JP2010160022A - Living body observation system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sharp image stable in the position of an observation region even if a dynamic living body having rapid behavior is observed. <P>SOLUTION: This living body observation system 1 is equipped with: imaging parts 7a and 7b for condensing the light from the living body A on a stage 2 by an objective optical system 10 to image the same; a movement mechanism 9 for relatively moving the objective optical system 10 and the stage 2; a noticeable point position detecting part 13 for detecting the position of the noticeable point in the image taken by the imaging parts 7a and 7b; a history memory part 14 for storing history wherein the position detected by the noticeable point position detecting part 13 and the data related to the acquiring time of the image taken by the imaging parts 7a and 7b are allowed to correspond to each other in the state where the movement mechanism 9 is stopped; a motion presuming part 15 for presuming the motion of the living body A after a predetermined time on the basis of the present position of the noticeable point acquired by the imaging parts 7a and 7b and the history stored in the history memory part 14; and a control part 16 for controlling the movement mechanism 9 so as to move the objective optical system 10 and the stage 2 according to the motion estimated by the motion estimating part 15. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a living body observation apparatus.

  In recent years, visualization of ion concentration, membrane potential, and the like using a fluorescent probe has been performed using an optical microscope. For example, biological functions such as nerve cells are observed as specimens, in particular, dynamic behaviors are observed. As an apparatus for observing such a dynamic behavior, a living body observation apparatus as disclosed in JP-A-2005-300308 is known. Japanese Laid-Open Patent Publication No. 2006-209698 is known as a method for observing an observation target site.

JP-A-2005-3000278 JP 2006-209698 A

However, when observing a dynamic living body, if the focus and field of view of the objective lens are kept constant, the living body moves in the depth direction and the image becomes unclear or the observation site is out of the field of view. There is.
In addition, the living body observation apparatus of Patent Document 1 and the observation method of Patent Document 2 have a problem that they cannot cope with the case where the observation region moves quickly in the three-dimensional direction due to pulsation or pulsation of the living body.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a living body observation apparatus that can always obtain a clear image in which the position of an observation site is stable even in a living body having a fast behavior. Yes.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a stage on which a living body is placed, an objective optical system that collects light from the living body, an imaging unit that captures light collected by the objective optical system, the objective optical system, and the stage. A moving mechanism for relatively moving; a target point position detecting unit for detecting a position of a target point in the image of the living body imaged by the imaging unit; and the target point position in a state where the moving mechanism is stopped. A history storage unit that stores a history in which the position of the point of interest detected by the detection unit is associated with information related to the acquisition time of image acquisition by the imaging unit, and the point of interest in the image acquired by the imaging unit Based on the current position and the history stored in the history storage unit, the motion estimation unit that estimates the motion of the living body after a predetermined time, and the objective optical system according to the motion estimated by the motion estimation unit The stay To provide a biological observation apparatus and a control unit which controls the moving mechanism so as to relatively move and.

  According to the present invention, light from the living body on the stage is collected by the objective optical system, and an image of the living body is acquired in the imaging unit. Then, the position of the target point in each image is detected by the target point position detection unit, and a history in which the position is associated with the time of image acquisition is stored in the history storage unit.

  After this, when the moving mechanism is operated and the image of the living body is acquired and the position of the target point is detected, the motion estimation unit compares the current position with the history already stored in the history storage unit, The motion up to the next image acquisition time of the point is estimated. The control unit controls the moving mechanism to move the objective optical system and the stage relative to each other in the same manner as the estimated operation, and then acquires the next image. The living body can be observed by repeating the operation of the moving mechanism from this image acquisition.

  That is, information on the past trajectory of the target point is accumulated in the history storage unit from the history in which the position of the target point and the time at the time when the image is acquired are associated with each other. Then, by comparing the current position of the attention point with the past trajectory, the position on the trajectory where the attention point will exist at the next image acquisition time is estimated, and the operation up to that position, that is, the movement direction And the amount of movement can be calculated.

In addition, by relatively moving the objective optical system and the stage in accordance with the operation estimated at every imaging, the position of the target point of the living body and the position of the objective optical system are kept relatively constant, Even if the attention point moves due to the behavior of the living body, the position of the attention point can be stopped in the image.
Therefore, according to the present invention, it is possible to obtain a clear image in which the position of the observation site is stable even when a dynamic living body having a fast behavior is observed.

In the above invention, the target point position detection unit may detect within a partial range in the image.
The range of movement of the attention point is grasped by preliminary observation. By detecting the position of the attention point only within the moving range of each image, the amount of data to be handled and the calculation value can be reduced. As a result, the image acquisition interval can be shortened to improve the control speed of the moving mechanism and can be accurately followed by the movement of the point of interest, that is, an image with a more stable position of the observation site can be acquired.

Moreover, in the said invention, the said attention point may be the feature point of the said biological body.
By doing in this way, a position can be detected without giving a substance like a marker to the living body as a point of interest from the outside of the living body.

In the above invention, the history storage unit stores a history of the behavior of the biological body that exhibits a periodic behavior, and the motion estimation unit statistically estimates the behavior of the biological body from the history. May be.
By doing this, the locus of irregular behavior of the living body is also stored, and even when the living body behaves like this, the degree to which the position of the observation site is shifted and the probability thereof are reduced, and the observation site is further reduced. An image with a stable position can be acquired.

  According to the present invention, there is an effect that a clear image with a stable position of an observation site can be obtained even when a dynamic living body having a fast behavior is observed.

It is a whole lineblock diagram showing the living body observation device concerning a 1st embodiment of the present invention. It is a figure which shows the structure of the moving mechanism control system which concerns on 1st Embodiment. It is a figure which shows the movement amount of the attention point by the pulsation of a biological body. It is the figure which showed a mode that an attention point moved with the beat of a biological body. It is a whole block diagram which shows the biological examination apparatus which concerns on the 2nd Embodiment of this invention. It is a figure which shows the structure of an image detection system. It is a figure which shows the structure of the moving mechanism control system which concerns on 2nd Embodiment.

A living body observation apparatus 1 according to a first embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the biological sample observation apparatus 1 according to the present embodiment is based on an upright microscope, and includes a stage 2 on which a living body A is placed, and an imaging optical system 4 disposed inside the housing 3. The support column 5 and the arm 6 that support the housing 3, the imaging units 7 a and 7 b and the light source 8 that are disposed outside the housing 3, and an objective lens that is disposed on the bottom of the housing 3 via the moving mechanism 9 ( Objective optical system) 10 and a movement mechanism control system 11.

The housing 3 is movable in the vertical direction by an arm 6 that moves along a support column 5 fixed vertically to the stage 2.
The imaging units 7a and 7b are, for example, first and second CCD cameras (hereinafter also referred to as CCD cameras 7a and 7b). The first CCD camera 7 a is connected to the moving mechanism control system 11, and the second CCD camera 7 b is connected to a monitor (not shown) via the controller 12.

The imaging optical system 4 includes a plurality of wavelength filters 4a, 4b, 4c, dichroic mirrors 4d, 4e, and a lens group (not shown).
Of the light emitted from the light source 8, only the light having the wavelength set by the first wavelength filter 4a in the housing 3 is reflected by the first dichroic mirror 4d, and the second dichroic mirror 4e and the objective lens are 10 The light passes through and is irradiated onto the living body A.

  In addition, a part of the light collected by the objective lens 10 passes through the second dichroic mirror 4e, and then the light having the wavelength transmitted through the first dichroic mirror 4d and the second wavelength filter 4b is the first. 1 is detected by one CCD camera 7a. The other part of the light condensed by the objective lens 10 is reflected by the second dichroic mirror 4e, and then the light having the wavelength transmitted through the third wavelength filter 4c is reflected by the second CCD camera 7b. Detected.

  The moving mechanism 9 includes an X-axis stage 9a and a Y-axis stage 9b that move the objective lens 10 in the horizontal direction, and a Z-axis stage 9c that moves in the focus direction (Z direction).

  The movement mechanism control system 11 is, for example, a general computer or CPU, and as shown in FIG. 2, an attention point position detection unit 13 that detects the position of the attention point in the image, and a history about the detected position. Is stored, a motion estimation unit 15 that estimates the next motion of the point of interest, and a control unit 16 that controls the moving mechanism 9. The attention point position detector 13 is connected to the first CCD camera 7 a and the controller 16 is connected to the moving mechanism 9.

The point-of-interest position detection unit 13 extracts the point of interest by image processing and detects the position of the center of gravity as the X and Y positions. The attention point position detection unit 13 calculates the amount of movement in the Z direction from changes in the size and inclination of the attention point. The initial position in the Z direction is used as the origin of the Z position, and the amount of movement from the initial position in the Z direction is detected as the Z position.
The history storage unit 14 assigns the order in which each image was acquired (information related to the acquisition time) as an address to the X, Y, Z position of the point of interest in each detected image, and uses these as a history. It comes to memorize.

The operation of the biological observation apparatus 1 according to the present embodiment configured as described above will be described below.
In this embodiment, a living body A that periodically beats, such as a living mouse, is an observation target, and a marker B such as a fluorescent bead on the living body A is a point of interest. FIG. 3 is a diagram showing how the position of the marker B moves according to the pulsation of the living body A, with the horizontal axis representing time and the vertical axis representing the amount of movement of the marker B position. The position of the marker B is stable at the positions of the reference signs a1 and a2, and the position of the marker B is moved greatly by the pulsation at the position of the reference sign b. FIG. 4 is a diagram showing how the marker B moves in the horizontal direction. The marker B exists in the field of view t1 of the CCD camera at the position of the signs a1 and a2 where the movement of the living body A is stable. Represents a state in which the position moves to the outside of the visual field t1.

  In order to observe the living body A using the living body observation apparatus 1 according to this embodiment, the living body A is placed on the stage 2 and the living body A is illuminated by the light source 8. Then, the reflected light from the surface of the living body A or the fluorescence excited by the illumination light is condensed by the objective lens 10, and the image detected by the second CCD camera 7b is observed on the monitor while the arm 6 and the living body are observed. The position of A is moved to adjust the field of view and focus of the objective lens 10.

  Next, preliminary observation is performed with the moving mechanism 9 stopped. The behavior in which the marker B moves from the position of the symbol a1 to the position of the symbol a2 via the position of the symbol b1 is defined as one cycle, and the living body A is observed for one cycle, and the first CCD camera 7a is used at a predetermined time interval. Get an image. The acquired image is sent to the point-of-interest position detection unit 13 to detect the X, Y, and Z positions of the marker B in each image, and the history storage unit 14 stores the history of one cycle of the behavior of the marker B. .

  Subsequently, the moving mechanism 9 is operated to start the main observation. When the image of the living body A is acquired, the X, Y, and Z positions of the marker B in the image are detected, and this is compared with the preliminary observation history stored in the history storage unit 14 in the motion estimation unit 15. Then, the position where the marker B will have moved by the next image acquisition time is estimated, and the respective movement amounts in the X, Y, and Z directions up to that position are calculated. The control unit 16 acquires the next image by controlling the moving mechanism 9 so that the objective lens 10 moves by the calculated movement amount. By repeating the operations from the image acquisition to the movement of the objective lens 10, the living body A can be observed.

  That is, since the image acquisition interval of the first CCD camera 7a is constant, the history of the position coordinates of the marker B at a constant time interval is stored in the history storage unit 14. Thereby, the information of the past locus for one cycle of the marker B having a periodic behavior can be acquired. Then, by comparing the current position of the marker B with the past locus, it is possible to estimate to which position the marker B has moved on the locus at the next image acquisition time.

  Since the objective lens 10 is also moved in the same manner as the estimated movement of the point of interest, the objective lens 10 observes the living body A while being moved on the same locus as the marker B. That is, since the relative position of the objective lens 10 and the marker B is kept constant, the position of the marker B is stationary within the field of view of the objective lens 10.

Moreover, since the objective lens 10 is moved to the next position of the marker B every time an image is acquired, an image can be acquired following the movement even if the marker B moves quickly.
Therefore, according to the present embodiment, even in the case of the living body A that has a fast behavior in a three-dimensional direction such as a pulsation, the observation site moves in the horizontal direction and the focus direction, and is out of the field of view or out of focus. In other words, it is possible to obtain an image in which the observation site is always focused and the position is stable.

In the above embodiment, the target point position detection unit may detect within a partial range in the image.
The movement range of the marker B is previously grasped by preliminary observation, and the position of the marker B is detected only within the movement range in each image. By narrowing the detection range in this way, the amount of data to be processed and the calculation value are reduced. Therefore, the image acquisition interval is shortened, the control speed of the moving mechanism 9 is improved, and the behavior of the marker B is more accurately matched. The objective lens 10 can be moved. That is, an image in which the position of the marker B is more stable can be obtained.

Moreover, in the said embodiment, although the said attention point was mentioned as a marker, it may replace with this and may be feature points, such as a high-intensity part in an image, and a part which has a characteristic shape.
By doing so, the marker B becomes unnecessary, and the influence on the living body A due to the application of the marker B can be further reduced.

  In the above embodiment, the history of one cycle of the behavior of the attention point is stored as a preliminary observation. Instead, the history of a plurality of cycles is stored, and the motion estimation unit statistically operates the biological motion from the history. It may be estimated.

  By doing this, it is also possible to store a history when the marker B has an irregular behavior, and to estimate the movement even if such a behavior is present in this observation. Thereby, the extent to which the position of the marker B shifts within the field of view and the probability thereof can be reduced, and an image with a more stable position of the observation site can be obtained.

In the above embodiment, an upright microscope is used as a base and a CCD camera is used as an imaging unit. Instead, a laser scanning microscope is used as a base and a photomultiplier (PMT) is used as an imaging unit. May be used.
In this case, the imaging optical system 4 further includes a two-dimensional scanning unit such as a galvano scanner or a mems scanner, and uses a laser light source as the light source 8.

  The laser light output from the laser light source is moved pixel by pixel by the galvano scanner and scans the surface of the living body A. Then, the fluorescence excited on the surface of the living body A is collected by the objective lens 10 and detected by the PMT, and the luminance of the detected fluorescence and the scanning position are associated with each other by an arithmetic unit (not shown), for example, a computer. And an image is acquired. Thereafter, similarly to the above-described operation, a series of processing and control are performed from detection of the position of the attention point to control of the moving mechanism 9, and the living body A can be observed.

  In the present embodiment, the history is stored by storing the position of the attention point extracted from the image acquired at the predetermined time interval in the preliminary observation together with the order in which the images are acquired. The history may be associated with the image acquisition time or relative time.

A living body observation apparatus 1 according to a second embodiment of the present invention will be described below with reference to FIGS.
In the description of the present embodiment, portions having the same configuration as those of the live observation apparatus 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

  As shown in FIG. 5, the biological sample observation apparatus 1 according to the present embodiment includes an XY stage 9d disposed on a base 17 via a Z-axis stage 9c, a light source 8 that illuminates the living body A obliquely from above, An imaging optical system 4 disposed inside the housing 3, an objective lens 10 and an imaging unit 7 disposed outside the housing 3, and a moving mechanism control system 11 are provided.

  In the first embodiment, the first and second CCD cameras 7a and 7b are used as the imaging units 7a and 7b, and the uses are distinguished from each other for detecting the position of the target point and for observing the image. In the present embodiment, the second CCD camera 7b is the same as that in the first embodiment. However, instead of the first CCD camera 7a, an imaging function unit 7c of the CCD camera is used, as shown in FIG. An image detection system 18 having this configuration and the point-of-interest position detection unit 13 in one configuration is provided.

  The point of interest position detector 13 detects the X and Y positions of the point of interest as in the first embodiment. Further, the point-of-interest position detection unit 13 obtains the displacement amount in the Z direction from the length and direction of the shadow of the attention point generated by illuminating from obliquely above, and detects the displacement amount from the initial position in the Z direction as the Z position. . Then, the detected X, Y, and Z positions are output to the outside of the image detection system 18 in the order in which the images are acquired.

  A part of the light condensed by the objective lens 10 is reflected by the second dichroic mirror 4e, and then only the light having the wavelength transmitted through the third wavelength filter 4c is detected by the CCD camera 7b. In the other part of the light collected by the objective lens 10, only the light having a wavelength transmitted through the second dichroic mirror 4 e and the second wavelength filter 4 b is detected by the image detection system 18.

The XY stage 9d is a stage 2 on which the living body A is placed, and at the same time, is a moving mechanism 9 that moves relative to the objective lens 10.
The field of view of the objective lens 10 is selected by moving the living body A by controlling the XY stage 9d in the horizontal direction and the Z-axis stage 9c in the focus direction.

As shown in FIG. 7, the movement mechanism control system 11 includes a history storage unit 14, an operation estimation unit 15, and a control unit 16, and an image detection system 18, an XY stage 9 d that is the movement mechanism 9, and a Z axis. It is connected to the stage 9c.
The history storage unit 14 attaches to the three-dimensional position information sent from the image detection system 18 an image number corresponding to the order in which each piece of information is sent (information related to the acquisition time) as an address. Store as history.

  In order to observe the living body A using the living body observation apparatus 1 according to the present embodiment, the living body A is placed on the XY stage 9d and illuminated by the light source 8. Since the living body A is illuminated obliquely from above, the illumination light is reflected at an angle corresponding to the unevenness of the living body A. The reflected light is collected by the objective lens 10 and detected by the CCD camera 7b, and the image of the living body A is observed by a monitor (not shown). When the preliminary observation and the main observation are performed as in the first embodiment, the living body A can be observed.

  According to the embodiment described above, the amount of information transmitted to the moving mechanism control system 11 can be reduced from one image to X, Y by configuring the imaging function unit 7c and the target point position detection unit 13 of the CCD camera as one configuration. , Z position information can be reduced only. Thus, the processing speed of the data transmission and movement mechanism control system 11 can be increased, the imaging time of the CCD camera 7b can be shortened and blurring in the image can be suppressed, and at the same time, the imaging function unit 7c of the CCD camera can be controlled. An image with a more stable position of the marker B can be acquired by shortening the imaging interval.

DESCRIPTION OF SYMBOLS 1 Living body observation apparatus 2 Stage 3 Housing | casing 4 Imaging optical system 4a, 4b, 4c Wavelength filter 4d, 4e Dichroic mirror 5 Support | pillar 6 Arm 7a 1st imaging part (1st CCD camera)
7b Second imaging unit (second CCD camera)
7c Imaging function part of CCD camera 8 Light source 9 Moving mechanism 9a X axis stage 9b Y axis stage 9c Z axis stage 9d XY stage 10 Objective lens (objective optical system)
DESCRIPTION OF SYMBOLS 11 Movement mechanism control system 12 Controller 13 Attention point position detection part 14 History storage part 15 Motion estimation part 16 Control part 17 Base 18 Image detection system A Living body B Marker

Claims (4)

A stage for placing a living body,
An objective optical system for condensing light from the living body;
An imaging unit for imaging light collected by the objective optical system;
A moving mechanism for relatively moving the objective optical system and the stage;
An attention point position detection unit for detecting the position of the attention point in the image of the living body imaged by the imaging unit;
A history storage unit that stores a history in which the position of the target point detected by the target point position detection unit and the information about the acquisition time of image acquisition by the imaging unit are associated with the movement mechanism stopped;
A motion estimation unit that estimates a motion of the living body after a predetermined time based on a current position of the attention point in the image acquired by the imaging unit and a history stored in the history storage unit;
A living body observation apparatus comprising: a control unit that controls the moving mechanism so as to relatively move the objective optical system and the stage according to the motion estimated by the motion estimation unit.   The living body observation apparatus according to claim 1, wherein the attention point position detection unit detects within a partial range in the image.   The living body observation apparatus according to claim 1, wherein the attention point is a feature point of the living body. The history storage unit stores a history of a plurality of cycles of the living body exhibiting a periodic behavior,
The living body observation apparatus according to any one of claims 1 to 3, wherein the movement estimation unit statistically estimates the movement of the living body from the history.

Images