20  of the second imaging device  20 , to the region control portion  35 . Namely, the optical-path-difference changing portion  50  has the function to change the position of the first imaging region  22 A and the set position of the second imaging region  22 B, through the region control portion  35 . 
         [0068]    The optical-path-difference changing portion  50  uses the above-described functions to change the position of the first imaging device  18 , the position of the second imaging device  20 , or, the set positions of the first imaging region  22 A and the second imaging region  22 B, thereby changing the optical path difference between the first optical path L 1  and the second optical path L 2 . When an optical path difference changing process by the optical-path-difference changing portion  50  is performed based on a predetermined target focus interval dz, it becomes feasible for the first imaging device  18  to capture the first images in focus around positions shifted by the target focus interval dz in the depth direction of the sample S. Namely, the image capturing apparatus M becomes able to capture Z-stack images consisting of a plurality of first images in the depth direction (the Z-direction) of the sample S. A specific method for it will be described below. 
         [0069]      FIG. 13  is a drawing used for explaining the Z-stack images captured by the image capturing apparatus M, showing a cross-sectional shape of the sample S and stage  1  cut along a plane (XZ plane) parallel to both of the scanning direction of the stage  1  (X-direction) and the depth direction of the sample S (Z-direction). In the same drawing, line Z 0  indicated by a curve represents a line along the surface shape (relief) of the sample S. Furthermore, lines Z 1  and Z 2  represent lines resulting from movement of the line Z 0  by the predetermined target focus interval dz each in the Z-direction. In the image capturing apparatus M, as described above, while the focus control portion  34  controls the focus position of the image pickup by the first imaging device  18 , the stage control portion  37  scans the stage  1  (i.e., performs the dynamic focus). Therefore, the image capturing apparatus M captures the Z-stack images consisting of images of sections along curved surfaces (e.g., lines Z 0 , Z 1 , Z 2 , and so on) approximately similar to the surface shape (relief) of the sample S, different from the images of sections of the sample by XY planes parallel to the stage plane (cf.  FIG. 16 ). In this manner, the image capturing apparatus M can capture the images in focus at respective layers. As described above, the dynamic focus is carried out with focus at respective layers inside the sample S, whereby the Z-stack images can be captured in focus with cells inside the sample S. When compared to the Z-stack images captured by the conventional image capturing apparatus, more information can be obtained about the sample S in the depth direction by a smaller number of images (e.g., three layers of the lines Z 0 , Z 1 , and Z 2 ). 
         [0070]    In the description hereinbelow, for simplicity of description, let us define an initial state as a state in which each of the devices is arranged so that a locus of focus positions (hereinafter referred to as “focus locus”) of the first image captured by the operation in which the stage control portion  37  scans the stage  1  while the focus control portion  34  controls the focus position of the image pickup by the first imaging device  18 , follows the line Z 0 . Then, let us explain the optical-path-difference changing process carried out by the optical-path-difference changing portion  50  for the first imaging device  18  to capture the first image in focus around the positions indicated by the line Z 1  resulting from parallel movement of the line Z 0  by the target focus interval dz in the depth direction of the sample S. 
       First Example 
       [0071]    The optical-path-difference changing portion  50  moves the optical-path-difference producing member  21  and the second imaging device  20  by a moving distance calculated according to Formula (1) below, along the optical-axis direction of the second optical path L 2 , from the initial state. For example, in a case where the Z-stack images are captured in the opposite direction (arrow A in  FIG. 2 ) to the traveling direction of light along the optical axis, the optical-path-difference producing member  21  and the second imaging device  20  are moved in the opposite direction (arrow B in  FIG. 2 ) to the traveling direction of light along the optical axis. In a case where the Z-stack images are captured in the traveling direction of light along the optical axis (the opposite direction to the arrow A), the optical-path-difference producing member  21  and the second imaging device  20  are moved in the traveling direction of light along the optical axis (the opposite direction to the arrow B). Therefore, the relationship of the moving direction of the optical-path-difference producing member  21  and second imaging device  20  with the capturing direction of the Z-stack with respect to the traveling direction of light along the optical axis is the same direction. 
         [0000]      Moving distance=the target focus interval  dz ×(the square of an optical magnification in the second optical path  L 2)  (1)
 
         [0072]    Here, the optical magnification in the second optical path L 2  is determined by the product of magnifications of the objective lens  15  and the view-field adjustment lens  19  via which the light emitted from the light source  12  travels up to the second imaging device  20  through the second optical path L 2  while being reflected by the beam splitter  16 . 
       Second Example 
       [0073]    The optical-path-difference changing portion  50  moves the first imaging device  18  by a moving distance calculated according to Formula (2) below, along the optical-axis direction of the first optical path L 1 , from the initial state. For example, in a case where the Z-stack images are captured in the opposite direction (arrow A) to the traveling direction of light along the optical axis, the first imaging device  18  is moved in the opposite direction (arrow C in  FIG. 2 ) to the traveling direction of light along the optical axis. In a case where the Z-stack images are captured in the traveling direction of light along the optical axis (the opposite direction to the arrow A), the first imaging device  18  is moved in the traveling direction of light along the optical axis (the opposite direction to the arrow C). Therefore, the relationship of the moving direction of the first imaging device  18  with the capturing direction of the Z-stack with respect to the traveling direction of light along the optical axis is the same direction. 
         [0000]      Moving distance=the target focus interval  dz ×(the square of an optical magnification in the first optical path  L 1)  (2)
 
         [0074]    Here, the optical magnification in the first optical path L 1  is determined by the product of magnifications of the objective lens  15  and the image forming lens  17  via which the light emitted from the light source  12  travels up to the first imaging device  18  through the first optical path L 1  while passing though the beam splitter  16 . 
       Third Example 
       [0075]    The optical-path-difference producing member  21  to be used is a member having a portion whose thickness continuously varies along the in-plane direction of the imaging area  20   a . Furthermore, the optical-path-difference changing portion  50  changes, through the region control portion  35 , the set positions of the first imaging region  22 A and the second imaging region  22 B, based on a rate of change in thickness of the optical-path-difference producing member  21  in the in-plane direction of the imaging area  20   a  and the target focus interval dz, from the initial state. For example, a case using the optical-path-difference producing member  21 B shown in  FIG. 5 , as the optical-path-difference producing member  21  will be specifically described using  FIG. 14 . 
         [0076]    (a) of  FIG. 14  is a drawing showing the set positions of the first imaging region  22 A and the second imaging region  22 B in the initial state. Distance S 1  indicates a distance from one end of the second imaging device  20  (the end on the side where the thickness of the optical-path-difference producing member  21 B opposed to the imaging area  20   a  is small) to the central position of the first imaging region  22 A, in the in-plane direction of the imaging area  20   a . Angle θ indicates an angle (acute angle) between the plane opposed to the imaging area  20   a  of the second imaging device  20  and the inclined plane, in the optical-path-difference producing member  21 B. Here, the angle θ acts as a parameter indicating the rate of change in thickness of the optical-path-difference producing member  21  in the in-plane direction of the imaging area  20   a.    
         [0077]    (b) of  FIG. 14  is a drawing showing a state after each of the set positions of the first imaging region  22 A and the second imaging region  22 B is changed to a position distant by a change distance ΔS 1  along the in-plane direction (the same direction as the arrow A and arrow C) of the imaging area  20   a , from the initial state. Distance S 1 ′ indicates a distance from the one end of the second imaging device  20  to the central position of the first imaging region  22 A after the change, in the in-plane direction of the imaging area  20   a . The distance S 1 ′ is represented by the sum of the distance S 1  and the change distance ΔS 1  (S 1 ′=S 1 +ΔS 1 ). Furthermore, thickness t 1 ′ indicates the thickness of the optical-path-difference producing member  21 B where the second optical image to enter the first imaging region  22 A after the change passes. Thickness difference Δt 1  is represented by a difference between the thickness t 1  after the change and the thickness t 1  before the change (Δt 1 =t 1 ′−t 1 ). 
         [0078]    Here, the change distance ΔS 1  of the set positions of the first imaging region  22 A and the second imaging region  22 B is calculated according to Formula (3) below, where an index of refraction of the optical-path-difference producing member  21 B is defined as the index of refraction n. 
         [0000]      Δ S 1= A/B   (3)
 
         [0079]    A=the target focus interval dz×(the square of the optical magnification in the second optical path L 2 ) 
         [0080]    B=(1−1/n)×tan θ 
         [0081]    After the optical-path-difference changing portion  50  changes the optical path difference between the first optical path L 1  and the second optical path L 2  by any one method described in the foregoing first example to third example, the stage control portion  37  scans the stage  1  while the focus control portion  34  controls the focus position of the image pickup by the first imaging device  18 , whereby the focus locus comes to follow the line Z 1  shifted by the target focus interval dz in the depth direction of the sample S from the line Z 0 . This allows the first imaging device  18  to capture the first image in focus around the positions indicated by the line Z 1  shifted by the target focus interval dz in the depth direction of the sample S. 
         [0082]    Therefore, as the focus control by the focus control portion  34 , the stage scanning by the stage control portion  37 , and the capturing of the first images by the first imaging device  18  are carried out every change of the optical path difference between the first optical path L 1  and the second optical path L 2  by any of the methods described in the above examples, the apparatus can capture the first images in focus at the respective layers of the sample S (e.g., the line Z 0 , line Z 1 , line Z 2 , and so on). The optical-path-difference changing portion  50  for changing the optical path difference between the first optical path L 1  and the second optical path L 2  does not have to be limited to those in the first to third examples, but we may adopt a configuration wherein an optical member capable of changing the optical path length, such as a liquid crystal lens, is arranged in the optical path and the optical member is controlled, or other configurations. 
         [0083]    Formulae (1) to (3) described in the above respective examples are theoretical formulae, and the optical magnification in the first optical path L 1 , the optical magnification in the second optical path L 2 , and others can be slightly different depending upon the actual device configuration. Therefore, measurement with some samples may be carried out to acquire correction values and calibration may be performed based on the correction values. 
         [0084]    The operation of the image capturing apparatus M described above will be described below. 
         [0085]      FIG. 15  is a flow chart which shows an operation of the image capturing apparatus M. As shown in the flow chart, at the image capturing apparatus M, at first, a macro image of the sample S is captured by the macro image capturing device M 1  (Step S 01 ). The captured macro image is binarized by using, for example, a predetermined threshold value and, thereafter, displayed on a monitor  32 . A scope for capturing micro images from macro images is set by automatic setting based on a predetermined program or manual setting by an operator (Step S 02 ). 
         [0086]    Then, the sample S is transferred to the micro image capturing device M 2  and focusing conditions are set (Step S 03 ). Here, as described above, a waiting time W before a start of image pickup at the second imaging region  22 B is set based on a scanning velocity v of the stage  1  and an interval d between the first imaging region  22 A and the second imaging region  22 B. It is more preferable that consideration is given to exposure time el of image pickup at the first imaging region  22 A, time st from release of setting of the first imaging region  22 A to setting of the second imaging region  22 B etc. 
         [0087]    After the focusing conditions have been set, scanning of the stage  1  is started to capture a micro image for each of the divided regions  40  of the sample S by the micro image capturing device M 2  (Step S 04 ). In capturing the micro image by the first imaging device  18 , at the second imaging device  20 , a deviating direction of the objective lens  15  with respect to the sample S is analyzed based on a difference in contrast value between the front focus and the rear focus by the first imaging region  22 A and the second imaging region  22 B, thereby adjusting a position of the objective lens  15  in real time. After micro images have been captured completely for all the divided regions  40 , the captured micro images are synthesized to produce a virtual micro image (Step S 05 ). The processes of steps S 04  and S 05  result in capturing the virtual micro image at one layer forming the Z-stack images (e.g., an image in focus around the positions indicated by the line Z 0  shown in  FIG. 13 ). 
         [0088]    Subsequently, it is determined whether all the images forming the Z-stack images (virtual micro images) have been captured (step S 06 ). Here, all the images forming the Z-stack images refer to, for example, images at all the depth positions of the sample S with the predetermined target focus interval dz, which was set in advance, in between. Unless all the images forming the desired Z-stack images have been captured (step S 06 : NO), the optical path difference between the first optical path L 1  and the second optical path L 2  is changed by any one of the aforementioned methods (or by a combination thereof), based on the predetermined target focus interval dz (step S 07 ). Thereafter, the processes of steps S 04  and S 05  are again carried out to capture an image in focus at different depth positions of the sample S (e.g., the image in focus around the positions indicated by the line Z 1  shown in  FIG. 13 ). After all the images forming the Z-stack images have been captured, the processing is terminated (step S 06 : YES). 
         [0089]    As described above, the image capturing apparatus M has the optical-path-difference changing portion  50  for changing the optical path difference between the first optical path L 1  and the second optical path L 2 . The focus position of the image pickup by the first imaging device  18  varies (or moves) in the depth direction of the sample S according to the optical path difference changed by the optical-path-difference changing portion  50 . Therefore, every time the optical-path-difference changing portion  34  changes the optical path difference based on the predetermined target focus interval dz, while the focus control portion  34  controls the focus position of the image pickup by the first imaging device  18 , the stage control portion  37  scans the stage  1  and the first imaging device  18  captures the first image, whereby the apparatus can readily capture the Z-stack images consisting of the first images in the depth direction of the sample S according to the change of the optical path difference. 
         [0090]    Specifically, the optical-path-difference changing portion  50  moves the second imaging device  20  by the moving distance calculated by the aforementioned Formula (1) including the target focus interval dz as a parameter, along the optical-axis direction of the second optical path L 2 , so as to change the optical path difference between the first optical path L 1  and the second optical path L 2 , whereby the first images can be captured at a plurality of focus positions with the target focus interval dz in between. 
         [0091]    The optical-path-difference changing portion  50  moves the first imaging device  18  by the moving distance calculated by the foregoing Formula (2) including the target focus interval dz as a parameter, along the optical-axis direction of the first optical path L 1 , so as to change the optical path difference between the first optical path L 1  and the second optical path L 2  as well, whereby the first images can also be captured at a plurality of focus positions with the target focus interval dz in between. 
         [0092]    When the optical-path-difference producing member  21  to be used is the optical-path-difference producing member  21  having the portion whose thickness continuously varies along the in-plane direction of the imaging area  20   a , and configured to give the optical path difference to the second optical image along the in-plane direction of the imaging area  20   a  (e.g., the optical-path-difference producing member  21 B), the optical-path-difference changing portion  50  changes the set positions of the first imaging region  22 A and the second imaging region  22 B by the change distance determined based on the rate of the change of the thickness in the in-plane direction of the imaging area  20   a  and the target focus interval dz, so as to change the optical path difference between the first opt L 1  and the second optical path L 2  as well, whereby the first images can also be captured at a plurality of focus positions with the target focus interval dz in between. 
         [0093]    The above-described embodiment showed the apparatus for producing the virtual micro images by way of illustration, but it should be noted that the image capturing apparatus according to the present invention can be applied to a variety of devices as long as they are apparatuses for capturing images while scanning the sample at a predetermined speed by the stage or the like. 
       REFERENCE SIGNS LIST 
       [0094]      1  stage;  12  light source;  14  light guiding optical system;  15  objective lens;  16  beam splitter (light dividing unit);  18  first imaging device (first imaging unit);  20  second imaging device (second imaging unit);  20   a  imaging area;  21  ( 21 A,  21 B) optical-path-difference producing member;  22 A first imaging region;  22 B second imaging region;  34  focus control portion (focus control unit);  35  region control portion (region control unit);  37  stage control portion (stage control unit);  50  optical-path-difference changing portion (optical-path-difference changing unit); L 1  first optical path; L 2  second optical path; M image capturing apparatus; M 1  macro image capturing device; M 2  micro image capturing device; S sample