Abstract:
The present application includes a culture chamber, an observation chamber having an optical system of observation to observe samples to be cultured in the culture chamber, and a movement stage provided at a boundary which separates the culture chamber and the observation chamber, functioning as a wall to maintain an environment of both of the chambers, bearing the samples, and moving the samples on a light axis of observation of the optical system of observation. Due to such an arrangement, a culture observation equipment which has good response and excellent environmental resistance can be achieved, preventing problems such as the overall size of the device became large, maintenance work was difficult, and device cost became expensive.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a Continuation Application of International Application No. PCT/JP2007/000171, filed Mar. 6, 2007, designating the U.S., in which the International Application claims a priority date of Mar. 14, 2006, based on prior filed Japanese Patent Application No. 2006-069030, the entire contents of which are incorporated herein by reference. 
     BACKGROUND 
     1. Field 
     The present application relates to a culture observation equipment which has an optical device such as a microscope. 
     2. Description of the Related Art 
     In recent years, research and development of cell culture has been spread widely in the field of regenerative medicine and drug development, where culture devices (incubators) which culture cells and observation equipments such as a microscope to observe cell situation are used. Conventionally, when samples during culture in an incubator are observed, a holder containing samples is taken out from a culture chamber temporarily to be placed on an observation stage for observation, and thereafter the holder is returned to the culture chamber again. 
     Further, in Patent Document 1 (Japanese Unexamined Patent Application Publication No. 2004-180675), a microscope is placed in a shield case separating the microscope from the culture chamber so that the environment in the culture chamber is not fluctuated, hence optical devices are not damaged. 
     In Patent Document 1, a microscope unit can be moved in the three dimensional direction, X, Y and Z, so as to advance towards the observation part, accordingly, working distance can be shortened. However, such a stage mechanism is required that has a large driving power to drive the whole microscope unit in the three dimensional directions, X, Y and Z. Therefore, when moving among the observation points, there is a task to improve quick response. 
     In view of the above task, it is the proposition to provide a culture observation equipment with high response performance and excellent environmental resistance. 
     An culture chamber includes an culture chamber, an observation chamber having an optical system of observation to observe samples cultured in the culture chamber, and a movement stage provided at a boundary which separates the culture chamber and the observation chamber, functioning as a wall to maintain an environment of both chambers, bearing the samples, and moving the samples on a light axis of observation of the optical system of observation in an orthogonal direction thereto. 
     Particularly, the movement stage is arranged to be coupled to a connecting material which is elastic and forms a boundary between the culture chamber and the observation chamber. 
     Further, the connecting material is made up of an elastic material having elasticity. 
     Alternatively, the connecting material includes a plate material which supports the movement stage and shielding liquid which lies between the movement stage and the plate material. 
     Particularly, the elastic material is made up of rubber provided with flexure being elastic in all circumferential directions. 
     Further, a section of the elastic material in a direction of expanding and shrinking is formed to have a rectangular shape. 
     In addition, the culture observation equipment includes driving units to drive the movement stage in horizontal direction so as to move a target point of observation of the sample, and optical driving units to drive the optical system of observation in vertical direction so as to focus on the sample. 
     Particularly, the movement stage is made up of transparent material and a fixed partition around the movement stage is made up of light shielding material. 
     Further, the connecting material is made up of low reflection and light shielding material. 
     SUMMARY 
     A movement stage, on which samples are placed and which is coupled to the fixed partition with an elastic material, can be moved freely while the culture chamber and the observation chamber are separated, and therefore a culture observation equipment which has good response as well as excellent environmental resistance can be achieved. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a front view of a culture observation equipment. 
         FIG. 2  is a top plan view of the culture observation equipment. 
         FIG. 3  is a top plan view of the culture observation equipment in a state where an elastic material is removed. 
         FIGS. 4(   a ) and  4 ( b ) are diagrams for illustrating a shape of the elastic material. 
         FIGS. 5(   a ) and  5 ( b ) are diagrams for illustrating a state of the elastic material being elastic. 
         FIGS. 6(   a ) and  6 ( b ) are diagrams for illustrating a state of the elastic material being elastic. 
         FIGS. 7(   a ) and  7 ( b ) are diagrams for illustrating a state of the elastic material being elastic. 
         FIGS. 8(   a ) to  8 ( c ) are cross sections showing a sectional shape of the elastic material. 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     Hereinafter, detailed description of a culture observation equipment will be described with reference to attached drawings. 
     First Embodiment 
       FIG. 1  is a front view of a culture observation device  101  according to the first embodiment, and  FIG. 2  is a top view thereof. The culture observation device  101  according to the first embodiment is arranged to have an culture chamber  102  in the upper part thereof, and an observation chamber  105  that includes a personal computer  103 , a controller  104  and the like in the lower part thereof. Between the culture chamber  102  and the observation chamber  105 , a base plate  106  is fixed to a housing case  108  via a gasket  107 , so that the humidity in the culture chamber  102  does not diffuse into the observation chamber  105 . 
     In  FIG. 2 , an external door  109  of the culture chamber  102  is provided with an external small door  113  for carrying a carrier  112  shown in  FIG. 1  into the culture chamber  102 . In addition, heat-resistant gaskets  114  are provided respectively between the housing case  108  and the external door  109  as well as between the external door  109  and the external small door  113 . Further a glass door  115  is provided inside of the external door  109 , and in the glass door  115 , at the corresponding position to the external small door  113 , a small glass door  116  is attached. Gaskets  117  for seal-up are attached at the outer circumference of the glass door  115  and the glass small door  116 . 
     As described above, complete seal-up of the culture chamber  102  achieves such a structure that the fluctuation of environment within the culture chamber  102  is suppressed as small as possible. 
     The environment in the culture chamber  102  is maintained at 37 degree C. in temperature, 90 percent in humidity, and ambient atmosphere of CO2. Temperature management is carried out by temperature-controlled air flow within the housing case  108 . 
     In  FIG. 1 , firstly holders  111 , on which containers  110  having cells ready to start culture are placed, are put down on a carrier  112  as a batch of multiple holders and carried into a transferring rack  118 , while the external small door  113  and the glass small door  116  of the culture chamber  102  are opened. Note that the carrier  112  has a role of a carrying case in carrying the holders  111 . 
     Subsequently, the holder  111 , on which the container  110  is placed, is taken out from the carrier  112  that was carried into the transferring rack  118  by an automatic transportation unit, and is stored in a holder storage cabinet  119  that is arranged at an internal side wall of the culture chamber  102 . Here, the holder storage cabinet  119  includes the carriers  112  that can accommodate multiple holders  111 , and samples such as cells are placed in the holder storage cabinet  119  to be cultured. Note that the holder  111  contains necessary culture fluid. 
     The automatic transportation unit includes driving parts for the triaxial direction of X axis direction, Y axis direction and Z axis direction, and a rotating direction. On the base plate  106 , there is a driving part of Y axis direction, and Y axis moving stage  122  is attached to guide axis  120  and a driving shaft  121 , the driving shaft  121  being rotated by a motor  123  to move the Y axis moving stage  122 . As for Z axis direction, a Z axis moving stage  126  is attached to a guide axis  124  and a driving shaft  125  on the Y axis moving stage  122 , and the Z axis moving stage  126  is moved by rotation of the driving shaft  125  with a motor  127 . 
     A lower side of the X axis moving stage  129  is attached onto the Z axis moving stage  126  via a rotating shaft (not shown), and a rotating motor  128  can rotate the lower side of the X axis moving stage  129 . In other words, the lower side of the X axis moving stage  129  can be turned toward the side of the transferring rack  118  or toward the side of the holder storage cabinet  119 . An upper side of the X axis moving stage  130  placed on a guide rail  131  on the lower side of the X axis moving stage  129  can be moved in the direction of X axis through driving a rack  132  provided on a side of the upper side of the X axis moving stage  130  by a pinion  134  of a motor  133 . 
     In addition, an arm  135  is attached on a top point of the X axis moving stage  130 . The arm  135  enters under a rib portion of the holder  111  to support the holder  111  so as to transfer the holder  111  having the container  110  thereon. 
     Now, a series of carrying-in procedures of the container  110  that holds cells to be cultured into the culture chamber  102  will be described. An operator opens the external small door  113  and the glass small door  116 , loads the container  110  placed on the holder  111  in the carrier  112 , places the whole carrier  112  on the transferring rack  118  in the culture chamber  102 , and closes the external small door  113  and the glass small door  116 . 
     Subsequently, the automatic transportation unit is operated to move the Y axis moving stage  122  to meet a Y axis coordinate position of the holder  111  stacked in the carrier  112  on the transferring rack  118 . Next, the Z axis moving stage  126  is moved in the direction of Z axis to the height of the holder  111  stacked in the carrier  112  on the transferring rack  118 . The height thereof is equal to the height where the arm  135  at the upper side of the X axis moving stage  130  can be inserted under the rib of the holder  111  with a slight gap. In addition, the upper side of the X axis moving stage  130  has been already turned to the direction of the transferring rack  118 . 
     In that state, the upper side of the X axis moving stage  130  is moved in the direction of X axis to insert the arm  135  under the rib of the holder  111  stacked in the carrier  112  on the transferring rack  118 . In that situation, the Z axis moving stage  126  is raised a little so that the arm  135  can bring up the holder  111  slightly. When the arm  135  comes to support the holder  111 , the upper side of the X axis moving stage  130  is drawn back from the transferring rack  118  to take out completely the holder  111  from the carrier  112  on the transferring rack  118 . 
     After taking out the holder  111 , the upper side of the X axis moving stage  130  is turned towards the holder storage cabinet  119 . Following that, the Y axis moving stage  122  is moved to meet a position on the Y axis of a targeted carrier  112  in the holder storage cabinet  119 . Then the Z axis moving stage  126  is moved in the direction of Z axis up to a height of the targeted carrier  112  in the holder storage cabinet  119 . This height is such that the bottom of the holder  111  has a small gap above the shelf position of the targeted carrier  112 . In this situation, the upper side of the X axis moving stage  130  is moved towards the side of the holder storage cabinet  119  in the X axis direction to bring in the holder  111  into the targeted carrier  112 . After carrying the holder  111  into the targeted carrier  112 , the Z axis moving stage  126  is lowered until the bottom of the holder  111  comes into contact with the shelf of the targeted carrier  112 . When the arm  135  is detached from the rib of holder  111 , the upper side of the X axis moving stage  130  is withdrawn from the holder storage cabinet  119  in a departing direction to complete a storage of the holder  111  to the carrier  112  in the holder storage cabinet  119 . 
     Now, when the cells under culture in the holder storage cabinet  119  is to be observed, the holder  111  is taken out from the holder storage cabinet  119  and moved onto the transparent glass stage plate  136 , which is an observation stage. Procedure of the automatic transportation unit in that event is identical with the procedure where the holder  111  is stored in the holder storage cabinet  119  from the transferring rack  118 , accordingly the description thereof is omitted. Electric heat wires (heater) for condensation proof are stuck on the rear surface of the transparent glass stage plate  136 , the wires being disposed in a toric form so as not to disturb effective luminous flux in a center of light axis for observation. 
     Next, an observation chamber  105  of the culture observation device  101  will be described. Referring to  FIG. 1 , in the observation chamber  105  as an optical system of observation, an observation support stand  137  is fixed to the base plate  106  and optical components are disposed on the observation support stand  137 , and thus the microscope is formed. The objective lens  138  of the microscope can be exchanged with a guide rail  140  on an objective lens support stand  139 , and the objective lens support stand  139  is provided with an upper and lower mechanism for focus adjustment. Further behind the objective lens  138 , a plurality of middle variable lenses  141  are provided on an electrically driven turret, so that a lens can be exchanged by electrical arrangement. In addition, a fluorescent lighting unit  142  is attached under the electrically driven turret. 
     In addition, a CCD support stand  143 , in which optical material such as lenses, reflectors are assembled, is attached to the observation support stand  137  located under the fluorescent lighting unit  142  so that imaging is performed in a CCD camera  144 . In the lower part of the observation chamber  105 , a personal computer  103  for processing the observation images shot by the CCD camera  144  and a controller  104  in which a device for maintaining the environment of the culture chamber  102 , power supply and the like are stored, are provided. 
     Moreover, there is an opening for observation by the microscope in a part of the base plate  106  between the culture chamber  102  and the observation chamber  105 , and around the opening, a stage support stand  145  integrated onto the base plate  106  via a gasket  107  is fixed. 
     In addition, on the stage support stand  145 , a stage plate  149 , an X stage  146  and a Y stage  147  are provided. Aluminum material is used for weight saving as structural material of each stage. Note that the stage support stand  145 , the stage plate  149 , X stage  146  and Y stage  147  have also openings for observation by the microscope, and to the opening portion of the upper-most Y stage  147 , a transparent glass stage plate  136  is stuck, which is to be an observation stage, and thereby airtightness is conserved. By the way, the electric heat wires of the transparent glass stage plate  136  are for heating the observation stage not to cause dew condensation in the culture chamber  102 , which is in high humidity. Further, an elastic material  148  is attached between the Y stage  147  and the stage support stand  145  as a connecting material to couple the both, thereby providing such an arrangement as to separate atmosphere completely between the culture chamber  102  and the observation chamber  105 . Incidentally, a column  156  is integrated onto a part of the stage support stand  145  to attach a lighting part. 
     Next, structure of the X stage  146  and the Y stage  147  will be described.  FIG. 3  is a top plan view when the elastic material  148  of  FIG. 2  is removed. The stage plate  149  fixed on the stage support stand  145  lies in the very bottom and the X stage  146  is mounted on two guide rails  150  on the stage plate  149 . As for the X stage  146 , a motor  152 , to which a pinion  151  on the stage plate  149  is attached, rotates to drive a rack  153  provided on a side of the X stage  146 , and thus the X stage  146  is moved in the X axis direction. The Y stage  147  is mounted on two guide rails  154  and a driving shaft  155 , and a motor  156  rotates the driving shaft  155  to move the Y stage  147  in the Y axis direction. 
     The holder  111 , which has been transferred by the automatic transportation unit, is positioned on the transparent glass stage plate  136  and can be observed by the objective lens  138  of the microscope. As for lighting from the upper part in an occasion of observation, an LED  157  is mounted in a seal-up portion fixed to the base plate  106  via a pole  156 . The light emitted from the LED  157  passes through a rectangular aperture  158 , a phase ring  159  and a lighting objective lens  160  and lights cells in the container  110  on the holder  111 . 
     Now, an elastic material  148 , which is a structural element of the culture observation device  101 , will be described in detail.  FIGS. 4(   a ) and  4 ( b ) are diagrams showing an enlarged circumference area of the elastic material  148  in  FIG. 1 .  FIG. 4(   a ) is a cross section view in the X axis direction when the top view of  FIG. 4(   b ) is cut along a cross section A. The elastic material  148  is provided between the Y stage  147  and the stage support stand  145  and is elastic in the direction of X axis, Y axis and in a diagonal direction. Note that the connecting position of the fixing side of the elastic material  148  is not always on the stage support stand  145  but any portion will do, which is fixed part to the housing case  108  and where seal-up can be executed. Similarly, the connecting position of the moving side is not always on the Y stage  147  but any portion will do, which is movable and where seal-up can be executed such as the transparent glass stage plate  136 . 
     Next, a situation where the elastic material  148  is expanded will be described.  FIGS. 5(   a ) and  5 ( b ) show a situation when the Y stage  147  and the transparent glass stage plate  136  are moved to the left side in the X axis direction.  FIG. 5(   a ) is a cross section view in the X axis direction when  FIG. 5(   b ) is cut along a cross section B. The left side of the elastic material  148  is in a shrinkage situation where flexure is compressed, and conversely the right side of the elastic material  148  is in an extended situation where flexure is expanded. As described above, the transparent glass stage plate  136 , which is integrated with the Y stage  147 , can be moved towards the left side while keeping the culture chamber  102  and the observation chamber  105  completely separated from each other. As a result, the objective lens  138  comes to a situation of being moved towards right side relatively, allowing the objective lens  138  of the microscope to be aligned to the right side of the sample and thus observation can be performed. 
       FIGS. 6(   a ) and  6 ( b ) show a situation where the Y stage  147  and the transparent glass stage plate  136  are moved to the right side in the X axis direction.  FIG. 6(   a ) is a cross section view in the X axis direction when  FIG. 6(   b ) is cut along a cross section C. Conversely to the situation of  FIG. 5(   a ), the right side of the elastic material  148  is in a shrinkage situation where flexure is compressed, and conversely the left side of the elastic material  148  is in an extended situation where flexure is expanded. As described above, the transparent glass stage plate  136 , which is integrated with the Y stage  147 , can be moved towards the right side while keeping the culture chamber  102  and the observation chamber  105  completely separated from each other, and observation can be performed while the objective lens  138  of the microscope is aligned to the left side of the sample. 
       FIG. 7(   a ) shows a situation of the elastic material  148  where the Y stage  147  and the transparent glass stage plate  136  are moved toward the upper side in the Y axis direction. The upper side of the elastic material  148  is in a shrinkage situation where flexure is compressed, and conversely the lower side of the elastic material  148  is in an extended situation where flexure is expanded. Similarly to the previous case, the transparent glass stage plate  136 , which is integrated with the Y stage  147 , is moved toward the upper side while keeping the culture chamber  102  and the observation chamber  105  completely separated from each other, and observation can be performed while the objective lens  138  of the microscope is aligned to the lower side of the sample. 
       FIG. 7(   b ) shows a situation of the elastic material  148  where the Y stage  147  and the glass heater are moved towards right and lower side diagonally. The lower side, the right side, and the diagonally lower and the right side of the elastic material  148  are in a shrinkage situation where flexure is compressed, and conversely the upper side, the left side and the diagonally upper and the left side of the elastic material  148  is in an extended situation where flexure is expanded. Similarly to the other cases, the transparent glass stage plate  136 , which is integrated with the Y stage  147 , is moved towards the right and lower side diagonally while keeping the culture chamber  102  and the observation chamber  105  being completely separated from each other, and observation can be performed while the objective lens  138  of the microscope is aligned to the left and upper side of the sample. 
     The elastic material  148 , as described in  FIG. 6  and  FIG. 7 , is elastic in all circumferential directions in a plane according to the movement of the X stage  146  or the Y stage  147 , and therefore robust and soft material such as rubber is required to be used. In addition, material that does not give any effect on cell culture is required and material such as nitrile rubber that has low gas permeation performance is used in an embodiment. Further, the elastic material  148  is made up of such material having low reflection and light-shielding performance, hence the light when observing samples does not cause diffused reflection, neither permeates into the observation chamber  105 . 
     Moreover, the elastic material  148  has to take the “flexure” effectively with expanding and shrinking due to driving stroke of X and Y stages. Therefore, the section shape of the elastic material  148  in the direction of expanding and shrinking is required to have bellows shape. The outer shape of the elastic material  148  in  FIG. 7  was a rectangular shape similarly with the shape of the transparent glass stage plate  136 . However, the outer shape may be round shape or elliptical shape and so on so that four corners of the elastic material  148  are not burdened when the transparent glass stage plate  136  is moved in X and Y direction. 
     Next, the bellows shape of the elastic material  148  will be described using  FIGS. 8(   a ) to  8 ( c ).  FIG. 8(   a ) is a diagram showing a situation of a section in the direction of expanding and shrinking the elastic material  148  according to the first embodiment, the section of which is a rectangular shape. In general, a bellows shape used for photograph devices such as camera is triangular shape as the elastic material  348  shown in  FIG. 8(   c ), but the stroke when stretched in expanding direction is shorter than that of the rectangular shape and the load required when expanded becomes heavier, resulting in more load in a driving motor. In an embodiment, the rectangular shape shown in  FIG. 8(   a ) is employed in order to secure flexure amount when expanded. As shown in the elastic material  248  of  FIG. 8(   b ), angled corners of the rectangular may be rounded to be circular arc shape. 
     As described so far, in the culture observation device  101  according to the present embodiment, driving part such as XY stages for observation is arranged in a side of the observation chamber  105  side separated completely from the culture chamber  102  by a fixed partition including the base plate  106 , the stage support stand  145  and the like, and a moving partition (movement stage) including the Y stage  147 , and the transparent glass stage plate  136  coupled with the elastic material  148  and the like. Therefore the driving part is not exposed in a high humidity environment of the culture chamber  102 . Particularly, as for a stage for observation, stainless steel (martensite) is usually used for the guide rails that perform critical function to obtain stage accuracy. When the XY stages for observation are placed in the culture chamber  102  as in a conventional method, it has been difficult to shut off rust in a high humidity environment of the culture chamber  102 . In the present embodiment, however, since the observation chamber  105  is isolated from the culture chamber  102 , grease can also be used, which has not been possible to use in the conventional method due to effects on cell culture, and resultantly rust and the like can be prevented. 
     Moreover, in the present embodiment the fixed partition and the moving partition are coupled with each other via the elastic material  148 , however, instead of the elastic material  148 , the fixed partition and the moving partition may be arranged so that one of them is put on top of another and a part of the both partition slides on each other, and injection of shielding liquid such as oil and the like in the sliding part enables to separate the culture chamber and the observation chamber. 
     In addition, motors of the XY stages do not require any countermeasure against the high humidity, and dust generated by sliding of mechanical parts can be shut off. Further, since the section shape of the elastic material  148  in the direction of expanding and shrinking is arranged to be a rectangular shape, the part to be driven in the XY stages is easy to expand as well as causing less load on motors. Particularly it is not necessary to move the heavy microscope as a whole as in a conventional method, thus enabling downsizing and cost-reduction. 
     Furthermore, the moving partition and the transparent glass stage plate  136  are integrated, therefore working distance of the objective lens  138  of the microscope can be shortened, and accordingly a bright view of observation with high NA can be obtained. 
     The many features and advantages of the embodiments are apparent from the detailed specification and, thus, it is intended by the appended claims to cover all such features and advantages of the embodiments that fall within the true spirit and scope thereof. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the inventive embodiments to the exact construction and operation illustrated and described, and accordingly all suitable modifications and equivalents may be resorted to, falling within the scope thereof.