JP5231610B2 - Microscope system and observation method - Google Patents

Description

  The present invention relates to a microscope system including a microscope autofocus device that is used in a device that automatically inspects and records an image using a microscope.

  In recent years, an inspection apparatus using a microscope has been automated in various functional aspects, and an autofocus function for focusing on a specimen is also an essential function of an automated item.

Microscope autofocus is also used for inspection devices for specimens enclosed in a slide glass. For example, in Japanese Patent Application Laid-Open No. 58-83906, an infrared reflecting film is applied to a slide glass or a cover glass in which a specimen is enclosed, and the film is applied to the film. On the other hand, a method for focusing is described. Japanese Patent Laid-Open No. 8-82747 specifically describes the configuration of the infrared reflecting film. Furthermore, Japanese Patent Application Laid-Open No. 2001-91821 describes a method of accurately focusing on a specimen using a passive AF method after focusing on a slide glass or cover glass by an active AF method.
JP 58-83906 A JP-A-8-82747 JP 2001-91821 A

  When the autofocus system as described above is used to analyze the movement of an object in a specimen over a long period of time using a high-magnification objective lens, the following problems arise.

  FIG. 13A is a model diagram in a case where a specimen having several nuclei is placed on a slide glass provided with an infrared reflecting film, and one of the nuclei is observed.

  As shown in FIG. 13A, an infrared reflecting film 202 is provided on the slide glass 201, and a cell 203 as a specimen is placed on the infrared reflecting film 202. The cell 203 includes a nucleus 204 to be examined. In FIG. 13A, a nucleus 204 indicated by a mesh represents that it is a nucleus of interest.

  In the methods described in JP-A-58-63906 and JP-A-8-82747, the focused core shown in FIG. 13A is used to focus on the infrared reflecting film indicated by the one-dot chain line in FIG. 13A. Since it is outside the focal depth region indicated by the two-dot chain line in FIG. 13A, there is a possibility that it cannot be observed.

  Further, in the method using passive AF described in Japanese Patent Application Laid-Open No. 2001-91821, when the nucleus 204 indicated by the broken line in FIG. 13B moves as indicated by the solid line, the maximum contrast position is moved from the position indicated by the broken line. For example, since it moves to the position of an alternate long and short dash line, there is a possibility that the movement of the target nucleus cannot be observed.

  Furthermore, when the portion of the specimen to be emitted is different due to the excitation light irradiated as in fluorescence observation, for example, the portion 205 and the portion 206 shown in FIG. 13C, the position of interest changes for each excitation light. It will be.

    If the autofocus system as described above is not used, observation becomes even more difficult. For example, the ambient temperature of the objective lens changes and the objective lens itself changes in temperature. Even if the lens is fixed, it will be out of focus.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a microscope system capable of reliably measuring the movement of a specimen in long-term observation analysis.

The microscope system of the present invention includes a stage on which an observation sample composed of a transparent body and an observation body placed on the transparent body is placed, and the stage so as to face the observation specimen placed on the stage. An objective lens disposed below, a focusing drive unit that drives at least one of the stage and the objective lens in a direction parallel to the observation optical axis, an observation light source that illuminates the observation sample, and the observation light source A focus detection light source that emits autofocus detection light to the observation sample from below the objective lens, and the detection light reflected from the transparent body in the observation sample through the objective lens. A light receiving means for receiving light through the light receiving means, and an autofocus for automatically focusing to a predetermined position of the transparent body by controlling the focusing drive unit from the result of the light receiving means. A debris portion, an offset amount recording means for recording a distance away from the predetermined position in the optical axis direction, an imaging device for taking an image of the observation sample via the objective lens, and an intermittent of the imaging device A time lapse measurement interval setting means for setting a time lapse measurement interval, which is a photographing interval, and a photographing control means for controlling the imaging device and the autofocus section, and the transparent body is controlled by the autofocus section by the photographing control means. After performing autofocus on the predetermined position, which is the position where the focus detection light is reflected from, the position is separated from the predetermined position recorded in the offset amount recording means by a predetermined amount. The observation sample is photographed at each time lapse measurement interval recorded in the time lapse measurement interval setting means.
The microscope system according to the present invention includes a stage on which an observation sample including a transparent body and an observation body placed on the transparent body is placed, and the stage in the XY direction perpendicular to the observation optical axis. XY stage driving means for driving and controlling, an objective lens disposed below the stage so as to face the observation sample placed on the stage, and at least one of the stage and the objective lens A focusing drive unit that is driven in a direction parallel to the observation optical axis, an observation light source that illuminates the observation sample, and a position at which photographing is performed with respect to the observation body are set to
An imaging position setting means for performing a plurality of settings in the Y direction, a focus detection light source that emits detection light for autofocusing from below the objective lens to the observation sample with a light source different from the observation light source, and the observation Light receiving means for receiving the detection light reflected from the transparent body in the sample through the objective lens, and autofocusing to a predetermined position of the transparent body by controlling the focusing drive unit from the result of the light receiving means An autofocus unit that performs recording, an offset amount recording unit that individually records a distance a predetermined amount away from the predetermined position in the optical axis direction for each imaging position, and imaging of the observation sample via the objective lens An image capturing apparatus that performs a time lapse measurement interval setting means for setting a time lapse measurement interval that is an intermittent shooting interval of the image capturing apparatus, the image capturing apparatus, and the auto And an imaging control means for controlling the focus section and the XY stage driving means, and when the time lapse observation is started by the imaging control means, each time lapse measurement interval recorded in the time lapse measurement interval setting means. In the XY direction by the electric stage to the shooting position set in the shooting position setting means,
The autofocus unit performs autofocus on the predetermined position, which is a position where the focus detection light is reflected from the transparent body, and then records the offset amount recording unit for each photographing position. Taking an image of the observation sample at a predetermined distance away from a predetermined position, if there is an unphotographed imaging position within the same time lapse measurement interval, moving to the next imaging position and imaging, When shooting at all the shooting positions set in the shooting position setting means is completed, a cycle of waiting until the next time lapse measurement interval is repeated.

  ADVANTAGE OF THE INVENTION According to this invention, the microscope system which can measure the movement of a sample reliably in long-time observation analysis is provided.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

First Embodiment This embodiment is directed to a microscope system including an upright microscope. FIG. 1 shows the configuration of the microscope system according to the first embodiment of the present invention. As shown in FIG. 1, a microscope system 100 according to the present embodiment includes a stage 101 on which an observation sample 102 is loaded, and an objective lens 109 arranged so as to face the observation sample 102 placed on the stage 101. I have.

  The objective lens 109 is attached to the revolver 108. The revolver 108 can be electrically driven. By controlling the rotation of the revolver 108 by a revolver drive control unit (not shown), the objective lens 109 having a desired magnification can be arranged in the optical path.

  The stage 101 can be electrically controlled at the position in the XYZ direction. The microscope system 100 includes a stage XY control unit 115 that controls the position of the stage 101 in the XY direction, and the stage 101 in the Z direction. And a stage Z drive control unit 116 for controlling the position. The stage Z drive control unit 116 constitutes a focusing drive unit that drives at least one of the stage and the objective lens for focusing.

  For example, as shown in FIG. 2A, the observation sample 102 includes a slide glass 102A, a cover glass 102B, and an observation body enclosed between them. The observation body includes, for example, a specimen (for example, a cell) and a culture solution. Therefore, the observation body is located between the slide glass 102A and the cover glass 102B, and the objective lens 109 is located above the cover glass 102A.

  Returning to FIG. 1, the microscope system 100 includes a known microscope active autofocus unit 118. The autofocus unit 118 controls the focusing drive unit, that is, the stage Z drive control unit 116 by a so-called TTL (Through The Lens) method. That is, the autofocus unit 118 optically detects the focus position of the observation optical system including the objective lens 109 via the objective lens 109 and sends the information to the stage Z drive control unit 116 by a known technique. The stage Z drive control unit 116 moves the observation sample 102 to the focus position in accordance with information from the autofocus unit 118.

  Furthermore, the microscope system 100 includes a light source 103 for transmitting and illuminating the observation sample 102 or a light source 103 ′ for performing fluorescent illumination. In the transmission observation, the illumination light from the light source 103 is irradiated to the observation sample 102 through the ND filter 104 for dimming, the field stop (FS) 105, and the capacitor 106 incorporating the aperture stop (AS). On the other hand, in the fluorescence observation, the illumination light from the light source 103 ′ is turned back by the fluorescent cube 107 and applied to the observation sample 102 as excitation light. Each optical element can be electrically converted by a respective drive control unit (not shown). Thereby, the illumination optical system for fluorescence observation including the light source 103 ′ and the fluorescence cube 107 can selectively irradiate excitation light having different wavelengths. In other words, the illumination optical system for fluorescence observation can select excitation light.

  A transmitted illumination image or a fluorescence image from the observation sample 102 passes through the objective lens 109, and a part thereof is guided to the eyepiece lens 111 by the lens barrel 110, and the other light flux enters the TV camera 112.

  Control of the microscope is performed from the host PC 113 via the microscope controller 114, and the microscope controller 114 performs actual drive control via the respective control units on the electric control part.

  The light source 103 and the ND, AS, and FS can be controlled from the microscope controller 114 by respective control units (not shown), and the light source voltage and the aperture diameter are controlled.

  On the other hand, the image of the observation sample 102 captured by the TV camera 112 is acquired by the host PC 113 by the video capture board 117. The host PC 113 can store a plurality of images in an image memory (not shown) for acquired images.

  FIG. 2A is a so-called time lapse model for observing the movement of a specimen for a long time. FIG. 2A shows that the specimen (or a specific part such as a nucleus in the specimen) enclosed in the slide glass 102A and the cover glass 102B moves from position A to position B over time. For observation, the stage is driven in the Z direction by a predetermined amount from the center position of the shooting area in FIG. 2A indicated by the one-dot chain line, and shooting is set at the position indicated by the two-dot chain line from the upper limit of the shooting area to the lower limit of the shooting area. It is performed every intermittent time.

  FIG. 3 shows a model of an observation image photographed during the period from photographing time t0 to t4 under the above conditions. In FIG. 3, each piece of image information in the vertical axis direction indicates the distance that the sample indicated by the shaded circle has moved in the stage Z direction, indicating that the sample has moved by Z4 between t0 and t4. Show. Similarly, the XY position in each image indicates that the specimen has moved in the XY direction. In the model of FIG. 3, the specimen that was present at the center of the image at t0 is moved to the right until t4. Indicates that it has moved.

  FIG. 2B shows a setting screen of the host PC 113 in the microscope system 100 for performing shooting as described above. The setting screen includes G1 that displays a sample image from the TV camera 112, an operation display unit G2 that relates to control of the microscope, and a display unit G3 that sets imaging conditions for the sample image.

  An imaging condition setting method will be described with reference to FIGS. 2A and 2B.

  When setting the shooting conditions, first, auto-focus (AF) is performed with the G4 button on the slide glass 102A or the cover glass 102B. FIG. 2A shows a case where AF is performed on the cover glass 102B. When the AF is completed, the stage 101 becomes the reference position in FIG. 2A. The observer moves the stage 101 up and down using the G5 button in order to focus on the specimen to be imaged. G6 displays the address of the current stage position, and the distance from the cover glass position (the position where AF is completed) to the position of the sample can be read. After focusing on the specimen, the G10 button is used to register the center position of the shooting area in the stage Z direction for shooting as shown by shooting count 3 in FIG. 2A. The distance between the cover glass position and the center position is an offset. After registering the center position of the imaging area, the G7 and G9 buttons are used to set the pitch in the Z direction and the number of shots for performing Z-direction slice imaging of the specimen as shown by the imaging Z interval and imaging count in FIG. Set. The number of shots and the pitch can be set by the range and the number of shots in the Z direction. In this case, settings are made using the G8 and G9 buttons. When the range in the Z direction to be photographed is set beyond the slide glass 102A or the cover glass 102B, a warning is displayed for the setting condition. After setting the number of Z shots and the two-way pitch, the shooting time interval is set using G11, and the shooting period is set using G12. When the shooting interval is set shorter than the shooting time in the Z direction set by G7 and G9, a warning display for setting the shooting interval is performed.

  In the present embodiment, the position of the object to be photographed is set after performing autofocus on the cover glass 102B. However, even if the position of the object to be photographed is set, autofocus is performed on the cover glass 102B. It is the same.

  The operation of the microscope system 100 set as described above will be described with reference to FIG.

  When measurement is started (S1), it is checked whether or not a predetermined number of shots have been taken and time lapse measurement according to the shooting time (total) set in the host PC is completed (S2). When the measurement is finished, the process is finished (S10). If the measurement has not been completed, the process waits until the time lapse measurement interval set at the time lapse interval set by the host PC is reached (S3).

  When the measurement interval is reached, AF control is performed on the cover glass 102B, and position control is performed on the reference position shown in FIG. 2A (S4). After driving to the reference position, the stage 101 is driven by the center position of the imaging region that is preset by the host PC, that is, the offset amount (S5).

  That is, after the autofocus unit 118 performs autofocus on the cover glass 102B, the stage Z drive control unit 116 moves the stage 101 by a predetermined fixed amount, that is, an offset amount.

  Furthermore, in order to acquire a Z direction image, it drives to an imaging | photography area upper limit (S6), and imaging | photography is performed (S7). In S5 and S6, the S5 operation can be omitted (internally processed) by calculating the upper limit of the shooting area from the set value of the center position of the shooting area in advance. In addition, it is desirable that the position of these drives be controlled from one direction so that a position shift due to so-called backlash does not occur as much as possible.

  After shooting is performed, driving is performed for the number of shooting counts set by the host PC, for the same shooting Z interval set by the host PC (S8, S9), and this operation is repeated until the measurement is completed ( S2).

  In the microscope system 100 of the present embodiment configured and controlled as described above, by applying autofocus to the cover glass 102B, the reference position is fixed even when the objective lens focal position changes due to a change in the ambient temperature. Since the specimen photographing position is set with an offset in the state, the movement of the specimen can be reliably measured.

  Although the microscope system 100 according to the present embodiment is configured to move the stage 101 up and down, the microscope system 100 may be configured to move the objective lens 109 up and down, and in that case, the same effect can be obtained. In the microscope system 100 of the present embodiment, the microscope is an upright microscope, but it may be a so-called inverted microscope in which an objective lens is installed under the stage 101. It is possible to obtain the same effect. Furthermore, in the present embodiment, the autofocus is an active autofocus, but may be a known passive autofocus, and in this case, the same effect can be obtained.

  In the present embodiment, various modifications can be made without departing from the purpose of photographing after performing autofocus once on the cover glass 102B or the slide glass 102A and then driving a predetermined amount.

  In the present embodiment, the stage 101 is not necessarily movable in the XY direction. That is, the stage 101 can be moved only in the Z direction, and the stage XY control unit 115 may be omitted. Further, the objective lens 109 is not necessarily exchangeable. In other words, the microscope does not have to have a revolver function that holds a plurality of objective lenses rotatably and can selectively place one of them in the optical path.

Second Embodiment The present embodiment is directed to time-lapse measurement for a plurality of measurement points having different XY positions. Since the configuration of the microscope system of this embodiment is the same as that of the first embodiment, a description thereof will be omitted. In the present embodiment, an offset driving operation is performed for each XY position of the sample.

  FIG. 5 shows a model having a plurality of measurement points XY1, XY2, and XY3 in the observation body enclosed by the slide glass 102A and the cover glass 102B. In this model, the Z-direction positions C1, C2, and C3 of the measurement points XY1, XY2, and XY3 with respect to the cover glass 102B are different from each other. A procedure for time-lapse measurement of the movement of each of these measurement points will be described.

  FIG. 6 is a host PC setting screen for setting offset driving for each XY position shown in the present embodiment, and FIG. 6 shows some functions added to the first embodiment. Explain the function.

  G20 is an entire image of the slide glass 102A, and the observer uses the G23 button to move the stage 101 in the X and Y directions to determine measurement points such as a nucleus for time-lapse observation. Information on the XY position of the stage 101 is displayed on G24, and determination of the measurement point is performed with the button of G21 after the stage XY position is stopped. When the position is set, the registered measurement points are indicated by X points in the entire G20 observation body image. The offset amount setting and photographing condition setting for each measurement point are performed in the same manner as the procedure shown in the first embodiment. If the same shooting conditions as those already set at another measurement point are set, the G22 button can be called if the shooting conditions are registered, and the shooting conditions for all measurement points or specific measurement points can be set. It is possible to unify.

  The operation of the microscope system set as described above will be described with reference to FIG.

  When the measurement is started (S20), it is checked whether or not a predetermined number of shots are completed and the time lapse measurement corresponding to the shooting time (total) set by the host PC is completed (S21). When the measurement is finished, the process is finished (S31). If the measurement has not ended, the process waits until the time lapse measurement interval set by the time lapse interval set by the host PC is reached (S22).

  When the measurement interval is reached, stage XY driving is performed for setting a plurality of measurement points (S23). When driven to the stage XY position of each measurement point, AF control is performed on the cover glass 102B at the stage XY position, and position control is performed on the reference position that is the cover glass position (S24). After driving to the reference position, the stage 101 is driven to each photographing center region position preset by the host PC, that is, by an offset amount (S25).

  That is, after the autofocus unit 118 performs autofocus on the cover glass 102B, the stage Z drive control unit 116 moves the stage 101 by a predetermined fixed amount, that is, an offset amount.

  Furthermore, in order to acquire a Z direction image, it drives to an imaging | photography area upper limit (S26), and imaging | photography is performed (S27). In S25 and S26, the S25 operation can be omitted (internally processed) by calculating the upper limit of the shooting area from the set value of the center position of the shooting area in the same manner as in the first embodiment.

  After shooting is performed, driving is performed by the number of shooting counts set by the host PC, by the shooting Z interval set by the host PC (S28, S29), and shooting at all measurement points is completed, and The aperture is continuously reduced until the time lapse measurement end condition is reached (S30, S31).

  The microscope system of the present embodiment configured and controlled as described above applies autofocus to the cover glass 102B for each of a plurality of measurement points, so that even when the focus position of the objective lens changes due to a change in ambient temperature. Since the specimen photographing position is set with an offset while the reference position is fixed, the movement of the specimen can be reliably measured even with a plurality of measurements.

  In addition, at a plurality of measurement points XY, for example, if the autofocus conditions acquired at the previous timelabs, such as a region for determining the range for searching the specimen, are updated during the AF control shown in S24 of FIG. AF control can be performed with high reliability.

  In the present embodiment, the objective lens 109 is not necessarily exchangeable. In other words, the microscope does not have to have a revolver function that holds a plurality of objective lenses rotatably and can selectively place one of them in the optical path.

Third Embodiment This embodiment is directed to time lapse measurement including replacement of an objective lens. Since the configuration of the microscope system of this embodiment is the same as that of the first embodiment, a description thereof will be omitted.

  When conversion of the objective lens is included as one of the conditions for the time lapse measurement, since the imaging lens is different, the offset amount may have to be changed. In this embodiment, time lapse measurement is performed by setting an offset amount for each objective lens.

  The offset amount for each objective lens is set by an offset amount setting button for each objective lens (not shown) on the host PC screen as in the first or second embodiment.

  FIG. 8 shows operation control of the microscope system in which the offset amount for each objective lens is set.

  When the measurement is started (S40), it is checked whether or not a predetermined number of shots are completed and the time lapse measurement corresponding to the shooting time (total) set by the host PC is completed (S41). When the measurement is finished, the process is finished (S51). If the measurement has not been completed, the system waits until the time lapse measurement interval set by the time lapse interval set by the host PC is reached (S42).

  When the measurement interval is reached, the set objective lens is inserted into the optical path (S43). When the objective lens is inserted in the optical path, AF control is performed on the cover glass 102B, and position control is performed on the reference position which is the cover glass position (S44). After driving to the reference position, the stage 101 is driven by an offset amount set for each objective lens preset by the host PC (S45).

  That is, after the autofocus unit 118 performs autofocus on the cover glass 102B, the stage Z drive control unit 116 moves the stage 101 by a predetermined fixed amount, that is, an offset amount.

  Furthermore, in order to acquire a Z direction image, it drives to an imaging | photography area upper limit (S46), and imaging | photography is performed (S47).

  After shooting is performed, driving is performed for the number of shooting counts set on the host PC, and also for the shooting Z interval set on the host PC (S48, S49), and shooting with all the set objective lenses is completed. In addition, the diaphragm is continuously returned until the time lapse measurement end condition is reached (S50, S51).

  The microscope system of the present embodiment configured and controlled as described above performs autofocus using the objective lens in each optical path when observation with a plurality of objective lenses is included in the time lapse measurement condition, Even when there is a focus shift including the autofocus side between the objective lenses, the specimen shooting position is set with an offset while the reference position is fixed, so the movement of the specimen is reliably measured even with multiple measurements. It becomes possible.

  In the present embodiment, the stage 101 is not necessarily movable in the XY direction. That is, the stage 101 can be moved only in the Z direction, and the stage XY control unit 115 may be omitted.

Fourth Embodiment The present embodiment is directed to time lapse measurement including excitation light conversion. Since the configuration of the microscope system of this embodiment is the same as that of the first embodiment, a description thereof will be omitted.

  When fluorescence cube conversion, that is, excitation light conversion, is included as one of the conditions for time lapse measurement, the amount of offset must be changed because the light emission site of the specimen is different. In the present embodiment, time lapse measurement is performed by setting an offset amount for each excitation light conversion.

  FIG. 9 shows operation control of the microscope system in which the offset amount for each excitation light conversion is set.

  When the measurement is started (S60), it is checked whether or not a predetermined number of shots are completed and the time lapse measurement corresponding to the shooting time (total) set in the host PC is completed (S61). When the measurement is finished, the process is finished (S71). If the measurement has not ended, the process waits until the time lapse measurement interval set by the time lapse interval set by the host PC is reached (S62).

  When the measurement interval is reached, the set fluorescent cube is inserted into the optical path (S63). When the fluorescent cube is inserted into the optical path and the excitation light is switched, AF control is performed on the cover glass 102B to indicate the cover glass position. Position control is performed on the reference position (S64). After driving to the reference position, the stage 101 is driven by an offset amount set for each excitation light set in advance by the host PC (S65).

  That is, after the autofocus unit 118 performs autofocus on the cover glass 102B, the stage Z drive control unit 116 moves the stage 101 by a predetermined fixed amount, that is, an offset amount.

  Furthermore, in order to acquire a Z direction image, it drives to an imaging | photography area upper limit (S66), and imaging | photography is performed (S67).

  After shooting is performed, driving is performed for the number of shooting counts set on the host PC, and also for the shooting Z interval set on the host PC (S68, S69), and shooting with all set excitation light ends. And the process is repeated until the time lapse measurement end condition is reached (S70, S71).

  The microscope system according to the present embodiment configured and controlled as described above is used when the movement of an object having a different emission point of a specimen for each excitation light is observed under the time-lapse measurement conditions of fluorescence observation. By applying autofocus to 102B, the specimen imaging position is set as an offset while the reference position is fixed, so that the movement of the specimen can be reliably measured for each excitation light.

  In this embodiment, each time observation is performed, auto-focusing is performed on the slide glass 102A or the cover glass 102B. However, by performing this only once every time the intermittent time elapses, the shooting time can be shortened. Good.

  So far, the offset for each stage XY of the second embodiment, the offset for each objective lens of the third embodiment, and the offset for each excitation light of the fourth embodiment have been described as being performed independently. Any two may be combined, or all three of them may be combined. In that case, more accurate measurement can be performed.

Fifth Embodiment This embodiment is directed to AF control performed on the slide glass 102A or the cover glass 102B. Since the configuration of the microscope system of this embodiment is the same as that of the first embodiment, a description thereof will be omitted.

  FIG. 10 is a model diagram of the slide glass 102A used in the present embodiment. As shown in FIG. 10, the slide glass 102A has AF markings 102C. The AF marking 102C is located in a range that does not affect the observation body 102D, and is subjected to processing for improving the accuracy of autofocus control. In the present embodiment, active AF is taken as an example, and the AF marking 102C is coated with a film that reflects laser light used in active AF with high reflectivity.

  FIG. 11 illustrates operation control of the above-described microscope system.

  When measurement is started (S80), it is checked whether or not a predetermined number of shots have been completed and time lapse measurement corresponding to the shooting time (total) set by the host PC is completed (S81). The process ends (S81). If the measurement has not been completed, the system waits until the time lapse measurement interval set by the time lapse interval set by the host PC is reached (S82).

  When the measurement interval is reached, stage XY drive is performed at the AF marking position (S83). When moving to the AF marking position, autofocus control is performed, and position control is performed to the reference position which is the cover glass position (S84). After performing the position control to the reference position, the stage is moved to the stage XY position where the actual measurement is performed (S85). After driving to the reference position, the stage 101 is driven by an offset amount set in advance by the host PC (S86).

  That is, after autofocusing is performed on the AF marking 102C of the slide glass 102A by the autofocus unit 118, the stage 101 is moved by a predetermined fixed amount, that is, an offset amount by the stage Z drive control unit 116.

  Furthermore, in order to acquire a Z direction image, it moves to an imaging | photography area upper limit (S87), and imaging | photography is performed (S88).

After shooting, the same number of shooting counts set on the host PC is also used.
Driving is performed for the imaging Z interval set in C (S89, S90), and the operation is repeated until the imaging with all the set excitation light ends and the time-lapse measurement end condition is reached (S90, S81).

  In the microscope system of the present embodiment, highly accurate AF can be performed on the slide glass 102A, so that the measurement accuracy can be improved. Furthermore, since the target of autofocus is a well-known one, the optimum autofocus condition can be set. This makes it possible to shorten the focusing time and improve the measurement throughput.

  Further, in the present embodiment, an example in which autofocus is an active AF and the AF marking 102C is a reflective film corresponding to this is described. However, the autofocus may be a passive AF, In this case, the AF marking 102C may be a high-contrast marking, and autofocus control such as a known hill-climbing method may be performed.

Sixth Embodiment This embodiment is directed to a microscope system including an inverted microscope. FIG. 12 shows the configuration of the microscope system according to the sixth embodiment of the present invention.

  As shown in FIG. 12, the microscope system 600 of the present embodiment includes a stage 621 for placing the observation sample 610 and an objective lens 631 disposed so as to face the observation sample 610 placed on the stage 621. And an observation optical system.

  The observation sample 610 includes an observation body 611 and a container 612 (so-called dish) that accommodates the observation body 611. The observation body 611 includes, for example, a specimen (for example, a cell) and a culture solution. The container 612 includes a dish-shaped transparent container body 613 having an opening at the bottom, and a cover glass 614 that closes the opening of the container body 613. Therefore, the observation body 611 is located on the cover glass 614, and the objective lens 631 is located below the cover glass 614.

  The stage 621 is movable in the XY direction. Since the microscope system 600 electrically controls the position of the stage 621 in the XY direction, the stage driving motor 622 for moving the stage 621 in the XY direction and the position of the stage 621 in the XY direction are controlled. And a stage XY control unit 623.

  The objective lens 631 is attached to the electric revolver. The electric revolver includes a revolver body 632 that can hold a plurality of objective lenses 631, a revolver motor 633 for rotating the revolver body 632, and a revolver motor drive unit 634 for controlling the revolver motor 633. Yes.

  The electric revolver can selectively place one of the objective lenses 631 held by the revolver body 632 on the optical path by controlling the rotation of the revolver body 632 by the revolver motor drive unit 634.

  The revolver body 632 has a plurality of objective lens attachment holes to which the objective lens 631 can be attached, and the electric revolver further has an objective of the revolver body 632 to which the objective lens 631 disposed on the optical path is attached. An attachment hole position detection unit 618 for detecting a lens attachment hole is provided.

  The revolver body 632 is movable along the observation optical axis, and the microscope system 600 further includes an aiming motor 636 for moving the revolver body 632 along the observation optical axis for aiming, and an aiming motor 636. And a focusing motor driving unit 637 for controlling. The aiming motor 636 and the focusing motor driving unit 637 constitute a focusing driving unit that drives at least one of the stage and the objective lens for focusing.

  The microscope system 600 includes an active AF device based on an active pupil division method. The AF apparatus includes a reference light source 671, a laser driving unit 672, a collimator lens 673, a light projection side stopper 674, a polarization beam splitter (PBS) 675, a condenser lens group 676, a chromatic aberration correction lens group 677, The λ / 4 plate 678, a dichroic mirror 679, a light receiving sensor 681, a signal processing unit 682, a chromatic aberration correction lens group driving motor 683, and a chromatic aberration lens driving unit 684 are configured.

  The microscope system 600 includes a microscope controller 691 for controlling the stage XY control unit 623, the revolver motor drive unit 634, the focusing motor drive unit 637, and the chromatic aberration lens drive unit 684. The microscope controller 691 is a well-known CPU circuit. An operation unit 692 having various operation SW is connected to the microscope controller 691. The observer can perform operations such as AF start / stop and switching of the objective lens, input of necessary information regarding AF such as glass thickness, and the like via the operation unit 692. In addition, a jog dial 693 is connected to the microscope controller 691 via a pulse counter 695 and a jog encoder 694. The observer can move the revolver body 632 up and down by the jog dial 693.

  The microscope system 600 includes a transmission illumination optical system for transmitting illumination of the observation sample 610. The transmission illumination optical system includes an illumination light source 641, a lens 642, a mirror 643, and a lens 644.

  The microscope system 600 includes an illumination optical system for fluorescence observation. The fluorescent illumination optical system includes a light source 651 that emits excitation light and a fluorescent cube 652. The fluorescent cube 652 can be replaced by electric control. Thereby, the illumination optical system for fluorescence observation can selectively irradiate excitation light having different wavelengths. In other words, the illumination optical system for fluorescence observation can select excitation light.

  The microscope system 600 does not necessarily include both the transmission illumination optical system and the fluorescence observation illumination optical system, and may include only one of the illumination optical systems suitable for the observation purpose. .

The microscope system 600 further includes a TV camera 661 for capturing an observation image, a video capture board 662 and a host PC 663. In transmitted illumination observation, illumination light from the illumination light source 641 passes through a lens 642. , And reflected by the mirror 643 and collected by the lens 644 to illuminate the observation sample 610 from above. The light transmitted through the observation sample 610 passes through the objective lens 631, passes through the dichroic mirror 679, and enters the TV camera 661.

  Further, in the fluorescence observation, the excitation light emitted from the light source 651 is reflected by the fluorescent cube 652 and irradiated to the observation sample 610 through the objective lens 631. The fluorescence generated from the observation sample 610 passes through the objective lens 631, is wavelength-selected by the fluorescence cube 652, passes through the dichroic mirror 679, and enters the TV camera 661.

  The image captured by the TV camera 661 is acquired by the host PC 663 by the video capture board 662 as in the first embodiment. The host PC 663 can store a plurality of images in an image memory (not shown) for acquired images.

  In the microscope system 600 of this embodiment, the active pupil division AF device performs focusing by irradiating the observation sample with laser light and detecting the reflected light.

  The reference light source 671 is composed of a light source that emits light in a wavelength region that is not visible, for example, an infrared laser. The reference light source 671 is controlled by the laser driving unit 672. The laser driver 672 performs pulse lighting of the reference light source 671 and controls the strength of the reference light source 671.

  The laser light emitted from the reference light source 671 is converted into a parallel light beam by passing through the collimating lens 673, and half of the light beam diameter is cut by the projection-side stopper 674. Only the component is reflected.

  The light beam from the polarization beam splitter (PBS) 675 is once converged by the condenser lens group 676 and then passes through the chromatic aberration correction lens group 677. The light that has passed through the chromatic aberration correction lens group 677 is polarized by 45 degrees when passing through the λ / 4 plate 678 and enters the dichroic mirror 679. Since the dichroic mirror 679 reflects only light in the infrared region, the laser beam is reflected. The reflected light beam passes through the objective lens 631 and irradiates the observation sample 610 to form a light spot.

  The light beam reflected by the observation sample 610 enters the objective lens 631 and is reflected by the dichroic mirror 679. The reflected light beam is further polarized by 45 degrees when passing through the λ / 4 plate 678 again, and changes to an S-polarized light component. The light beam further passes through the chromatic aberration correction lens group 677 and the condenser lens group 676 and enters the PBS 675. Since the light beam is an S-polarized component, it passes through the PBS 675. The transmitted light beam passes through the condenser lens group 680 and then forms an image on the light receiving sensor 681.

  The light receiving sensor 681 is a two-divided photodiode having two adjacent light receiving portions, and is arranged so that the optical axis is positioned on the boundary line between the two light receiving portions. The signal processing unit 682 acquires a so-called S-shaped curve that can determine the focus position and the focusing direction based on the output from the light receiving sensor 681. The microscope controller 691 performs autofocus control of the observation optical system by controlling the focusing motor driving unit 637 based on the information (S-curve) obtained by the signal processing unit 682.

This AF apparatus can correct the chromatic aberration of the infrared laser by moving the chromatic aberration correction lens group 677 along the optical axis by the chromatic aberration correction lens group drive motor 683, and can shift the focus position. It can be driven. Thereby, for example, it is possible to compensate for the difference in focus drift between the observation optical system and the AF optical system caused by a temperature change, in other words, it is possible to make both the focus drifts the same.

  In the microscope system 600 of the present embodiment, as in the first embodiment, after autofocusing is performed on the cover glass 614 by active AF, the revolver body 632 is moved by the focusing motor driving unit 637 and the objective lens 631 is moved. Is moved by a predetermined fixed amount, that is, an offset amount. Thereby, the same advantage as the first embodiment can be obtained.

  Furthermore, the second embodiment to the fifth embodiment are applied to the microscope system 600 of the present embodiment in the same manner as the second embodiment to the fifth embodiment are applied to the first embodiment. May be. In those cases, the same advantages as those of the second to fifth embodiments can be obtained.

  So far, several embodiments of the present invention have been described with reference to the drawings, but the present invention is not limited to these embodiments, and various modifications and changes can be made without departing from the scope of the present invention. May be applied.

  That is, the microscope system of the present invention performs auto-focusing once on a transparent body (slide glass 102A, cover glass 102B, or cover glass 614), and drives the offset amount to a preset measurement point to perform imaging. The control is not limited to the above-described embodiment, and various modifications are possible.

1 shows a configuration of a microscope system according to a first embodiment of the present invention. A time-lapse model for observing the movement of the observation object for a long time is shown. 3 shows a setting screen of a host PC in the microscope system of the first embodiment of the present invention. The model of the observation image image | photographed during the predetermined | prescribed imaging | photography period is shown. It is a flowchart which shows operation | movement of the microscope system of 1st embodiment of this invention. A model having a plurality of measurement points in an observation body enclosed with a slide glass and a cover glass is shown. The host PC setting screen in the microscope system of 2nd embodiment of this invention is shown. It is a flowchart which shows operation | movement of the microscope system of 2nd embodiment of this invention. It is a flowchart which shows operation control of the microscope system of 3rd embodiment of this invention. It is a flowchart which shows operation control of the microscope system of 4th embodiment of this invention. It is a model figure of the slide glass in the microscope system of 5th embodiment of this invention. It is a flowchart which shows operation control of the microscope system of 5th embodiment of this invention. The structure of the microscope system of 6th embodiment of this invention is shown. It is a model figure which observes with respect to one of the some nucleus inside the sample mounted on the slide glass which gave the infrared reflective film. This shows how the position with the maximum contrast moves as the nucleus of interest in the observation moves. It shows a state in which the portion of the specimen that emits light is different in the fluorescence observation.

DESCRIPTION OF SYMBOLS 100 ... Microscope system, 101 ... Stage, 102 ... Sample, 102A ... Slide glass, 102B ... Cover glass, 102C ... Marking for AF, 103 ... Light source, 104 ... ND filter, 106 ... Condenser, 107 ... Fluorescent cube, 108 ... Revolver , 109 ... objective lens, 110 ... lens barrel, 111 ... eyepiece, 112 ... camera, 113 ... host PC, 114 ... microscope controller, 115 ... stage XY control unit, 116 ... stage Z drive control unit, 117 ... video Capture board, 118 ... auto focus unit, 600 ... microscope system, 610 ... observation sample, 611 ... observer, 612 ... container, 613 ... container body, 614 ... cover glass, 618 ... mounting hole position detector, 621 ... stage, 622... Motor for driving the stage, 623 Stage XY control unit, 631 ... objective lens, 632 ... revolver body, 633 ... revolver motor, 634 ... revolver motor drive unit, 636 ... aiming motor, 637 ... focusing motor drive unit, 641 ... illumination light source 642 ... Lens, 643 ... Mirror, 644 ... Lens, 651 ... Light source, 652 ... Fluorescent cube, 661 ... TV camera, 662 ... Video capture board, 663 ... Host PC, 671 ... Reference light source, 672 ... Laser driver, 673 ... collimating lens, 674 ... projection side stopper, 675 ... polarizing beam splitter, 676 ... condensing lens group, 677 ... chromatic aberration correction lens group, 678 ... λ / 4 plate, 679 ... dichroic mirror, 680 ... condensing lens group, 681... Light receiving sensor, 682... Signal processing unit, 683. Motor, 684 ... chromatic aberration lens drive unit, 691 ... microscope controller, 692 ... operation unit, 693 ... jog dial, 694 ... jog encoder, 695 ... pulse counter.

Claims (9)

A stage for placing an observation sample comprising a transparent body and an observation body placed on the transparent body;
An objective lens disposed below the stage so as to face the observation sample placed on the stage;
A focusing drive unit for driving at least one of the stage and the objective lens in a direction parallel to the observation optical axis;
An observation light source for illuminating the observation sample;
A focus detection light source that emits detection light for autofocus from the lower side of the objective lens to the observation sample with a light source different from the observation light source;
A light receiving means for receiving the detection light reflected from the transparent body in the observation sample through the objective lens;
An autofocus unit that performs autofocus on a predetermined position of the transparent body by controlling the focusing drive unit from the result of the light receiving means;
Offset amount recording means for recording a distance away from the predetermined position in the optical axis direction by a certain amount;
An imaging device that photographs the observation sample via the objective lens;
A time lapse measurement interval setting means for setting a time lapse measurement interval which is an intermittent shooting interval of the imaging device;
Photographing control means for controlling the imaging device and the autofocus unit;
After the autofocusing is performed on the predetermined position, which is a position where the focus detection light is reflected from the transparent body by the autofocus unit by the photographing control unit, the autofocus unit records the offset amount on the offset amount recording unit. The microscope system characterized in that photographing of the observation sample at a position away from the predetermined position by a predetermined amount is performed for each time lapse measurement interval recorded in the time lapse measurement interval setting means. In claim 1,
Display means for displaying image information of the observation object imaged by the imaging device or / and coordinate information in the optical axis direction of the microscope system;
An offset amount setting means for a user to record in the offset amount recording means a position away from the transparent body by a certain amount in the optical axis direction;
From the information by the display means, by the offset amount setting means, a predetermined distance away from the transparent body, which is a position for photographing the observation body, before the user starts photographing in advance at every time lapse measurement interval. The microscope system characterized in that the position of the current position is recorded in the offset amount recording means. A stage for placing an observation sample comprising a transparent body and an observation body placed on the transparent body;
XY stage driving means for driving and controlling the stage in the XY direction perpendicular to the observation optical axis;
An objective lens disposed below the stage so as to face the observation sample placed on the stage;
A focusing drive unit for driving at least one of the stage and the objective lens in a direction parallel to the observation optical axis;
An observation light source for illuminating the observation sample;
Photographing position setting means for setting a plurality of positions for photographing on the observation body in the XY direction;
A focus detection light source that emits detection light for autofocus from the lower side of the objective lens to the observation sample with a light source different from the observation light source;
A light receiving means for receiving the detection light reflected from the transparent body in the observation sample through the objective lens;
An autofocus unit that performs autofocus on a predetermined position of the transparent body by controlling the focusing drive unit from the result of the light receiving means;
Offset amount recording means for individually recording a distance away from the predetermined position in the optical axis direction for each photographing position;
An imaging device that photographs the observation sample via the objective lens;
A time lapse measurement interval setting means for setting a time lapse measurement interval which is an intermittent shooting interval of the imaging device;
An imaging control unit that controls the imaging device, the autofocus unit, and the XY stage driving unit;
When the time lapse observation is started by the photographing control means, the motorized stage moves the XY to the photographing position set in the photographing position setting means at every time lapse measurement interval recorded in the time lapse measurement interval setting means. The auto focus unit performs auto focus on the predetermined position, which is a position where the focus detection light is reflected from the transparent body by the auto focus unit. When the observation specimen is photographed at a predetermined distance from the recorded predetermined position and there is an unphotographed photographing position within the same time lapse measurement interval, the movement to the next photographing position is performed. When shooting is completed at all shooting positions set in the shooting position setting means, the next time Microscope system and repeating the cycle to wait until TOPS measurement interval. In claim 3,
Display means for displaying image information of the observation object photographed by the imaging device and / or coordinate information in the optical axis direction and / or XY direction of the microscope system;
An offset amount setting means for a user to record in the offset amount recording means a position that is separated by a certain amount in the optical axis direction from the transparent body for each of the plurality of photographing positions in the XY direction;
The position at which the observation object is photographed at each of the plurality of photographing positions in the XY direction before the user starts photographing at each time lapse measurement interval by the offset amount setting means based on the information from the display means. A microscope system, wherein a position that is a fixed amount away from the transparent body in the optical axis direction is recorded in the offset amount recording means.   5. The microscope system according to claim 4, wherein the display unit has a function of displaying an entire image of the observation body and / or a marker display function indicating the plurality of photographing positions in the XY directions.   6. The microscope system according to claim 1, wherein the objective lens is exchangeable, and the fixed amount is set for each objective lens in the optical path. In any one of Claims 1-6,
The observation body consists of a specimen and a culture solution,
The transparent body is a container for storing the specimen and the culture solution;
The microscope system according to claim 1, wherein the predetermined position is a bottom surface of the container. In any one of Claims 1-7,
A correction lens disposed in the autofocus optical system for autofocus;
The focus position of the autofocus optical system is shifted by moving the correction lens along the optical axis of the autofocus optical system, and an optical offset between the autofocus optical system and the observation optical system for the imaging device is set. A microscope system comprising correction lens driving means for performing the correction. In any one of Claims 1-8,
Has a fluorescent cube for fluorescence observation,
The microscope system, wherein the observation light source is a light source that emits excitation light for fluorescence observation.

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