Cell observing apparatus and cell incubation method

User's time and labor required for performing manipulations with respect to cells which exist in an incubation container, are reduced. To achieve the above, a cell observing apparatus includes an observation stage supporting an incubation container that houses cells, a micro imaging optical system forming, on an imaging device for micro imaging, an image of the cell in the incubation container disposed at an observing position of the observation stage, a macro imaging optical system forming, on an imaging device for macro imaging, an image of an area wider than that captured by the micro imaging optical system in the incubation container, and a controlling unit controlling an operation of a manipulation needle that manipulates the cells in the incubation container, in which the micro imaging optical system is disposed on a side facing the macro imaging optical system with the observation stage being located therebetween.

BACKGROUND

The present application relates to a cell observing apparatus and a cell incubation method.

2. Description of the Related Art

A process of generating induced pluripotent stem cells (iPS cells) is disclosed in, for example, Non-Patent Document 1 (Center for iPS Cell Research and Application, Institute for Integrated Cell-Material Sciences, “Generation of Human induced Pluripotent Stem Cells”, Kyoto University, Mar. 5, 2009).

In the process in Non-Patent Document 1, a feeder cell layer is first formed on a bottom surface of an incubation container that accommodates culture fluid, human adult skin cells (fibroblasts) are seeded on the layer, and then four genes called as Yamanaka factors are introduced into those cells (retroviral vectors for introducing the four genes are added). Thereafter, when the incubation is continued while changing the culture fluid, a cell colony in which the four genes are introduced and nothing else happens (Non-iPS cell colony) and a cell colony in which after the four genes are introduced, a differentiation potency is exhibited (iPS cell colony) appear, on the feeder cell, so that by picking up only the latter among the above using a syringe, the generation of iPS cell line is realized.

In this process, the picking of cell colony is manually performed by a skilled researcher while looking through an eyepiece lens of a microscope. At that time, the researcher sets an observation magnification of the microscope to a low-power side to observe a relatively wide range of the incubation container, and searches for the iPS cell colony. Subsequently, a stage is moved to dispose the cell colony in a vicinity of an optical axis of an objective lens, the observation magnification of the microscope is then set to a high-power side, and after the cell colony is confirmed as the iPS cell colony, the observation magnification of the microscope is returned to the low-power side, and a tip of the syringe is inserted into a dish to perform a picking of the cell colony.

However, when the cell colony was the Non-iPS cell colony, there was a need to reset the observation magnification of the microscope to the low-power side, to again search for a cell colony which seems like the iPS cell colony.

SUMMARY

The present invention has been made to solve the problems of the related art described above. A proposition of the present invention is to provide a cell observing apparatus and a cell incubation method effective for saving user's time and labor required for performing manipulations (injection, patch clamp, picking and so on) with respect to cells which exist in an incubation container.

A cell observing apparatus includes an observation stage supporting an incubation container that houses cells, a micro imaging optical system forming, on an imaging device for micro imaging, an image of the cell in the incubation container disposed at an observing position of the observation stage, a macro imaging optical system forming, on an imaging device for macro imaging, an image of an area wider than that captured by the micro imaging optical system in the incubation container, and a controlling unit controlling an operation of a manipulation needle that manipulates the cells in the incubation container, in which the micro imaging optical system is disposed on a side facing the macro imaging optical system with the observation stage being located therebetween.

Note that it is also possible that the controlling unit moves the manipulation needle to a position at which a picking of the cell in the incubation container can be performed based on a wide image obtained by the imaging device for macro imaging and a partial image obtained by the imaging device for micro imaging.

Further, it is also possible that the controlling unit decides a focused cell being a cell to be focused among the cells in the incubation container based on an image analysis of a wide image obtained by the imaging device for macro imaging, calculates position coordinates of the focused cell, and then controls the manipulation needle based on the wide image obtained by the imaging device for macro imaging and a partial image obtained by the imaging device for micro imaging.

Further, it is also possible that the controlling unit moves the manipulation needle to the position coordinates of the cell being a manipulation target based on the wide image obtained by the imaging device for macro imaging and makes the manipulation needle to be positioned at the position coordinates of the cell based on the partial image obtained by the imaging device for micro imaging when controlling the manipulation needle.

Further, it is also possible that the micro imaging optical system and the macro imaging optical system are configured coaxially.

Further, it is also possible that the micro imaging optical system is disposed on a side of a bottom portion of the incubation container.

Further, it is also possible that the controlling unit controls the manipulation needle to perform a picking of a target cell from the incubation container based on the partial image obtained by the imaging device for micro imaging, and seeds the target cell obtained through the picking in another incubation container.

Further, a cell incubation method is a cell incubation method of incubating cells using the cell observing apparatus, increasing a number of the target cell by repeatedly conducting, a step seeding the target cell obtained through the picking in the other incubation container, and then transferring the other incubation container to an incubator, and a step incubating the seeded target cell for a certain period of time in the incubator, and then returning the other incubation container to the cell observing apparatus.

Note that the target cell may also be an iPS cell.

Further, a cell observing apparatus includes an observation stage supporting an incubation container that houses cells, a micro imaging optical system forming, on an imaging device for micro imaging, an image of the cell in the incubation container disposed at an observing position of the observation stage, a macro imaging optical system forming, on an imaging device for macro imaging, an image of an area wider than that captured by the micro imaging optical system in the incubation container, and a controlling unit controlling an operation of a manipulation needle that manipulates the cells in the incubation container, in which the controlling unit realizes both of an obtainment of a wide image by the imaging device for macro imaging and an obtainment of a partial image by the imaging device for micro imaging at a same time when controlling the manipulation needle.

Note that it is also possible that there is further provided an oblique illuminating optical system illuminating the incubation container on the observation stage with an illumination luminous flux which is not parallel to optical axes of the macro imaging optical system and the micro imaging optical system.

Further, it is also possible that the controlling unit displays, in real time, both of a wide dark-field image obtained by the imaging device for macro imaging during a period of time in which the oblique illuminating optical system is turned on, and a partial dark-field image obtained by the imaging device for micro imaging during the period of time.

Further, it is also possible that the cell observing apparatus further includes an excitation light illuminating optical system irradiating excitation light to the cells in the incubation container, and a storing unit obtaining, through the imaging device for micro imaging, partial fluorescence images from respective parts of the incubation container to which the excitation light is irradiated, and previously storing histories of the respective parts, in which the controlling unit reads the history of the part, in the incubation container, positioned on the optical axis of the micro imaging optical system, from the storing unit, and displays the history together with the partial dark-field image which is being displayed in real time.

Further, it is also possible that the controlling unit displays the history as a movie image.

Further, it is also possible that the controlling unit reads latest partial fluorescence images of the respective parts of the incubation container from the storing unit, and superimpose displays a guiding image in which the partial fluorescence images are connected, on the wide dark-field image which is being displayed in real time.

Further, it is also possible that the controlling unit automatically adjusts the observation stage to make a manipulation target candidate position on the optical axis of the micro imaging optical system when the manipulation target candidate in the incubation container is designated on the guiding image.

Further, it is also possible that, when a manipulation target candidate in the incubation container is designated on the guiding image and a completion notification of manipulation with respect to the manipulation target candidate is input, the controlling unit highlights the manipulation target candidate on the guiding image.

Further, it is also possible that the controlling unit simultaneously displays the wide image obtained by the imaging device for macro imaging, the partial image obtained by the imaging device for micro imaging, and the movie image.

Further, it is also possible that, when an arbitrary cell is designated from the wide image, the controlling unit displays the movie image of the designated cell.

A cell incubation method of incubating cells, increasing a number of good cell by repeatedly conducting a micro imaging step performing a micro observation of the cell which is being incubated in an incubation container, to obtain a partial image, a macro imaging step performing a macro observation of an area wider than that in the micro imaging step in the incubation container, to obtain a wide image, a judging step judging a state of the cell based on the partial image, a picking step controlling, based on the wide image and the partial image, a manipulation needle to perform a picking of the good cell whose state is judged as good, from the incubation container, a step seeding the good cell picked up by the manipulation needle, in another incubation container, and then transferring the other incubation container to an incubator, and an incubating step incubating the seeded good cell in the incubator for a certain period of time.

Note that it is also possible that in the picking step, an XY coordinate position of the manipulation needle is made to coincide with an XY coordinate position of the cell based on the wide image obtained in the macro imaging step, the manipulation needle is driven toward an XYZ coordinate position of the cell based on the partial image obtained in the micro imaging step, and the cell is picked up by the manipulation needle.

Further, the cell may also be an iPS cell.

DETAILED DESCRIPTION OF THE EMBODIMENTS

First Embodiment

Hereinafter, embodiments of a cell observing system will be described as embodiments of the present invention.

FIG. 1is a diagram explaining a configuration of a mechanical part of the present system. As illustrated inFIG. 1, in the present system, there are provided an inverted microscope10for observing cells in an incubation container30, a manipulator20for manipulating the cells in the incubation container30, an electrically-operated reserve stage60that supports a reserve container40, a manipulator controller21for driving the manipulator20, and a stage controller12for driving an observation stage11of the inverted microscope10. Note that the present system also includes a not-illustrated computer (explanation of the computer will be made later).

In the inverted microscope10, there are provided the transmission-type and electrically-operated observation stage11that supports the incubation container30, a macro imaging optical system (stereomicroscope)14that obtains an entire image of the incubation container30from above and front of the incubation container30, a micro imaging optical system (magnifying microscope)18that obtains a magnified image of a part of the incubation container30from below and front of the incubation container30, an oblique illuminating optical system15that illuminates the entire incubation container30from a diagonally upward direction of the incubation container30, a fluorescence epi-illumination optical system17that irradiates excitation light to a part of the incubation container30from below and front of the incubation container30through an objective lens18eof the micro imaging optical system18, and a focus knob13with which a user manually performs focusing of the objective lens18ewith respect to the incubation container30.

The incubation container30is, for example, a dish with a diameter of 100 mm. On a bottom surface of the incubation container30, a feeder cell layer is formed, and an upper part of the feeder cell layer is filled with culture fluid. Further, on the feeder cell layer, human adult skin cells (fibroblasts) are previously seeded, and to those cells, retroviral vectors for introducing four genes called as Yamanaka factors are added. Note that in these cells, a fluorescence gene that generates fluorescence of specific color (green color in this case) only when a differentiation potency is exhibited after the introduction of four genes, is previously introduced.

In this incubation container30, a cell adhered to a surface of the feeder cell layer is proliferated to form a cell colony. In order to observe the cell colony, in the aforementioned focusing, a focal plane of the objective lens18eof the micro imaging optical system18is positioned in the vicinity of the bottom surface of the incubation container30(in the vicinity of the feeder cell layer).

The observation stage11fixes and holds the incubation container30with a holder suitable for a shape of the incubation container30. Accordingly, even if the incubation container30is temporarily removed from the observation stage11for the purpose of the change of the culture fluid or the like, a posture and a disposed position of the incubation container30with respect to the observation stage11are reproduced. Further, the observation stage11is connected to the stage controller12, and when a user operates the stage controller12, the observation stage11makes, in accordance with the operation contents, the incubation container30move in directions (XY directions) along a mounting table of the observation stage11.

Note that although the observation stage11and the stage controller12may also be directly connected, here, for convenience of explanation, it is assumed that they are indirectly connected via a controlling circuit of the computer (FIG. 2).

The micro imaging optical system18includes an electrically-operated revolver18dthat holds a plurality of objective lenses, a light deflecting mirror18c, an imaging optical system18b, and an imaging device18a, and obtains a magnified image (an image magnified 10 times, for example) of an area (partial area), in the incubation container30, captured by a field of view of the objective lens18e. Note that an optical axis of the objective lens18eof the micro imaging optical system18is vertical to a reference plane of the observation stage11.

The revolver18dholds the plurality of objective lenses with different magnifications, and switches the objective lens18eof the micro imaging optical system18to another objective lens. Accordingly, an observation magnification of the micro imaging optical system18is switched between 10-power and 4-power, for example.

The macro imaging optical system14includes imaging lenses14band an imaging device14a, and obtains a reduced image (an image reduced to half, for example) of the entire incubation container30. With the imaging lenses14b, the vicinity of the incubation container30and an imaging area of the imaging device14aare coupled in a conjugate relation, and even if the focusing is not performed, it is possible to form the entire image of the incubation container30on the imaging area with a sufficient contrast. Note that an optical axis of the imaging lenses14bcoincides with the optical axis of the objective lens18eof the micro imaging optical system18.

The oblique illuminating optical system15includes a light source for oblique illumination15aformed of a white light source or the like, and illuminating lenses15b, and illuminates the entire incubation container30from a diagonal direction with a substantially uniform illuminance. Note that an optical axis of the illuminating lenses15bintersects the optical axis of the macro imaging optical system14in the vicinity of the mounting table of the observation stage11.

It is set that, out of the light emitted from the light source for oblique illumination15aand passing through the illuminating lenses15b, scattered light generated at the incubation container30is incident on the macro imaging optical system14and the objective lens18eof the micro imaging optical system18, but, non-scattered light (direct light) generated at the incubation container30is not incident on the macro imaging optical system14and the objective lens18eof the micro imaging optical system18almost at all.

Accordingly, when the light source for oblique illumination15ais turned on, the micro imaging optical system18can obtain a magnified dark-field image of the aforementioned partial area of the incubation container30(referred to as “micro dark-field image”, hereinafter). Further, when the light source for oblique illumination15ais turned on, the macro imaging optical system14can obtain a reduced dark-field image of the entire incubation container30(referred to as “macro dark-field image”, hereinafter).

The fluorescence epi-illumination optical system17includes an excitation light source17a, illuminating lenses17b, and a fluorescence block17c, and irradiates excitation light to the aforementioned partial area via the objective lens18eof the micro imaging optical system18. Note that an emission wavelength of the excitation light source17ais set to a wavelength for exciting a fluorescent material exhibited in a cell, and a detection wavelength of the fluorescence block17cis set to a wavelength same as a wavelength of fluorescence emitted by the fluorescent material (a wavelength of green color, in this case).

Accordingly, when the excitation light source17ais turned on, the micro imaging optical system18can obtain a magnified fluorescence image of the aforementioned partial area of the incubation container30(referred to as “micro fluorescence image”, hereinafter).

Note that the fluorescence block17cof the fluorescence epi-illumination optical system17is configured to be capable of being inserted/removed into/from an optical path of the micro imaging optical system18, and the insertion/removal is performed by a not-illustrated electrical mechanism. When the micro imaging optical system18obtains the micro dark-field image, the fluorescence block17cis removed from the optical path, and when the micro imaging optical system18obtains the micro fluorescence image, the fluorescence block17cis inserted into the optical path.

The manipulator20is, for example, a hydraulic manipulator, and a manipulation needle for manipulating cells in the incubation container30, is attached thereto. Here, it is assumed that a syringe22is attached as the manipulation needle. Note that a tip portion of the syringe22can be changed with a new one, in accordance with need.

The manipulator20is provided on a base common to the inverted microscope10, at a position separated from the inverted microscope10, and supports a pump part of the syringe22with the tip of the syringe22directed diagonally downward. The manipulator20rotates the syringe22around a rotating shaft20aparallel to the optical axis of the macro imaging optical system14, or makes the syringe22shift in a direction along the rotating shaft20a.

Further, the manipulator20can set a combination of a rotation position and a shift position of the syringe22to an observing mode (mode illustrated by a solid line inFIG. 1) which is previously determined according to need. Further, the manipulator20can set a combination of the rotation position and the shift position of the syringe22to a separating mode (mode illustrated by a dotted line inFIG. 1) which is previously determined according to need.

The observing mode illustrated by the solid line inFIG. 1is a mode in which the tip of the syringe22is disposed on the optical axis of the macro imaging optical system14, and the tip of the syringe22is positioned above the uppermost portion of the incubation container30. When the syringe22is in this observing mode, even if the observation stage11is tentatively moved in the XY directions, there is no chance that the syringe22is brought into contact with the incubation container30. Further, when the shift position of the syringe22is displaced downward from this observing mode, it is possible to make the tip of the syringe22dip in the culture fluid in the incubation container30.

The separating mode illustrated by the dotted line inFIG. 1is a mode in which the entire syringe22is completely separated from the inverted microscope10. The aforementioned reserve stage60disposes, at a position below the tip of the syringe22in the separating mode (a reserve position indicated by a reference numeral40ainFIG. 1), a reservoir of the reserve container40.

Further, the manipulator20is connected to the manipulator controller21, and when the user operates the manipulator controller21, the manipulator20drives the syringe22in accordance with the contents of the operation.

Note that although the manipulator20and the manipulator controller21may also be directly connected, here, for convenience of explanation, it is assumed that they are indirectly connected via the controlling circuit of the computer (FIG. 2).

Further, although the pump part of the syringe22may also be directly manually operated by the user, here, for convenience of explanation, it is assumed that the pump part is electrically operated, and is driven by the manipulator20. In this case, the user performs each of suction of fluid (here, the culture fluid including cells is called as “fluid”) into the syringe22, and ejection of the fluid from the syringe22, through the operation of the manipulator controller21.

The reserve stage60fixes and holds the reserve container40with the holder suitable for the shape of the reserve container40. In the reserve container40, a plurality of reservoirs40-1to40-8are formed by being arranged in an XY plane in a state of facing respective openings thereof upward.

Therefore, if the fluid is ejected from the syringe22when the syringe22is in the separating mode, the fluid can be reserved in the reservoir (available reservoir) disposed at the reserve position40a. Further, when the reserve stage60moves the reserve container40in the XY directions, it is possible to switch the available reservoir among the reservoirs40-1to40-8.

FIG. 2is a diagram explaining the computer of the present system. As illustrated inFIG. 2, a computer50of the present system includes a controlling circuit52, a CPU51, a storage memory53, a working memory54, and an interface circuit55.

Among the above, the controlling circuit52is connected to the observation stage11, the revolver18d, the imaging devices14aand18a, the fluorescence block17c, the light source for oblique illumination15a, the excitation light source17a, the reserve stage60, the stage controller12, and the manipulator controller21illustrated inFIG. 1.

Further, in the computer50, an operation program for the CPU51is previously installed. This operation program is stored in the storage memory53, and is read on the working memory54according to need, to be executed by the CPU51.

Further, to the computer50, input/output devices such as a keyboard56, a mouse57, and a display58are connected via the interface circuit55. The user can input various instructions into the CPU51of the computer50via the keyboard56or the mouse57. Note that the transmission/reception of information between the computer50and the user is set to be conducted through a well-known GUI utilizing the keyboard56, the mouse57, and the display58.

The information which is input into the computer50by the user includes an observing schedule of the incubation container30, a start instruction of observation, a start instruction of picking assistance, and the like.

The observing schedule indicates an observation frequency with respect to the incubation container30, and is set as “every 24 hours” or the like, for example. This observing schedule is stored in the storage memory53.

The start instruction of observation is an instruction which is input when a preparation of the incubation container30is completed and the incubation is started, and is an instruction for making the CPU51execute observation processing (which will be described later).

The start instruction of picking assistance is an instruction which is input when a picking of necessary cell (iPS cell colony, in this case) is performed after the incubation is conducted over a sufficient period of time, and is an instruction for making the CPU51execute picking assistance processing (which will be described later).

FIG. 3is a flow chart of the observation processing performed by the CPU51. Hereinafter, respective steps inFIG. 3will be described in order. Note that it is set that, when executing the observation processing, the tip of the syringe22is removed from the syringe22.

Step S11: The CPU51reads the observing schedule stored in the storage memory53, and compares the observing schedule with current time and date, to thereby judge whether an observation timing arrives or not. When the observation timing arrives, the process proceeds to step S12, and when the observation timing does not arrive, the CPU51stands by.

Step S12: The CPU51instructs the controlling circuit52to perform tiling shooting with oblique illumination. The controlling circuit52drives the fluorescence block17caccording to need to remove the fluorescence block17cfrom the optical path of the micro imaging optical system18, and drives the revolver18daccording to need to insert the objective lens for 10-power observation into the optical path of the micro imaging optical system18. Under this state, by turning on the light source for oblique illumination15a, and repeatedly driving the imaging device18awhile step-moving the observation stage11in the XY directions, the controlling circuit52obtains a plurality of micro dark-field images which individually covers respective partial areas of the incubation container30, and turns off the light source for oblique illumination15a.

The CPU51gives, to each of the plurality of micro dark-field images obtained by the controlling circuit52, coordinate information on the incubation container of the corresponding partial area (container coordinate information), and then writes those micro dark-field images into the storage memory53. Note that when performing the writing, the CPU51gives information of current time and date (observing time and date) to each of these micro dark-field images.

Step S13: The CPU51gives an instruction to the controlling circuit52to perform tiling shooting with the excitation light source. The controlling circuit52drives the fluorescence block17cto insert the block into the optical path of the micro imaging optical system18. Under this state, by turning on the excitation light source17a, and repeatedly driving the imaging device18awhile moving the observation stage11in a movement pattern same as that of step S13, the controlling circuit52obtains a plurality of micro fluorescence images which individually covers respective partial areas of the incubation container30, and turns off the excitation light source17a.

The CPU51gives, to each of the plurality of micro fluorescence images obtained by the controlling circuit52, coordinate information on the incubation container of the corresponding partial area (container coordinate information), and then writes those micro fluorescence images into the storage memory53. Note that when performing the writing, the CPU51gives information of current time and date (observing time and date) to these micro fluorescence images. Note that in this case, it is assumed that a portion with high brightness in the micro fluorescence image is represented by a color same as the color corresponding to the detection wavelength of the fluorescence block17c(green color, in this case).

Step S14: The CPU51judges whether or not a termination instruction is input by the user, in which when the instruction is not input, the process returns to step S11, and when the termination instruction is input, the flow is terminated.

Therefore, every time the observation timing arrives, the CPU51obtains the micro dark-field images and the micro fluorescence images regarding the respective partial areas of the incubation container30, and writes the images into the storage memory53. Accordingly, a history of the micro dark-field images and a history of the micro fluorescence images are gradually accumulated for each partial area of the incubation container30.

FIG. 4andFIG. 5are flow charts of the picking assistance processing performed by the CPU51. Hereinafter, respective steps inFIG. 4andFIG. 5will be described in order. Note that although the user performs attachment or change of the tip of the syringe22according to need, in the middle of the picking assistance processing, here, for simplification of explanation, it is assumed that the tip of the syringe22is attached when starting the picking assistance processing, and is then continuously used without change.

Step S20: The CPU51reads the images (the plurality of micro dark-field images and the plurality of micro fluorescence images) for each partial area stored in the storage memory53, and based on those images, the CPU51creates a time lapse movie image for each partial area, and stores it in the storage memory53. Note that the creation of time lapse movie image for each partial area is conducted in the following manner. Specifically, the CPU51synthesizes each of the two images covering the same partial area and having the same observing time and date, among the plurality of micro dark-field images and the plurality of micro fluorescence images, into one image, and connects a plurality of synthesized images obtained through such synthesis, in an order of the observing time and date. The time lapse movie image created as above corresponds to the time lapse movie image of the partial area.

Further, the CPU51gives an instruction to the controlling circuit52to set each part of the system to an initial state. The controlling circuit52drives the manipulator20according to need, to set the syringe22to be in the observing mode, and drives the observation stage11according to need, to dispose a center of the incubation container30on the optical axis of the macro imaging optical system14(=the optical axis of the objective lens18eof the micro imaging optical system18). Hereinafter, the optical axis is simply referred to as “optical axis”.

Further, the controlling circuit52drives the fluorescence block17caccording to need to remove the fluorescence block17cfrom the optical path of the micro imaging optical system18, and drives the revolver18daccording to need to insert the objective lens for 10-power observation into the optical path of the micro imaging optical system18.

Further, the controlling circuit52drives the reserve stage60according to need to set the available reservoir to a first reservoir (the reservoir40-1).

Step S21: The CPU51gives an instruction to the controlling circuit52to start a display of live image.

The controlling circuit52turns on the light source for oblique illumination15a, and starts continuous driving of both of the imaging device18aof the micro imaging optical system14and the imaging device14aof the macro imaging optical system18. Accordingly, the micro dark-field images and the macro dark-field images start to be obtained continuously and in a parallel manner.

The CPU51starts sequentially outputting the micro dark-field images sequentially obtained by the controlling circuit52, on a predetermined area on the display58as indicated by a reference numeral58binFIG. 6, and it starts sequentially outputting the macro dark-field images sequentially obtained by the controlling circuit52, on another predetermined area on the display58as indicated by a reference numeral58cinFIG. 6.

Therefore, on the display58, the live image58bof the micro dark-field image (referred to as “micro live image58b”, hereinafter) and the live image58cof the macro dark-field image (referred to as “macro live image58c”, hereinafter) start to be simultaneously displayed.

Further, the CPU51displays a magnification change button58b′, in the vicinity of the micro live image58bon the display58. The magnification change button58b′ is a button with which the user inputs an instruction of changing the observation magnification, into the computer50.

Note that at this moment, the syringe22is set to be in the observing mode, so that a dark-field image22′ of the syringe22is also captured in the micro live image58band the macro live image58c. If it is assumed that a center of the micro live image58bcorresponds to the optical axis, and a center of the macro live image58ccorresponds to the optical axis, a dark-field image of the tip of the syringe22is positioned at a center of each of the micro live image58band the macro live image58c. When only the observation stage11is driven (when the manipulator20is not driven) under this state, a dark-field image of the incubation container30moves on the micro live image58band the macro live image58c, and the dark-field image22′ of the syringe22does not move.

Step S22: The CPU51reads, out of the micro fluorescence images stored for each partial area in the storage memory53, the micro fluorescence images with the latest observing time and date, performs size reduction processing on the micro fluorescence images, and arranges the processed images in an order of the container coordinates, to thereby create a tiling fluorescence image. This tiling fluorescence image is used as a guiding image.

The CPU51performs brightness reduction processing on this tiling fluorescence image, and superimpose displays the processed tiling fluorescence image on the macro live image58c(a reference numeral58dinFIG. 6). Note that when superimpose displaying, the CPU51adjusts a superimposing position of the tiling fluorescence image58d, so that a center of the container in the tiling fluorescence image58dcoincides with a center of the container in the macro live image58c.

Therefore, on the macro live image58c, a current state (a texture, a size, and the like) of cell colonies dotted in the incubation container30and a recent degree of fluorescence of those cell colonies are simultaneously visualized, as illustrated inFIG. 7in an enlarged manner. Note that inFIG. 7, an area, in the tiling fluorescence image58d, which should be represented by green color (an area that exhibits fluorescence) is represented by filling the area (the same applies to the other respective drawings). The user can easily find out a cell colony which seems like an iPS cell colony, on such a macro live image58c.

Further, the CPU51of the present step highlights a partial area disposed at a position of the optical axis, out of the plurality of micro fluorescence images (the plurality of partial areas) that form the tiling fluorescence image58d, as an extraction target candidate of cell. Hereinafter, it is set that the highlighting of the partial area is performed by thickening a contour line of the partial area, as illustrated inFIG. 6andFIG. 7.

Note that when the user selects a partial area which is not highlighted on the tiling fluorescence image58d, it is possible to cancel the designation of the extraction target candidate at the present moment, and to designate the selected partial area as a new extraction target candidate.

Note that the designation of the partial area by the user is conducted in a manner that the user operates the mouse57or the keyboard56to move a cursor (not illustrated) on the display58to the partial area to be designated, and performs clicking operation of the mouse57(or pressing-down of an enter key of the keyboard56).

Meanwhile, the CPU51of the present step reads, as a history of the extraction target candidate, a time lapse movie image of the extraction target candidate from the storage memory53, and writes the time lapse movie image into an area for movie image display of the working memory54. Further, the CPU51starts displaying a still image of a predetermined frame (the latest frame, for example) of the time lapse movie image, as a sample image, on a predetermined area on the display58, as indicated by a reference numeral58ainFIG. 6.

Further, the CPU51displays a play button58a′, in the vicinity of the sample image58a(the sample image of the time lapse movie image) on the display58. The play button58a′ is a button with which the user inputs an instruction of playing the time lapse movie image, into the computer50.

Further, the CPU51of the present step creates a container image of the reserve container40, and displays the image on a remaining area of the display58, as indicated by a reference numeral58einFIG. 6. The container image58eis an image schematically representing an arrangement of the plurality of reservoirs of the reserve container40.

Further, the CPU51highlights a reservoir disposed at the aforementioned reserve position40a, among the plurality of reservoirs that form the container image58e, as a current ejection target candidate. Hereinafter, it is set that the highlighting of the reservoir is performed by thickening a contour line of the reservoir, as illustrated inFIG. 6.

Note that when the user selects a reservoir which is not highlighted on the container image58e, it is possible to cancel the designation of the ejection target candidate at the present moment, and to designate the selected reservoir as a new ejection target candidate.

Note that the designation of the reservoir by the user is conducted in a manner that the user operates the mouse57or the keyboard56to move the cursor (not illustrated) on the display58to the reservoir to be designated, and performs clicking operation of the mouse57(or pressing-down of the enter key of the keyboard56).

Step S23: The CPU51judges whether or not a new designation of the extraction target candidate is made, in which when the new designation is made, the process proceeds to step S24, and when the new designation is not made, the process proceeds to step S26.

Step S24: The CPU51calculates, based on container coordinates of the newly designated extraction target candidate and coordinates of the observation stage11at the present moment, target coordinates of the observation stage11for disposing the newly designated extraction target candidate on the optical axis, and gives an instruction of driving the observation stage11together with the target coordinates, to the controlling circuit52. The controlling circuit52drives the observation stage11to make actual coordinates of the observation stage11coincide with the target coordinates, to thereby dispose a center of the newly designated extraction target candidate on the optical axis.

Step S25: The CPU51updates the tiling fluorescence image58don the display58, the time lapse movie image on the area for movie image display, and the sample image58aon the display58, in the following manner.

The CPU51cancels the highlighting at the present moment on the tiling fluorescence image58d, and starts highlighting of the extraction target candidate newly designated on the tiling fluorescence image58.

Further, the CPU51displaces, in accordance with the displacement of the observation stage11in step S24, the superimposing position of the tiling fluorescence image58don the macro live image58c, to thereby make a center of the container in the tiling fluorescence image58dcoincide with a center of the container in the macro live image58c.

Further, the CPU51reads the time lapse movie image of the newly designated extraction target candidate from the storage memory53, and overwrites the time lapse movie image into the area for movie image display of the working memory54. Further, the CPU51starts displaying a predetermined frame (the latest frame, for example) of the time lapse movie image, instead of the sample image58awhich is being displayed.

Note thatFIG. 8illustrates an example of screen updated by the present step. A partial area separated from the center of the container is designated as the extraction target candidate on the macro live image58cillustrated inFIG. 8, the micro live image58bshows a micro dark-field image of the extraction target candidate, and the sample image58ashows a history of the extraction target candidate (a state at a time of the latest observation, in this case).

Step S26: The CPU51judges whether or not a new designation of the ejection target candidate is made, in which when the new designation is made, the process proceeds to step S27, and when the new designation is not made, the process proceeds to step S29.

Step S27: The CPU51calculates, based on a number of the newly designated ejection target candidate and coordinates of the reserve stage60at the present moment, target coordinates of the reserve stage60for disposing the newly designated ejection target candidate at the aforementioned reserve position40a, and gives an instruction of driving the reserve stage60together with the target coordinates, to the controlling circuit52. The controlling circuit52drives the reserve stage60to make actual coordinates of the reserve stage60coincide with the target coordinates, to thereby dispose the newly designated ejection target candidate at the reserve position40a.

Step S28: The CPU51cancels the highlighting at the present moment of the container image58e, and starts highlighting of the newly designated ejection target candidate. Note that a reference numeral58einFIG. 8indicates an example of the container image58eupdated by the present step.

Step S29: The CPU51judges whether or not the instruction of changing the observation magnification is input, in which when the instruction is input, the process proceeds to step S30, and when the instruction is not input, the process proceeds to step S31.

Step S30: The CPU51instructs the controlling circuit52to change the observation magnification. The controlling circuit52drives the revolver18dto switch the observation magnification of the micro imaging optical system18.

Step S31: The CPU51judges whether or not the instruction of playing the time lapse movie image is input, in which when the instruction is input, the process proceeds to step S32, and when the instruction is not input, the process proceeds to step S33.

Step S32: The CPU51displays (plays and displays) the time lapse movie image written on the area for movie image display, instead of the sample image58a. Through the play and display, the user can observe a growing process (a variation with time of an amount of fluorescence and the like) of the cell colony existed in the extraction target candidate, and can accurately judge whether or not the cell colony is an iPS cell colony. When the user judges that the cell colony is the iPS cell colony, it is only required to operate the stage controller12while looking the micro live image58b, to thereby make the iPS cell colony to be slowly approximated to the side of the tip of the syringe22, as indicated by an arrow mark inFIG. 9.

Step S33: The CPU51judges, via the controlling circuit52, whether or not the stage controller12is operated, in which when the operation is made, the process proceeds to step S34, and when the operation is not made, the process proceeds to step S35.

Step S34: The CPU51gives a driving signal generated by the stage controller12to the observation stage11via the controlling circuit52. Accordingly, the observation stage11is driven as the user desires. Note that in this case, it is assumed that a moving range of the observation stage11is limited to a very small range, which is small enough to prevent the tip of the syringe22from being separated from the extraction target candidate in the incubation container30. When the user judges that the iPS cell colony sufficiently approaches the tip of the syringe22on the micro live image58b, he/she stops the driving of the observation stage11, and starts the operation of the manipulator controller21.

Step S35: The CPU51judges, via the controlling circuit52, whether or not the manipulator controller21is operated, in which when the operation is made, the process proceeds to step S36, and when the operation is not made, the process proceeds to step S39.

Step S36: The CPU51gives a driving signal generated by the manipulator controller21to the manipulator20via the controlling circuit52. Accordingly, the manipulator20is driven as the user desires. For example, when the user makes the syringe22shift downward, the tip of the syringe22is brought into contact with the iPS cell colony, the iPS cell colony is sucked into the syringe22, and after the syringe22is set to be in the separating mode, the iPS cell colony is ejected to the outside from the syringe22.

Step S37: The CPU51judges, based on the driving signal generated by the manipulator controller21, whether or not the fluid is ejected from the syringe22in the separating mode (whether or not the picking is completed), in which when the fluid is ejected (when the picking is completed), the process proceeds to step S38, and when the fluid is not ejected (when the picking is not completed), the process proceeds to step S39.

Step S38: As illustrated inFIG. 10, the CPU51displays the extraction target candidate on the tiling fluorescence image58din a more highlighted manner (performs reverse display, for example), and gives an extraction completion mark58d′ to the extraction target candidate. Further, the CPU51displays the ejection target candidate on the container image58ein a more highlighted manner (performs reverse display, for example), and gives an ejection completion mark58e′ to the ejection target candidate.

Here, in order to clarify the correspondence between the extraction target candidate which is already extracted and the ejection target candidate in which the ejection is already made, the CPU51provides relevance between the extraction completion mark58d′ and the ejection completion mark58e′. For example, the CPU51recognizes the number of times of picking with respect to the incubation container30, based on the number of times of execution of the present step up to the present moment, and the like, and applies a number representing the number of times, to both of the extraction completion mark58d′ and the ejection completion mark58e′. Therefore, the user can intuitively know that the cell of Which partial area is reserved in which reservoir, on the display58.

Note that the extraction completion mark58d′ given to the tiling fluorescence image58dis kept given to the same partial area on the tiling fluorescence image58d, even if the superimposing position of the tiling fluorescence image58don the macro live image58cis changed thereafter. Therefore, the user can avoid a mistake of designating again the partial area which is already extracted as the extraction target candidate, a mistake of reserving a cell extracted from a different cell colony in the same reservoir, and the like.

Note that when, after displaying the extraction completion mark58d′, the user selects a partial area to which the extraction completion mark58d′ is not given on the tiling fluorescence image58d, the partial area can be designated as a new extraction target candidate.

Further, when, after displaying the ejection completion mark58e′, the user selects a reservoir to which the ejection completion mark58e′ is not given on the container image58e, the reservoir can be designated as a new ejection target candidate.

Step S39: The CPU51judges whether or not the termination instruction is input by the user, in which when the instruction is not input, the process returns to step S23, and when the instruction is input, the flow is terminated. Therefore, the user can repeatedly perform a picking of cell colonies until the ejection completion marks58e′ are given to all of the reservoirs of the container image58e.

As described above, the present system includes the macro imaging optical system14and the micro imaging optical system18that observe the incubation container30from mutually opposite sides, and the oblique illuminating optical system15that illuminates the incubation container30from the diagonal direction, as illustrated inFIG. 1, so that it is possible to simultaneously observe a brief state of the entire incubation container30(the macro dark-field image) and a detailed state of a part of the incubation container30(the micro dark-field image).

Furthermore, since the computer50of the present system simultaneously displays, on the display58, both of the live image of the macro dark-field image (the macro live image58c) and the live image of the micro dark-field image (the micro live image58b) arranged side by side, the user does not have to switch the objective lens between when searching for the cell colony which seems like the iPS cell colony among the plurality of cell colonies in the incubation container30, and when observing the cell colony in detail, and the user is only required to transfer his/her gaze on the display58between the macro live image58cand the micro live image58b.

Further, the computer50of the present system previously stores the time lapse movie image of each partial area of the incubation container30in the storage memory53. Further, the computer50reads the time lapse movie image of the partial area (the extraction target candidate) positioned on the optical axis of the micro imaging optical system18from the storage memory53, and displays it, together with the micro live image58b, on the display58, so that the user can simultaneously check the detailed state of the cell colony and the history of the cell colony.

Further, since the computer50of the present system performs superimposing display of the latest tiling fluorescence image58dof the incubation container30on the macro live image58c, the user can simultaneously observe the current state (the texture, the size, and the like) of the plurality of cell colonies dotted in the incubation container30, and the recent degree of fluorescence of those cell colonies.

Further, the computer50of the present system drives the observation stage11in accordance with the designation of the extraction target candidate made by the user, and automatically disposes the extraction target candidate on the optical axis (step S24), so that it is possible to minimize user's time and labor required for operating the stage controller12.

Further, the computer50of the present system judges whether or not the fluid is ejected from the syringe22in the separating mode, via the manipulator controller21, and when the fluid is ejected, the computer50highlights (reverse display) the extraction target candidate on the tiling fluorescence image58d, so that the user can intuitively know that the cell of which partial area is already extracted, on the tiling fluorescence image58d.

Second Embodiment

Hereinafter, an embodiment of another cell observing system will be described as an embodiment of the present invention, with reference toFIG. 11toFIG. 15. Note that in a second embodiment, members denoted by the same reference numerals as those of the first embodiment are the same members as those of the first embodiment including the configurations and the operations, so that explanation thereof will be omitted.

FIG. 11andFIG. 12are configuration diagrams of the present system. A characteristic configuration in the second embodiment is a configuration in which the cell observing system is automated, which is, concretely, a configuration in which the manipulator20automatically controls the syringe22based on a wide-area image (an image of the entire incubation container) obtained by the macro imaging optical system14, and a partial-area image (an image of a focused cell) obtained by the micro imaging optical system18.

The inverted microscope10, the manipulator20, and the reserve stage60are disposed on the same base. The change in the observing position in the incubation container30is conducted by moving the incubation container30in the XY plane by using the observation stage11. As illustrated inFIG. 12, the observation stage11is provided with an observing position detecting unit4formed of an X-direction position detecting encoder4X and a Y-direction position detecting encoder4Y, and by detecting XY coordinates of the observation stage11, observing position coordinates (corresponding to a coordinate system of cell) (X, Y) in the incubation container30are detected. Further, regarding a Z coordinate at the observing position in the incubation container30, by detecting a vertical motion of the objective lens18emade by the focus knob13, using an observing position detecting unit4formed of a Z-direction position detecting encoder4Z, the observing position coordinate Z in the incubation container30is detected. Accordingly, coordinate data (X, Y, Z) at the observing position in the incubation container30is detected, and the coordinate data is registered in a memory of the CPU51(a personal computer PC or the like, which is described as PC, hereinafter) being a controlling device.

The manipulator20has a motor that changes a rotation angle φ of the syringe22(manipulation needle), a motor that changes a swing angle θ of the syringe22, and a motor that changes a movement amount Z of the syringe22in the optical axis direction.

A coordinate system of the manipulator20is detected by a manipulator coordinate detecting unit formed of position detecting encoders in the X′ direction, Y′ direction, and Z′ direction disposed in the manipulator20. Further, coordinates of the tip of the syringe22fixed to the manipulator20are registered in the memory of the PC51as coordinate data (X′, Y′, Z′).

Further, as illustrated inFIG. 11, in the vicinity of the manipulator20, there is disposed a needle tip position detecting unit100that detects the tip of the syringe22fixed to the manipulator20. The needle tip position detecting unit100is a camera using a low-power imaging lens and imaging device (a CCD camera or the like, for example) (described as low-power camera100, hereinafter), and as the lens of the low-power camera100, it is desired to use a lens with a numerical aperture of 0.2 or more and a field number of 1.5 mm or more, for performing positioning of the tip of the syringe22at a set position in an observation field of view of the objective lens18e, with high accuracy. Further, it is also possible that the needle tip position detecting unit100is formed of, not the camera, but a simple optical sensor that detects whether or not the needle tip reaches predetermined position coordinates.

Coordinate data at the set position in the field of view of the objective lens18eand coordinate data of the tip of the syringe22, and a set position in a field of view of the low-power camera100are relatively associated via the PC51. As a result of this, the tip of the syringe22is set at the set position in the field of view of the low-power camera100, and thereafter, it is driven by the manipulator20via the controlling unit of the PC51to be positioned at the set position in the field of view of the objective lens18e. As described above, calibration of the coordinate position of the tip of the syringe22is conducted. Note that at the time of performing the calibration, the incubation container30is not placed on the observation stage11. Further, when a plurality of objective lenses with various magnifications are used, the calibration is conducted with respect to each of the objective lenses.

In like manner, coordinate data at a set position in a field of view of the macro imaging optical system14and the coordinate data of the tip of the syringe22, and the set position in the field of view of the low-power camera100are relatively associated via the PC51. As a result of this, the tip of the syringe22is set at the set position in the field of view of the low-power camera100, and thereafter, it is driven by the manipulator20via the controlling unit of the PC51to be positioned at the set position in the field of view of the macro imaging optical system14.

After an initial setting operation to be described below, the manipulator20is controlled by the controlling unit provided in the PC51, resulting in that the tip of the syringe22is moved from the set position in the field of view of the low-power camera100to the set position in the observation field of view of the objective lens18e, and set at a predetermined position of the incubation container30. Note that the set position is located in the vicinity of a center of the field of view, and is set as an observing position suitable for starting an operation of experiment.

Next, a positioning process of the tip of the syringe22will be described while referring to a flow chart executed by the PC51illustrated inFIG. 13.

First, an initial setting of home position of the coordinate system (X, Y, Z) of the inverted microscope10is conducted (S1).

As illustrated inFIG. 13, focusing of the objective lens18eof the inverted microscope10is performed. In the focusing, a bead with a diameter of several pal such as polystyrene placed on a cover glass is used, and focusing is performed on the bead in the center of the field of view of the objective lens18e. Concretely, an operator moves the bead to a position of the center of the field of view of the objective lens18e(utilizing cross hairs for shooting in the optical system, or the like), and operates the observation stage11and the focus knob13to perform focusing on the bead. Under this state, the observing position detecting units4(4X,4Y,4Z) detecting the XY movements of the observation stage11and the vertical motion of the objective lens18etransmit detected coordinate data (X0, Y0, Z0) to the PC51, and the PC51registers the coordinate data (X0, Y0, Z0) in the memory as home position data. Note that the focus knob13that moves the observation stage11and the objective lens18emay be electrically operated or manually operated.

Next, explanation will be made on the initial setting operation in which a relative positional relation between the tip position of the syringe22in the field of view of the objective lens18eand the tip position of the syringe22in the field of view of the low-power camera100is determined by the coordinate system (X′, Y′, Z′) of the manipulator20.

Through this initial setting operation, a correlation between the tip position of the syringe22at the set position in the field of view of the low-power camera100(the tip position is often set at the position of the center of the field of view) and the tip position of the syringe22at the set position in the observation field of view of the high-power (40-power, for example) objective lens18e(the tip position is often set at the center of the field of view), is registered in advance in the memory of the PC51.

Concretely, when the operator operates the manipulator20to move the tip of the syringe22to the predetermined set position in the field of view of the low-power camera100, coordinate data of the manipulator20at this time is registered in the PC51. Further, when the tip of the syringe22is moved to the predetermined set position in the field of view of the objective lens18e, coordinate data of the manipulator20at this time is registered in the PC51. Based on these two pieces of coordinate data, the PC51recognizes the correlation between the set position in the field of view of the low-power camera100and the set position in the field of view of the objective lens18e.

Description will be made further concretely. The operator drives the manipulator20in each direction of X′, Y′, and Z′, to move the tip of the syringe22to the set position in the field of view of the objective lens18e, and focuses the objective lens18eon the tip of the syringe22(S2).

At this time, a manipulator coordinate detecting unit22transmits detected coordinate data (X′0, Y′0, Z′0) of the manipulator20to the PC51, and the PC51registers the data in the memory (S3). Note that as a method of registration, it is possible to employ a method in which a manually-operated switch is used, or a method in which, when there is an automatic focusing device for detecting coordinates of the tip of the syringe22set at the set position in the field of view of the objective lens18e, the registration is made based on a focusing signal transmitted by the automatic focusing device.

Next, the operator drives the manipulator20in each direction of X′, Y′, and Z′, to move the tip of the syringe22to the set position in the field of view of the low-power camera100, and focuses the low-power camera100on the tip of the syringe22(54).

At this time, the manipulator coordinate detecting unit22transmits detected coordinate data (X′1, Y′1, Z′1) of the manipulator20to the PC51, and the PC51registers the data in the memory (S5). Note that as a method of registration, it is possible to employ a method in which a manually-operated switch is used, or a method in which, when there is an automatic focusing device in the low-power camera100, the registration is made based on a focusing signal transmitted by the automatic focusing device.

By using the above two pieces of coordinate data of the manipulator20, the PC51calculates a movement amount (φ, θ, Z) of the tip of the syringe22.

Regarding movement amount data of the tip from the set position in the field of view of the low-power camera100to the set position in the field of view of the objective lens18e, pieces of difference data among respective coordinates of the manipulator20, namely, δX′=X′1−X′0, δY′=Y′1−Y′0, and δZ′=Z′1−Z′0, are respectively calculated, and are registered, as the movement amount data (δX′, δY′, δZ′), in the memory of the PC51(S6). This is the end of the initial setting operation.

As described above, the initial setting operation, namely, the calibration is terminated.

Accordingly, when the tip of the syringe22is set at the set position in the field of view of the low-power camera100, the manipulator20is driven by the controlling unit of the PC51, and the tip of the syringe22is automatically set at the set position in the field of view of the objective lens18e.

(Explanation of Automatic Control of Manipulator20Based on Automatic Recognition of Cell Image)

FIG. 14is a configuration diagram of a cell production system. In the configuration of the cell production system inFIG. 14, an incubator300and a cell observing system500(the system of the first embodiment or the second embodiment) are connected by an incubation container transfer robot200. A space400in which the cell production system exists is managed to be put under a certain incubation environment. Note that the incubation environment mentioned here includes conditions of temperature, humidity, carbon dioxide and the like.

In this cell production system, an incubation container103in which cells are incubated is subjected to macro observation by the macro imaging optical system14to obtain a wide-area image, a position of cell is specified from the wide-area image and the cell is subjected to micro observation by the micro imaging optical system18, to thereby obtain a partial-area image in the wide-area image. Thereafter, a state of the cell is judged based on the partial-area image, and the cell whose state is judged as good (good cell) is picked up from the incubation container103. The picking is performed by controlling the tip of the syringe22based on the wide-area image and the partial-area image. Further, the cell picked up by the syringe22is seeded in a new incubation container103, and thereafter, the new incubation container103is transferred to the incubator300. The seeded cell is incubated for a certain period of time in the incubator. By repeating the routine, it is possible to incubate the good cell, and to increase the number of the good cell.

The control of picking performed by the syringe22is conducted in the following manner. Specifically, an XY coordinate position of the tip of the syringe22is made to coincide with an XY coordinate position of the cell based on the wide-area image obtained by the macro imaging optical system, and the tip is driven toward an XYZ coordinate position of the cell based on the partial-area image obtained by the micro imaging optical system, to thereby perform a picking of the cell using the syringe22. Further concrete description is as follows.

As illustrated inFIG. 15, the computer50inFIG. 2executes a predetermined program to carry out a control of automatically controlling the manipulator20based on cell images obtained by the macro imaging optical system14and the micro imaging optical system18, to pick up a focused cell, and incubating and proliferating the cell. The processing of the computer50will be described based onFIG. 15.

Step41: Cell images of wide area obtained by the macro imaging optical system14are stored in the storage memory53inFIG. 2. Based on the stored cell images (a plurality of cell images obtained through time lapse shooting), the CPU51performs processing of specifying an iPS cell colony.

The processing of specifying the iPS cell colony includes processing of detecting a coordinate position (XY coordinate value) of the focused cell based on the wide-area image, and processing of judging a noise component which is not the focused cell. For example, it is possible to read the fluorescence to specify the iPS cell colony as explained in the first embodiment, or, when the detection is made in a noninvasive manner, it is also possible to specify the iPS cell colony from morphological information based on a phase-contrast observation image.

The focused cell to be a candidate is specified based on the cell image of wide area. Specifically, cells in the incubation container include an air bubble, a dead cell, a colony cell in an early stage before the cell colony is formed, and so on, to be the noises. In order to remove these noise components, the morphological information of each of the cells scattered in the incubation container is extracted, based on a bright-field observation image (a transmission observation image, a phase-contrast observation image, an observation image obtained through oblique illumination, or the like, for example) obtained by the macro imaging optical system14. Based on the extracted morphological information of each of the cells, which is, for example, information regarding an area of the cell, information regarding a length of a long side of the cell, information regarding a circular degree of the cell, or the like, a cell which does not satisfy predetermined conditions is excluded from a candidate as a noise. If it is configured as above, it is possible to set only the iPS cell colony with good state, to a candidate cell.

Conversely, it is also possible to specify the cell to be the noise as the candidate cell, by performing the above-described processing based on the cell image of wide area. However, in that case, the subsequent processing corresponds to processing in which the cell to be the noise is picked up to leave only a good cell. Accordingly, in that case, the incubation can be continued without using a new incubation container.

Step42: Processing in which the focused cell (iPS cell colony) is specified from the candidate cell specified in step41, is carried out.

A coordinate position (XY coordinate value) of the focused cell is determined based on the wide-area image, the observation stage11is driven based on the coordinate position, and an image of the focused cell is obtained by the micro imaging optical system18(when there exist a plurality of focused cells, images of the respective focused cells are obtained). Further, processing in which a culturing state of the focused cell is judged based on the obtained partial-area image (the image of the focused cell), is conducted. For example, it is possible to read the fluorescence to specify the iPS cell colony as explained in the first embodiment, or, when the detection is made in a noninvasive manner, it is also possible to specify the iPS cell colony from the morphological information based on the phase-contrast observation image.

Note that when the morphological information of the iPS cell colony is used, a high-definition phase-contrast observation image obtained by the micro imaging optical system18is used. As a concrete method of specifying the iPS cell colony, there is a method, for example, in which well-known contour line extraction processing (binarization processing or differential processing, for example) for iPS cell colony is performed on the phase-contrast observation image, and when a variance value of brightness intensity of cell image within the contour line extracted through the processing is recognized to be less than a predetermined variance value (when a cell colony is recognized as a cell colony with uniformity), an iPS cell colony within the contour line is regarded as a good iPS cell colony.

Accordingly, the good iPS cell colony in the incubation container is specified. A processing timing of such steps41and42is previously set by the observing schedule, and, for example, steps S41and42are executed at predetermined time intervals during when the time lapse observation shooting is performed, or steps S41and42are executed after the shooting is performed a predetermined number of times. The processing timing is experientially set based on the incubation time of the iPS cell colony and the like.

Step43: When the iPS cell colony being the focused cell is detected, the manipulator20is automatically controlled based on the macro image58dand the micro image58b, as illustrated inFIG. 6. Concretely, the dark-field image22′ of the syringe captured on the macro image is first detected based on image information of the macro image58d, and a coordinate system of the manipulator20is calculated by the manipulator coordinate detecting unit22. Further, there is performed a control in which a coordinate position of the tip of the syringe is set at a coordinate position (XY coordinate value) of the focused cell determined in step41. Thereafter, the syringe is driven in the Z direction from the coordinate position (XY coordinate value) of the focused cell. When the tip of the syringe enters a predetermined range from the position of the focused cell, the micro imaging optical system can capture the tip, and accordingly, a minute control of the manipulator20is performed based on image information of the micro image58b. The micro image58bhas the image information with higher definition than that of the macro image58d, so that a coordinate position (XYZ coordinate value) of the focused cell and a coordinate position (XYZ coordinate value) of the tip of the syringe can be controlled in a micrometer unit.

Step44: The tip of the syringe approaches the focused cell, and the focused cell is picked up (sucked into the syringe).

Step45: The manipulator20is automatically controlled, and the picked-up focused cell is seeded in a new incubation container103inFIG. 11to be transplanted.

Step46: The transfer robot200inFIG. 15transfers the new incubation container103to the incubator300from the cell observing system500inFIG. 11. The transfer robot200grips the new incubation container103with its articulated transfer arm210, and transfers the incubation container103from an opening of the incubator300into a room in which the environment is maintained.

Step47: The incubation container103transferred to the incubator300is kept in an environment optimum for incubating the iPS cell colony, and the incubation is conducted for a predetermined period of time. An imaging device (a CCD camera or the like) provided in the incubator300performs time lapse shooting of the incubation container103, to obtain images of the iPS cell colony. Based on the images, a culturing state of the iPS cell colony is sequentially analyzed.

The iPS cell colony incubated in the incubator300is again transferred to the cell observing system500by the incubation container transfer robot200, after the elapse of predetermined period of time. Subsequently, the flow chart inFIG. 15is repeatedly executed as described above, in which the culturing state of the cell is analyzed and the cell incubation is repeatedly carried out. Accordingly, it is possible to proliferate only the good iPS cell colony.

Supplement to Embodiments

Note that in the system of each of the aforementioned embodiments, the number of partial areas which can be simultaneously designated as the extraction target candidates is set as one, but, the number may also be plural. In such a case, the computer50is only required to make the user newly designate one of a plurality of extraction target candidates in a state of being designated, and to drive the observation stage11so that the newly designated extraction target candidate is disposed on the optical axis.

Further, in the system of each of the aforementioned embodiments, a rough adjustment of the observation stage11is automatically conducted, and a fine adjustment of the observation stage11is manually conducted (by the stage controller12), but, all of the adjustment of the observation stage11may also be manually conducted (by the stage controller12).

In such a case, when the user designates the extraction target candidate on the tiling fluorescence image58d, only the superimposing position of the tiling fluorescence mage58dis shifted in a state where the contents of the macro live image58cdo not change, so that the user is only required to look the macro live image58cand the tiling fluorescence image58d, and to manually drive the observation stage11so that a dark-field image of cell colony group in the macro live image58cis superimposed on a fluorescence image of cell colony group in the tiling fluorescence image58d.

Further, in the system of each of the aforementioned embodiments, one oblique illuminating optical system15is shared by the macro imaging optical system14and the micro imaging optical system18, but, it is also possible to use an oblique illuminating optical system dedicated to the macro imaging optical system14, and an oblique illuminating optical system dedicated to the micro imaging optical system18. Note that in such a case, it is also possible to set at least one of the oblique illuminating optical system for the macro imaging optical system14and the oblique illuminating optical system for the micro imaging optical system18, as a dark-field epi-illumination optical system.

Further, in the system of the present embodiment, the macro imaging optical system14and the micro imaging optical system18are disposed to face each other with the observation stage11being located therebetween, but, the configuration is not limited to that, and, for example, it is also possible that both of the imaging optical systems are disposed on one side of the observation stage11. Concretely, it is only required that a micro imaging optical system is disposed at the position of the macro imaging optical system14illustrated inFIG. 1, a macro imaging optical system is disposed at the position of the oblique illuminating optical system15, and a transmission illuminating optical system is disposed at the position of the micro imaging optical system18.

Further, in the system of the present embodiment, the macro imaging optical system that obtains the wide-area image of the incubation container is formed of a low-resolution CCD sensor, and the micro imaging optical system that obtains the partial-area image in the wide-area image of the incubation container is formed of a high-resolution CCD sensor. Accordingly, in the micro imaging optical system, by performing trimming of a captured image, it is possible to obtain a partial-area image which is good enough.

Further, in the system of the present embodiment, the picking of the cell in the incubation container is explained, but, the system of the present embodiment is useful also when a predetermined medicine is dropped onto the cell.

Further, in the system of each of the above-described embodiments, the pump part of the syringe22is electrically operated, so that the computer50judges the presence/absence of the completion notification of the picking based on the operation contents of the manipulator controller21, but, when the pump part of the syringe22is not electrically operated, the user has to voluntarily input the completion notification of the picking.

Note that the input of the completion notification of the picking is performed through the aforementioned keyboard56and mouse57, or a separately prepared input device. Alternatively, the input is performed through a specific operation part provided to the manipulator controller21.

Further, although the micro imaging optical system18of each of the aforementioned embodiments detects only one type of the micro fluorescence image, it may also be modified to simultaneously detect a plurality of types of micro fluorescence images with different wavelengths. In such a case, the computer50synthesizes the plurality of types of micro fluorescence images obtained by the micro imaging optical system18, by using mutually different colors, to create a color micro fluorescence image, and after performing processing on the image as described above, the computer50displays the resultant on the display58.

Further, in the aforementioned embodiments, the operation of system when performing the picking of the cell is explained, but, the system can also be applied to manipulations other than the picking (injection, patch clamp and the like).

Further, in the system of each of the aforementioned embodiments, at least a part of the operation of the CPU51may also be executed by the controlling circuit52. Further, in the system of each of the aforementioned embodiments, at least a part of the operation of the controlling circuit52may also be executed by the CPU51.

Further, the inverted microscope10, the manipulator20, and the reserve stage60of the system of each of the aforementioned embodiments may also be disposed within an incubation apparatus. Note that the incubation apparatus corresponds to an apparatus with which a peripheral environment (carbon dioxide concentration, temperature, humidity and the like) of the incubation container is maintained as previously set.