Abstract:
A culture apparatus for microscopic viewing enables effective microscopic viewing at high magnification of samples such as cells suspended in a culture solution while the samples are maintained at a uniform temperature. The apparatus is used to microscopically view a sample placed on a microscope stage, and is provided with a housing unit that houses the vessel containing a culture solution and the sample; a lid that closes the aperture on the upper surface side of the housing unit; a transparent sheet top heater provided in the portion of the lid that corresponds to the viewing area; and a temperature detecting means where a thin wire detection unit penetrates into the interiors of the housing unit and a well plate to directly measure the temperature of the culture solution. Based on the temperature information, a controller controls the temperature of the transparent sheet top heater using a feedback method.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates generally to a culture apparatus for microscope viewing suitable for the observation of specimen such as cells. 
       BACKGROUND OF THE INVENTION 
       [0002]    Culture apparatus for microscope viewing of this type such as those described in Japanese Patent Laid-open (kokai) public disclosure 2008-259430 and shown in  FIG. 8 , are well known in the art. A culture apparatus for microscope viewing  100  shown in  FIG. 8  comprises a housing unit  103  and a lid  107  for closing an upper opening of the housing unit. A water tank  105  is provided along the inner side wall of the housing unit  103 . The interior space defined by the inner edge of the water tank  105  can accommodate a container such as a well plate W for holding culture media or medium A and a specimen or specimens B. The culture apparatus, which accommodates the well plate, are adapted to be rested on the stage of a microscope. 
         [0003]    In order to hold the specimen B such as cells within the culture solution A under the conditions suitable for making observation, it is required to keep a temperature of the culture solution A stable at or around 37° C. In the prior art, a heater plate  109  of thin aluminum sheet of high thermal conductivity is provided over the lower opening of the housing unit  103  for heating the solution A within the well plate. 
         [0004]    The heater plate  109  has a plurality of through holes  111  at positions corresponding to wells w of the well plate W for making the wells w remain for the microscope viewing. 
         [0005]    Recently, there has been used an inverted microscope for high magnification observation. In order to make observation operation in high magnification, the objective lens R is likely to be close to the lower surface of the housing unit  103 . Accordingly, if observation object is changed from one well to an adjacent well, as shown in arrow in the figure, the objective lens R is retracted from the one well, the housing unit  103  is moved in X-Y (two dimensional) directions on the on the microscope stage S in order that the objective lens S faces on the adjacent well, and the objective lens R is closed to the adjacent well, because the wells w is plated on the heater plate  109 . This is a cumbersome operation. Even if such operation can be effected automatically, complicated programming is required. Further, if there is used as the well plate, a multi-type one (for example, 96 wells), it is difficult to bring the objective lens R even close to the lower surface of unit  103 , because the hole web of the heater plate  108  is smaller. 
         [0006]    So called oil or water immersion lens in which the space between the exposed lower surface of the container and the objective lens R is filled with oil or water, is used to observe the specimen B for clear observation in high magnification. However, the liquid ball made on the top of the objective lens R, which fills the space is apt to be broken under the effect of the reciprocal movement of the objective lens R. 
         [0007]    On the other hand, when the heater plate  109 , instead, has one large though hole, which cover all of the wells w and heats the wells s by w by transferring heat energy from the web of the heater plate  109 , it is impossible to heat each well w equally, because of difference in distance between each well and the heater plate. In other words, the one wells w adjacent the web of the heater plate  108  is rich-heated, and the other wells w far away from the web thereof is poor-heated. 
       TECHNICAL LITERATURE OF PRIOR ART 
     Patent Literature 
       [0008]    Japanese Patent Laid-open (kokai) public disclosure 2008-259430 
       SUMMARY OF THE INVENTION 
     Problem or Problems to be Solved by the Invention 
       [0009]    The object of the present invention is to provide a culture apparatus for microscope viewing enabling effective microscopic viewing in high magnification a specimen such as cells dispersed in the culture solution, and the method for using the same. 
       The Means for Solving the Problem or Problems 
       [0010]    In accordance with the invention defined as a first aspect, there is provided a culture apparatus for microscope viewing enabling effective microscopic viewing a specimen placed on a stage of the microscope including a housing unit for accommodating a container holding a culture solution including the specimen therein, and a lid for closing an upper opening of the housing unit comprising: a transparent sheet top heater provided over a portion of the lid that corresponds to the viewing area; and a temperature detecting means in which a thin wire detector penetrates into the interior of the housing unit and further penetrates into the interior of a container to measure the temperature of the culture solution supplied thereto directly; wherein the temperature of the transparent sheet top heater is controlled through a feedback method on the basis of the temperature information obtained from the temperature detecting means. 
         [0011]    In accordance with the invention defined as a second aspect, there is provided a culture apparatus for microscope viewing according to the first aspect further comprising: an introducing means for introducing the detector of the temperature detecting means into the housing unit. 
         [0012]    In accordance with the invention defined as at third aspect, there is provided a culture apparatus for microscope viewing according to the first or second aspect, further comprising: a dedicated lid for the container; wherein the detector of the temperature detecting means is adapted to be inserted into the container through an introducing means provided on the container. 
         [0013]    In accordance with the invention defined as a fourth aspect, there is proved a culture apparatus for microscope viewing according to the third aspect, wherein: the lid for the container is made of a transparent glass material. 
         [0014]    In accordance with the invention defined as a fifth aspect, there is provided a culture apparatus for microscope viewing according to any of the first to fourth aspects wherein: within the housing unit is accommodated a container having a plurality of sections or a plurality of containers, the detector of the temperature detecting means being introduced into one of the sections or one of the containers to detect the temperature thereof, and remaining sections or containers being used for the microscopy of the specimen. 
       Effect to be Obtained from the Invention 
       [0015]    In accordance with a culture apparatus for microscope viewing of the present invention, effective microscopic viewing in high magnification the specimen such as cells dispersed in the culture solution kept in the constant temperature is accomplished efficiently. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]    Embodiments of the present invention will now be described more fully with reference to the accompanying drawings in which: 
           [0017]      FIG. 1  is an exploded perspective view illustrating an embodiment culture apparatus for microscope viewing according to the present invention; 
           [0018]      FIG. 2  is a perspective view of the  FIG. 1  culture apparatus from seen from the upper side; 
           [0019]      FIG. 3  is a perspective view of the  FIG. 1  culture apparatus from the lower side; 
           [0020]      FIG. 4  is a cross-sectional view illustrating the lid of the  FIG. 1  culture apparatus; 
           [0021]      FIG. 5  is an enlarged perspective view illustrating part of the housing unit to which a detector holder for guiding and securing the detector of the temperature control means of the culture apparatus for microscope viewing; 
           [0022]      FIG. 6  is an enlarged perspective view illustrating the side of the container relating to the temperature control means of the  FIG. 1  culture apparatus; 
           [0023]      FIG. 7  is a cross-sectional view illustrating the behavior of the objective lens when used in the  FIG. 1  culture apparatus; and 
           [0024]      FIG. 8  is a cross-sectional view illustrating the behavior of the objective lens when used in a prior art culture apparatus. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0025]    A culture apparatus  1  for microscope viewing in accordance with an embodiment of the present invention will now be described with reference to  FIGS. 1-7 . 
         [0026]    The culture apparatus  1  includes a housing unit  3  and a lid  53 . 
         [0027]    The structure of the housing unit  3  will now be described as follows. 
         [0028]    Referring to  FIG. 1 , reference numeral  5  is added to a body portion of the unit  3 . The body portion  5  has an upper opening  7  and a lower opening  9 . The lower opening  9  has an inward flange  11  inwardly extending from the side portion, whereby it is smaller than the upper opening. 
         [0029]    A substantially rectangular frame shaped adaptor  13  is fixedly fit, along its side surfaces, to the inner edges of the lower opening  9 . The adaptor  13  has an inner flange  17  extending inwardly from the lower opening  15 . Retaining springs  19  are removably connected at their base to four portions of the upper end surface of the adaptor  13 . The adaptor  13  is to be used for a well plate W, and when the well plate W is fit to the adaptor  13 , the well plate W can be retained therein stationary by means of spring force of the retaining springs  19 . 
         [0030]    The lower end surface of the adaptor  13  protrudes downwardly from the lower surface of the housing unit  3 , to make a stepped portion  21 . 
         [0031]    The space defined by the inner side surface of the body portion  5 , the outer side surface of the adaptor  13  and the inward flange  11  is adapted to be served as a water tank  23 . 
         [0032]    A pair of water supplying tubes  25  and a gas supplying tube  27  is adapted to be attached to the body portion  5 . The tips of the water supplying tubes  25  are extended into the water tank  23 , and the tip of the gas supplying tube  27  are extended into the water tank  23  and reaches to the bottom surface thereof. 
         [0033]    There is prepared a temperature detecting means  29  having a thin-wired thermocouple-type detector  31 . The housing unit  3  has, provided therewith, a detector holder  33  for guiding the detector  31 . The detector holder  33  is positioned between the water supplying tubes  25  and the gas supplying tube  27 . As can be seen from  FIG. 5 , the holder  33  includes a base block  35  and the retainer block  43 . The base block  35  of the holder  33  is secured onto the outer side surface of the body portion  5 . The base block  35  is provided with a pair of threaded holes  37  and a shallow groove  39  for passing the detector  31  there through. Further, a groove  41  is provided on the upper surface of the body portion  5  so as to align the groove  39  for guiding the detector  31  within the body portion  5  of the housing unit  3 . 
         [0034]    The retainer block  43  is also provided with a pair of holes  45  which will correspond to the holes  37  of the base block  35 . 
         [0035]    The detector  31  is passed through the groove  39  of the base block  35  and the groove  41  of the body portion  5 , and the retainer block  43  is placed on the base block  35  so as to squeeze the detector  31  between a protrusion protruding from the lower surface of the retainer block  43  and the grooves  39 ,  41 , and then the base block  35  and the retainer block  43  are secured by tightening the nuts  47 . Thus, the detector  31  is secured to the body portion  5  with placing the tip thereof into the body portion  5 . As can be seen from the above, the length of the detector  31  to be introduced within the body portion  5  can be adjusted so as to be enough that the tip of the detector  31  reaches a predetermined well w in the well plate W and is steeped therein. 
         [0036]    The body portion  5  is also provided with a holder  51  for connecting a pair of perfusion hoses so that the culture apparatus  1  can be adjusted for perfusion culture. 
         [0037]    The culture apparatus  1  is also provided with a lid  53  for closing the upper opening  7  of the housing unit  3 . 
         [0038]    As can be seen from  FIG. 4 , the lid  53  has a substantially rectangular shape. The lid  53  includes a frame  55  supporting a light permeable portion  57 . The light permeable portion  57  is formed by a pair of juxtaposed transparent glass plates  59 ,  59  spaced with each other. On the upper surface of the lower glass plate  59  is formed a transparent conductive film  61 . The transparent conductive film  61  is adapted to be served as a heater by providing electric energy thereto through the electric cord  63 . The air layer formed between the glass plates  59 ,  59  is expected to be served as an insulation layer. 
         [0039]    The cord  63  and the temperature detecting means  29  are both connected to the controller  65 . The controller  65  is adapted to supply the electric energy to the transparent conductive film  61  by means of feedback control system on the basis of information from the detecting means  29  and a predetermined target temperature. 
         [0040]    Although the well plate W to be installed in the adaptor  13  can be the one with or without lid, the well plate W of this embodiment is that with a lid  67 . In this connection, the lid  67  is provided with a means for introducing the tip of the detector  31  of the temperature detecting means  29  into the predetermined well w. 
         [0041]    The lid  67  is of transparent glass plate for suppressing fluorescence effect, and makes a box-shape. The lid  67  is adapted to mate with the stepped portion formed around the rim of the upper opening of the well plate W and fit thereinto to close the well plate W, as shown in  FIG. 6 . A small rectangular cutout is formed through the lid  67  along the side nearest to the holder  33 . Thus exposed upper surface of the rim of the side wall of the well plate W is provide with a pair of threaded holes  71 ,  71  and a shallow groove (not shown) disposed between holes. 
         [0042]    There is provided for securing the detector  31  to the well plate, a retainer plate  73  which be fit into the rectangular cutout  69 . The retainer plate  73  has a pair of threaded holes  75 ,  75  corresponding to the holes  71 ,  71 . 
         [0043]    In order to secure the detector  31  to the well plate W, the detector  31  is passed through the groove, the retainer plate  73  is fit to the cutout  69  and then set screws  77 ,  77  are fastened through the threaded holes  75 ,  75  and  71 ,  71 . 
         [0044]    The detector  31  may be bent so that after the detector  31  is extended within the well plate W over a predetermined well w, the detector  31  can be bent to descend the tip thereof in the vicinity of the bottom surface of the well w. 
         [0045]    The method for using the culture apparatus  1  for microscope viewing will now be described. 
         [0046]    A dummy well plate W (which has been closed by the lid  67 ) is set within the adaptor  13 , which has been installed preliminary to the body portion  5  of the housing unit  3 , and the housing unit  3  is closed by the lid  53  to make a closed culturing space. Then CO 2  gas is supplied through the gas supplying tube  27 , water is also supplied to the water tank  23  through the water supplying tube  25 , and electric current is supplied to the transparent conductive film  61  of the transparent sheet top heater through the cords  63  to heat the atmosphere within the housing unit  3 . Although not shown in the drawings, the bottom surface of the water tank  23  is also provided with a heater for evaporating the water contained within the water tank  23  to attain predetermined humidity within the closed culturing space. As can be seen, the gas supplying tube  27 , the water supplying tube  25 , and the heaters all fall within conditions under control of the controller  65  to provide the predetermined culturing condition. 
         [0047]    The housing unit  3  of thus obtained culturing space is set on the stage S of the microscope. The stepped portion  21  of the body portion  5  of the housing unit  3  is adapted to hook on the edge of the opening provided through the stage S of the microscope. 
         [0048]    On the other hand, a actual well plate W for microscope viewing is prepared. In one well w is filled the culture solution A, which is positioned along a side nearest to the holder  33 . The one well is that exclusive for detecting the temperature within the wall plate, and filled only with the culture solution A. Within the other wells w are filled the culture solution A with the specimen B. The tip of the detector  31  of the temperature detecting means  29  exposed by removing protective sheath there around is then steep into the exclusive well w down to the bottom thereof for immersing into the culture solution A. Then, the upper opening of the well plate W is closed by the lid  67  and the detector  31  is secured thereto through the above-described means. 
         [0049]    Upon completion of a preliminary run using the dummy well plate W for the predetermined period, the lid  53  is displaced, the dummy well plate W is removed from the housing unit  3 , the actual well plate W is set therein, the detector  31  of the temperature detecting means  29  is fixed by means of the detector holder  33 , and the housing unit  3  is again closed by the lid  53 . 
         [0050]    Since the culture solutions A with the specimens B within the wells w of the well plate W are kept in the predetermined humidity and temperature suitable being suitable for culturing, observation is possible for the specimens B. 
         [0051]    Observation of the specimens B contained within a plurality of wells w may be carried out parallelly by shifting the stage S of the microscope. 
         [0052]    The bottom surface portions of the well plate corresponding to the wells w are bared, and the spaces between wells w are also bared. In other words, the bottom surface of the well plate W defines flush surface which allows the stage S of the microscope to be shifted in the X-Y direction while keeping the objective lens R in the position nearest to the well w for making observation. 
         [0053]    Since the distance between the transparent sheet top heater and any one of the wells w is kept constant, the culturing solution A and the specimen B contained in every wells w are heated equally. 
         [0054]    The top heater is adapted to be energized on the basis of the information of the temperature of the culturing solution A detected directly through the detector  31  immersed within the culturing solution A, slight variation of the temperature is followed sequentially. In this connection, the temperature of the culturing solution A and the specimen B contained therein are be maintained always in the target temperature in high precision. 
         [0055]    It is to be appreciated that the present invention has been described hereinabove with reference to certain embodiment of the invention but that various additions, deletions, alterations and modifications may be made thereto without departing from the intended sprit and scope of the invention. 
         [0056]    For example, containers such as well plate are not limited to ones dedicated for the culture apparatus  1 , and separately sold container may be used. However, if it is intended to close the container, it is recommended to use the lid as described above for make it easier to introduce the detector  31  of the temperature detecting means  29 . 
         [0057]    The container cannot be limited to well plate and one or more dishes can be utilized instead therefor. If two dishes are used, the one is dedicated for detecting the temperature within the dishes. 
         [0058]    One dish or well is used only for detecting temperature, since the detector  31  may prevent observation and bring contaminants. Unless you mind such risks, the one dish or well may be used for detecting temperature as well as for observation. 
       INDUSTRIAL APPLICABILITY 
       [0059]    The culture apparatus for microscope viewing in accordance with an embodiment of the present invention can be used as an accessory of the microscope. 
       EXPLANATION FOR REFERENCE NUMERAL 
       [0000]    
       
           1  . . . culture apparatus  3  . . . housing unit  5  . . . body portion  7  . . . upper opening  9  . . . lower opening  11  . . . inward flange  13  . . . adaptor  15  . . . lower opening  17  . . . inner flange  19  . . . retaining spring  21  . . . water tank  25  . . . water supplying tubes  27  . . . gas supplying tube  29  . . . temperature detecting means  31  . . . detector  33  . . . detector holder  35  . . . base block  37  . . . threaded hole  39  . . . groove  41  . . . groove  43  . . . retainer block  45  . . . hole  47  . . . nut  49  . . . holder  51  . . . holder for perfusion culture  53  . . . lid  55  . . . frame  57  . . . light permeable portion  59  . . . transparent glass plates  61  . . . transparent conductive film  63  . . . electric cord  65  . . . controller  67  . . . lid (for well plate)  69  . . . rectangular cutout  71  . . . threaded hole  73  . . . retainer plate  75  . . . threaded hole  77  . . . screw W . . . well plate w . . . well A . . . culturing solution B . . . specimen S . . . stage of microscope R . . . objective lens