JP2010079222A - Living body observation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a living body observation apparatus which improves the handleability of a marker and has improved operability. <P>SOLUTION: The living body observation apparatus includes: an objective lens disposed in close contact with a specimen; an image pickup device that captures an image of the specimen through the objective lens; an objective lens drive device that drives the objective lens in the direction of correcting the displacement of the specimen; and a marker base carrying device that disposes a marker base onto the specimen within a range where the specimen is observed through the objective lens. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a living body observation apparatus.

  In recent years, in biological research, so-called in vivo observation has been performed in which an individual experimental animal is used as a specimen and its organs and the like are observed with an optical microscope while alive. In in-vivo observation, the observation object has movements such as pulsation and respiratory movement, so that the observation position is shifted and the focus is likely to be blurred. As a method of eliminating such displacement of the observation position and blurring of the focus, an apparatus (for example, Patent Document 1) that causes the entire microscope including the imaging unit to follow the movement of the observation target, or the movement of the observation target is used. There is known a method for tracking control of an apparatus such as an apparatus for tracking an objective lens together (for example, Patent Document 2).

When detecting the movement of the observation target, it is necessary to perform high-speed shooting. However, in the case of a biological sample such as an organ, the illumination is not so strong that the image is dark, and pattern matching image processing using a natural texture cannot accurately detect biological vibration. For this reason, a bright feature point (marker) that moves in the same manner as an organ in a dark screen is provided, and biological vibration is detected by detecting this marker.
JP-A-7-222754 JP 2007-187810 A

  Usually, the marker is sprinkled on the sample together with a liquid such as physiological saline. However, the conventional method has a problem that a marker does not exist in a desired place or there are too many markers in an observation image. Furthermore, the marker may be washed away by physiological saline or a liquid secreted from the living body, and the degree of adhesion with the living body may be reduced, so that the same movement as the living body may not be performed. Thus, it has been very difficult in the past to place a marker at a desired position, move the position of the marker, or remove the marker.

  In view of the above problems, an object of the present invention is to provide a living body observation apparatus with improved operability by facilitating handling of a marker.

In order to achieve the above object, the present invention provides the following means.
The present invention includes an objective lens disposed close to a sample, an imaging device that images the sample via the objective lens, an objective lens driving device that drives the objective lens in a direction to correct the displacement of the sample, Provided is a living body observation device including a marker substrate carrier device that arranges a marker substrate on a sample in an observation range by an objective lens.

  According to the present invention, it is possible to easily perform a detection operation using the marker base by including the marker base transporting device.

  In the said invention, a marker base | substrate conveying apparatus can be provided with the adsorption | suction mechanism which adsorb | sucks a marker base | substrate, and the positioning mechanism which adjusts the position which arrange | positions a marker base | substrate. In addition, the marker substrate transport device may include a tip portion that sucks and holds the marker substrate, and the tip portion may have a ring shape that surrounds the entire circumference of the observation range. Alternatively, the tip may have a shape that surrounds the entire circumference except a part of the periphery of the observation range.

  In one embodiment, the marker substrate transport device is a stabilizer that causes the marker substrate to closely contact the sample in the observation range.

  In one aspect, the marker-based transport device is separate from the biological observation device, and in another aspect, the marker-based transport device is provided integrally with the biological observation device.

  In one embodiment, the marker base is formed by adhering a plurality of markers to a transparent plate-like base. In another embodiment, the marker base is formed by containing a plurality of markers in a transparent sheet-like base.

  According to another aspect of the present invention, there is provided a marker base for photographing for vibration detection of a living body in which a plurality of markers serving as detection targets in living body observation are held on a transparent base. Moreover, the marker base | substrate conveyance apparatus for biological observation apparatuses which convey said marker base | substrate is provided.

  Further, the living body observation apparatus may be a living body observation microscope, or may be an endoscope or other observation apparatus.

  The sample observed in the living body observation apparatus may be a cell, tissue, or internal organ in the living body, and the minute behavior in the living sample is observed by correcting the dynamic behavior of the living body. It is possible.

  ADVANTAGE OF THE INVENTION According to this invention, a marker base | substrate conveyance apparatus is provided, By using a marker base | substrate, the handling of a marker can be made easy and the biological observation apparatus with improved operativity can be provided.

  In the present invention, a marker base manufactured by adhering a plurality of markers to a transparent plate-like base is used to facilitate handling of the marker. FIG. 1 shows a first embodiment of the marker substrate 100. The marker base 100 has a circular shape with a diameter of about 1 mm, for example. However, the present invention is not limited to this, and it may be formed in an arbitrary shape such as a circle, a quadrangle, a trapezoid, or an ellipse. It is preferable that the marker base 100 is sufficiently large for an observation target, for example, an organ. For example, it is the same size or larger than the organ. Thereby, a marker is arrange | positioned to the whole organ, and a detection position can be arbitrarily selected from the whole organ.

  The base 120 for the marker base 100 is a plate-like member formed of an arbitrary substance such as glass or resin, and is preferably made of glass.

  The marker 110 has an identifiable optical characteristic, and a marker made of a metal material or a fluorescent material is preferably used, but any marker that is usually used in the field can be used. In this embodiment, a thin film marker is preferably used.

  The marker 110 is adhered to one surface of the base 120 as described above by vapor deposition. The markers 110 are preferably arranged so that one or more markers exist in the field of view of the observation apparatus, and are arranged at a predetermined interval.

  The marker substrate 100 is arranged so that the surface to which the marker is adhered contacts the sample. This is because if the surface to which the marker is bonded is placed on the sample, a gap corresponding to the thickness of the substrate will be formed between the sample surface and the marker, and if one is focused, the other will be blurred. .

  The marker base may be exchanged depending on the magnification of the objective lens provided in the living body observation apparatus. As shown in FIG. 2, the marker base 103 with a relatively large marker 113 as shown in FIG. 2A is used at low magnification, and as shown in FIG. 2B at high magnification. It is possible to use a marker substrate 104 to which a relatively small marker 114 is adhered. The size of the marker and the marker base can be appropriately changed depending on the lens magnification.

  FIG. 3 shows a second embodiment of the marker base. In the second embodiment, a marker sheet 51 is used. The marker sheet 51 is formed by including the marker 50 in a transparent film-like substrate. The membranous substrate is formed to a thickness of about 0.1 mm by agar, for example. The marker sheet 51 is formed as thin as possible and has a thickness that does not cut or tear.

  The marker 50 has an identifiable optical characteristic similar to the marker used in the first embodiment, and a marker made of a metal material or a fluorescent material is preferably used. However, any marker that is usually used in the field can be used. Any of them can be used. The marker 50 used in the second embodiment may be an arbitrary shape such as a sphere, a cylindrical shape, or a thin film shape. The markers 50 are dispersed in the marker sheet 51 and are preferably arranged regularly. The marker for the thin film feature may be adhered on the membrane substrate.

  The marker sheet 51 can be manufactured, for example, by dispersing a marker in a liquid agar in a container, flowing it on a flat plate and drying it. Alternatively, liquid agar containing a marker can be poured into a desired mold and molded. Conditions such as temperature and humidity can be set as appropriate.

  The film-like marker sheet 51 in this embodiment has flexibility and can be in close contact with an observation sample, for example, an organ 52. Thereby, in addition to the high degree of adhesion between the marker and the observation target, there is an advantage that the marker is not easily displaced.

  The living body observation apparatus of the present invention includes a marker base transportation device, and uses the marker base as described above to improve the operability of the marker. As the living body observation apparatus, a microscope observation apparatus is preferably used, but is not limited thereto, and can be used as, for example, an endoscope observation apparatus or other appropriate apparatus. Hereinafter, for convenience of explanation, a microscope observation apparatus including two imaging apparatuses, that is, a detection imaging apparatus and an observation imaging apparatus will be described in detail as an example.

A microscope observation apparatus 1 according to a first embodiment of the present invention will be described with reference to FIG.
A microscope observation apparatus 1 according to this embodiment includes a stage 2 on which a sample S is mounted, an excitation light source 3 that emits excitation light E, and an illumination lens (illumination optical system) 4 that guides the excitation light E from the excitation light source 3. The excitation light E guided by the illumination lens 4 is guided to the sample S, while the objective lens 5 that collimates the fluorescence F generated in the sample S, and the fluorescence F collimated by the objective lens 5 from the excitation light E. A branching dichroic mirror 6, a beam splitter 7 for further branching the branched fluorescence F, and two CCD-like imaging devices for detecting the branched fluorescence F, that is, an observation imaging device 8 and a detection imaging device 9 is provided. Reference numeral 20 in the figure denotes an imaging lens.

  The excitation light source 3, the illumination lens 4, the dichroic mirror 6, the beam splitter 7, and the two imaging devices 8 and 9 are provided in the microscope body 10. On the other hand, the objective lens 5 is separated from the microscope body 10 and supported by the objective lens driving device 11.

  The objective lens driving device 11 is a first objective that moves the objective lens 5 disposed between the stage 2 and the microscope body 10 and the objective lens 5 held by the objective lens arm 12 in the vertical direction. Drive mechanism 13, second and third drive mechanisms 14, 15 for moving the objective lens 5 in two horizontal directions, and an axis along the two directions orthogonal to the optical axis of the objective lens 5. 4th and 5th drive mechanisms 16 and 17 which are swung around are provided. The detection image pickup device 9 processes the image acquired by the detection image pickup device 9 to calculate the displacement amount and direction of the sample S, and the drive mechanisms 13 to 17 of the objective lens drive device 11. On the other hand, a drive control device 18 that outputs a drive command for driving the objective lens 5 in a direction to correct the deviation of the optical axis of the objective lens 5 due to the displacement of the sample S is connected.

The microscope observation device 1 is further provided with a marker substrate transport device 30.
The marker substrate carrier device 30 includes an arm 35a attached to a support column 34 extending vertically from a base 37 via an elevating mechanism 31 and a positioning mechanism 36, and a tip portion 35b disposed at the tip of the arm 35a. And a suction pump 33 for sucking air through a tube 32 connected to the arm 35a. The arm 35 a can be moved up and down by the elevating mechanism 31, and can be moved in the XYZ directions by the positioning mechanism 36.

  The tip part 35b has a circular shape as shown in FIG. The arm 35a and the tip portion 35b have a hollow structure. A plurality of suction holes 38 are provided on the lower surface of the distal end portion 35b. When the suction pump 33 is operated, the air sucked from the suction hole 38 is sucked through the arm 35a and the tube 32, and the marker base can be sucked and held by the tip portion 35b.

The operation of the microscope observation apparatus 1 according to the present embodiment configured as described above will be described below.
In the microscope observation apparatus 1 according to this embodiment, the marker base is brought into contact with the distal end portion 35b of the arm 35a, and the suction pump 33 is operated. As a result, the marker base is sucked and held on the tip 35b. Next, the positioning mechanism 36 is operated to move the arm 35a in the XYZ directions, and the position where the marker base is to be arranged is determined. Subsequently, the marker base can be placed at a desired position by stopping the suction pump 33 and releasing the marker base.

  In this way, the marker can be easily transported and arranged by using it as a marker base instead of alone. Furthermore, even after the marker base is once arranged, the position of the marker base can be easily changed by sucking and holding it again. Furthermore, it is easy to collect the marker substrate after the observation.

  After arranging the marker base, a marker image is picked up by the detection image pickup device 9 in order to detect the displacement of the sample. The image information acquired by the detection imaging device 9 is sent to the drive control device 18 and subjected to image processing in the drive control device 18, whereby the displacement direction, the displacement amount, the swing angle, and the like are detected. The drive control device 18 calculates command signals to be sent to the drive mechanisms 13 to 17 of the objective lens drive device 11 based on the detection result, and the drive mechanisms 13 to 17 are driven based on the command signals. Thus, in the state in which the follow-up control for correcting the biological vibration is performed, the observation imaging device 8 captures an image for observing the sample.

  In the above embodiment, the tip 35b is circular, but it may be square, trapezoidal, or U-shaped, and may be a ring shape as shown in FIG. In the case of a U shape or a ring shape, the central portion preferably includes a transparent plate member such as glass. By providing the transparent plate at the center, the marker base can be stably held and the installation position of the marker base can be visually confirmed.

  In addition, when using a marker sheet | seat as a marker base | substrate, you may hold | maintain by wetting the contact surface of the front-end | tip part 35a of an arm, and sticking a sheet | seat.

  Moreover, in the said embodiment, although the microscope observation apparatus 1 and the marker base conveyance apparatus 30 were comprised as a different body, you may decide to comprise the marker base conveyance apparatus 30 integrally with the objective lens 5. FIG.

Next, a microscope observation apparatus 40 according to the second embodiment of the present invention will be described with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the microscope observation apparatus 1 according to the first embodiment described above, and the description thereof is omitted.

  In the second embodiment, the microscope observation apparatus 40 includes a stabilizer mechanism 60 having a marker-based transport function.

  The stabilizer mechanism 60 includes an arm 65a attached to a support column 64 extending vertically from a base 67 via an elevating mechanism 61 and a positioning mechanism 66, a distal end portion 65b disposed at the distal end of the arm 65a, and an arm 65a. And a suction pump 63 for sucking air through the connected tube 62. The arm 65a can be moved up and down by the elevating mechanism 61, and can be moved in the XYZ directions by the positioning mechanism 66.

  The distal end portion 65b has a ring shape. A plate-shaped member 69 made of a transparent material such as glass is provided in the ring-shaped central opening. The ring-shaped central opening is formed with a size that allows the observation range of the objective lens 5 to be accommodated.

  The arm 65a and the tip 65b have a hollow structure. A plurality of suction holes 68 are provided on the lower surface of the distal end portion 65b. When the suction pump 63 is operated, the air sucked from the suction hole 68 is sucked through the arm 65a and the tube 62, and the marker base can be sucked and held by the tip 65b.

  The operation of the microscope observation apparatus 40 according to the present embodiment configured as described above will be described below with reference to FIG.

  In the microscope observation apparatus 40 according to the present embodiment, the marker base 160 is brought into contact with the distal end portion 65b of the arm 65a, and the suction pump 63 is operated. Thereby, the marker base is sucked and held on the tip 65b. Next, the positioning mechanism 66 is operated to move the arm 65a in the XYZ directions, and the position where the marker base is to be arranged is determined. When the position is determined, the lifting mechanism 61 is operated to lower the arm 65a and bring the tip 65b into close contact with the surface of the sample S. In this state, focusing is performed by bringing the objective lens 5 of the microscope observation device 40 close to the upper surface of the plate-like member 69 corresponding to the circular observation range disposed in the central opening of the arm tip 65b of the stabilizer mechanism 60. , Observe.

  According to the microscope observation device 40 of the present embodiment, the dynamic behavior of the surface of the sample S is suppressed via the marker base 160 adsorbed to the arm tip 65b of the stabilizer mechanism 60, and only the adsorbing portion is stopped. It becomes a state. Therefore, the dynamic behavior is suppressed by the stabilizer, the marker can be arranged at a desired position, and the marker image can be easily acquired. In particular, since the field of view of the objective lens 5 (circular observation range) and the arrangement of the markers are always in a fixed positional relationship, position detection on the observation image is facilitated. Further, the marker base can be kept horizontal by pressing the marker base with a stabilizer.

  In the above embodiment, the tip portion 65b is ring-shaped, but it may be a quadrilateral shape, a trapezoidal shape, or a U-shape with an opening at the center.

  Further, the tip 65b may include an opening for adsorbing a sample in addition to the suction hole for adsorbing the marker base. By closely adhering the marker substrate to the sample surface and adsorbing the sample, the dynamic behavior of the sample S in the observation range can be more reliably suppressed.

  From another aspect of the present invention, a marker base as described above is provided. The marker substrate of the present invention can also be used in a biological observation apparatus that does not include a transport device. In this case, the marker base may be manually placed in a sample using tweezers or the like. Yet another aspect of the present invention provides a marker-based transport device for a living body observation device.

  In addition, about the objective lens drive device 11, it is not limited to what was described in the said embodiment, Arbitrary various mechanisms can be employ | adopted suitably.

  As described above, the configuration and operation of the present invention have been described using a microscope as an example. However, the scope of the present invention is not limited to a microscope. Any one is included in the present invention.

The schematic diagram which shows one Example of a marker base | substrate. The schematic diagram which shows the relationship between a lens magnification and a marker base | substrate. The schematic diagram which shows a marker sheet. The typical whole block diagram which shows the microscope observation apparatus which concerns on 1st Embodiment. The figure which shows one Embodiment of the front-end | tip part of a marker base | substrate conveyance apparatus. The figure which shows other embodiment of the front-end | tip part of a marker base | substrate conveyance apparatus. The typical whole block diagram which shows the microscope observation apparatus which concerns on 2nd Embodiment. The figure which shows the front-end | tip part of the marker-shaped carrier apparatus of a stabilizer shape.

Explanation of symbols

  DESCRIPTION OF SYMBOLS S ... Sample 1, 40 ... Microscope observation apparatus, 5 ... Objective lens, 8 ... Imaging apparatus for observation, 9 ... Imaging apparatus for detection, 10 ... Microscope main body, 11 ... Objective lens drive apparatus, 18 ... Drive control apparatus, 30 DESCRIPTION OF SYMBOLS ... Marker base conveyance apparatus, 31 ... Elevating mechanism, 32 ... Tube, 33 ... Suction pump, 35a ... Arm, 35b ... Tip part, 38 ... Suction hole, 50, 110 ... Marker, 51 ... Marker sheet, 52 ... Organ, 60 ... Stabilizer mechanism, 69 ... Plate member, 100, 160 ... Marker base, 120 ... Base.

Claims (13)

An objective lens placed close to the sample;
An imaging device for imaging a sample through the objective lens;
An objective lens driving device for driving the objective lens in a direction for correcting the displacement of the sample;
A marker substrate transport device for arranging a marker substrate on a sample in the observation range by the objective lens;
A living body observation apparatus.   The living body observation apparatus according to claim 1, wherein the marker substrate transport device includes an adsorption mechanism that adsorbs the marker substrate, and a positioning mechanism that adjusts a position where the marker substrate is disposed.   The microscope observation apparatus according to claim 1 or 2, wherein the marker substrate transporting device includes a tip portion that sucks and holds the marker substrate, and the tip portion has a ring shape that surrounds the entire circumference of the observation range.   The living body according to claim 1 or 2, wherein the marker substrate transporting device includes a tip portion that sucks and holds the marker substrate, and the tip portion has a shape that surrounds the entire circumference except a part of the periphery of the observation range. Observation device.   The living body observation device according to any one of claims 1 to 4, wherein the marker substrate transporting device is a stabilizer that closely attaches the marker substrate to a sample in the observation range.   The biological observation device according to any one of claims 1 to 5, wherein the marker-based transport device is separate from the biological observation device.   The living body observation apparatus according to any one of claims 1 to 5, wherein the marker-based transport apparatus is provided integrally with the living body observation apparatus.   The biological observation apparatus according to any one of claims 1 to 7, wherein the marker base is formed by bonding a plurality of markers to a transparent plate-like base.   The biological observation apparatus according to any one of claims 1 to 7, wherein the marker base is formed by including a plurality of markers in a transparent sheet-like base.   The living body observation apparatus according to any one of claims 1 to 9, wherein the living body observation apparatus is a microscope for living body observation.   The living body observation apparatus according to claim 1, wherein the sample is a cell, tissue, or viscera in a living body.   A marker base for imaging vibration detection, in which multiple markers, which are detection targets in living body observation, are held on a transparent base.   The marker base | substrate conveying apparatus for biological observation apparatuses which conveys the marker base | substrate of Claim 12.

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