JP2010014968A - Living body observation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a living body observation device, a control method for the living body observation device and a living body observation method by which a marker is prevented from being reflected in a camera for observation and living body information is prevented from being lost due to existence of the marker. <P>SOLUTION: The living body observation device includes: an illumination means which irradiates an observation portion of a living body and the marker with light; an observation imaging means which forms an observation image of the living body; a marker applying means which arranges the marker separately by a distance decided according to a numerical aperture of an observation optical system to an optical axis direction from the observation portion of the living body; a vibration-detection imaging means which forms an image with light from the marker; a control means which calculates displacement vector of the marker from the image picked up by the vibration-detection imaging means; and an objective lens drive means which is capable of driving according to the displacement vector. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a biological observation apparatus having a biological vibration suppression function that suppresses image blur due to biological vibration.

  In order to analyze biological functions and elucidate pathology, there is an increasing interest in techniques for observing living bodies. However, because the living body is alive, it has vibration due to breathing, pulsation, and pulsation. This vibration is a very big problem as a factor that obstructs observation, such as blurring of the observation image.

  As a technique for suppressing image blur, firstly, a technique for physically pressing a living body and suppressing vibration of the living body in an observation range has been developed (Patent Document 1). In Patent Document 1, a vibrated living body is physically pressed by a stabilizer, and the vibration itself in the observation range arranged inside the stabilizer is suppressed, whereby a clear image with less image blur can be obtained.

  Secondly, as a technique for suppressing image blur, a technique for suppressing image blur by causing the objective lens to follow the vibration of the living body instead of suppressing the vibration of the living body has been developed (Patent Document). 2). In Patent Document 2, a vibration detection camera, a control device, and an objective lens driving device are provided in order to cause the objective lens to follow the biological vibration. Image information captured by the vibration detection camera is processed by the control device, and the displacement amount of the biological vibration is converted into data. By driving the objective lens driving device in accordance with the amount of displacement, the objective lens is caused to follow the biological vibration.

In order for the objective lens to follow the vibration of the living body, it is necessary to accurately acquire the position information of the living body in real time. As one proposal, a method has been proposed in which a high-brightness marker such as a fluorescent dye is attached to a living body and position information is acquired from the marker (Non-patent Document 1). The bright marker does not lose its position information even if the frame detection (FPS) of the camera for vibration detection is set high and the exposure time of each imaging is shortened.
JP2007-279388 JP2007-187810A Official journal of the Society for Molecular Imaging, August 21, 2006, 198-240

  However, conventionally, since the marker is directly applied to the living body, there is a problem that the marker is reflected on the observation camera. For example, if the marker and the observation image are separated according to the wavelength and the light from the marker is not incident on the observation camera, the light from the marker can be blocked, but the biological information of the part where the marker exists is also lost. End up. When a marker is present at a desired observation site, it is not possible to obtain biological information of the desired observation site.

  Accordingly, an object of the present invention is to provide a living body observation apparatus, a living body observation apparatus control method, and a living body observation method in which the marker is not reflected on the observation camera and the living body information is not lost due to the presence of the marker. There is.

  As a result of earnest research, the inventor newly provided a marker providing means in the living body observation apparatus, and separated the marker from the observation part of the living body in the optical axis direction by a distance determined by the numerical aperture of the observation optical system. The arrangement has solved the above-mentioned problems.

  That is, according to the present invention, the observation imaging means for forming an observation image of the living body and the marker are arranged away from the observation portion of the living body by a distance determined by the numerical aperture of the observation optical system in the optical axis direction. Marker providing means capable of performing imaging, vibration detection imaging means for imaging light from the marker, control means for calculating a marker displacement vector from an image captured by the vibration detection imaging means, and the displacement There is provided a living body observation apparatus including an objective lens driving unit that can be driven according to a vector.

  In the present invention, the observation imaging means for forming the observation image of the living body and the marker are arranged away from the observation portion of the living body by a distance determined by the numerical aperture of the observation optical system in the optical axis direction. Marker providing means capable of performing imaging, vibration detection imaging means for imaging light from the marker, control means for calculating a marker displacement vector from an image captured by the vibration detection imaging means, and the displacement The objective lens driving means that can be driven according to the vector and the numerical aperture of the detection optical system are set lower than the numerical aperture of the observation optical system, so that the depth of focus of the imaging means for vibration detection becomes deeper. There is provided a living body observing apparatus comprising adjusting means for allowing light from a marker to be imaged on the vibration detecting image pickup means when the observation image pickup means is focused on a living body.

  Furthermore, the present invention provides an observation imaging unit that forms an observation image of a living body and a marker separated from the observation part of the living body by a distance determined by the numerical aperture of the observation optical system in the optical axis direction. Marker providing means that can be arranged, vibration detection imaging means for forming an image of light from the marker, control means for calculating a displacement vector of the marker from an image captured by the vibration detection imaging means, The objective lens driving means that can be driven according to the displacement vector and the numerical aperture of the detection optical system are set lower than the numerical aperture of the observation optical system, so that the depth of focus of the imaging means for vibration detection is deep. And a control method for the biological observation apparatus, comprising: an adjustment unit that allows the image from the marker to be imaged on the vibration detection imaging unit when the observation imaging unit is focused on the living body. When the observation imaging means is focused on a living body by the numerical aperture of the detection optical system set lower than the numerical aperture of the observation optical system, the vibration detection imaging means A step of forming an image of light, and a step of converting movement of a marker, which is disposed at a predetermined position of the living body and following the vibration of the living body, into a displacement vector of the marker by the vibration detection imaging means and the control means, There is provided a control method for a living body observation apparatus, comprising: a step of translating the objective lens driving unit according to the displacement vector; and a step of displaying an observation image of a living body imaged by the imaging unit for observation.

  Furthermore, the present invention provides an observation imaging unit that forms an observation image of a living body and a marker separated from the observation part of the living body by a distance determined by the numerical aperture of the observation optical system in the optical axis direction. Marker providing means that can be arranged, vibration detection imaging means for forming an image of light from the marker, control means for calculating a displacement vector of the marker from an image captured by the vibration detection imaging means, The objective lens driving means that can be driven according to the displacement vector and the numerical aperture of the detection optical system are set lower than the numerical aperture of the observation optical system, so that the depth of focus of the imaging means for vibration detection is deep. And using a living body observation apparatus comprising adjustment means that allows light from a marker to be imaged on the vibration detection imaging means when the observation imaging means is focused on a living body. A living body observation method, the step of setting the numerical aperture of the detection optical system lower than the numerical aperture of the observation optical system, the step of bringing the marker applying means into contact with a living body (however, excluding humans), The step of focusing the imaging means for observation on the living body, and the movement of the marker arranged at a predetermined position of the living body and following the vibration of the living body are performed by the imaging means for vibration detection and the control means. There is provided a living body observation method including a step of converting into a displacement vector, a step of translating the objective lens driving unit according to the displacement vector, and a step of obtaining an observation image of a living body by the imaging unit for observation.

  The present invention can provide a living body observation apparatus, a living body observation apparatus control method, and a living body observation method in which the marker is not reflected on the observation camera and the living body information is not lost due to the presence of the marker.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In addition, each embodiment shown below is only what was shown in illustration, in order to demonstrate the structure of this invention in detail. Therefore, the present invention should not be construed as being limited based on the description described in the following embodiments. The scope of the present invention includes all embodiments including various modifications and improvements of the following embodiments as long as they are within the scope of the invention described in the claims.

<First Embodiment>
FIG.
FIG. 1 is a schematic diagram of a living body observation apparatus 100 in the first embodiment. The living body observation apparatus 100 of the present invention includes an illuminating means 150 for irradiating light to the observation part of the sample A and the marker 9, a high-sensitivity camera 110 for forming an observation image of the living body, A stabilizer 1 that can be arranged away from the site in the optical axis direction by a distance determined by the numerical aperture of the observation optical system, a high-speed camera 120 that forms an image of light from the marker 9, and the high-speed The microscope apparatus includes a control unit 130 that calculates a displacement vector of a marker from an image captured by the camera 120, and an objective lens driving unit 140 that can be driven according to the displacement vector. Note that the present invention can be applied to bright field observation (transmitted light, reflected light, interference light, etc.) observation and dark field observation (fluorescence, light emission), so that the illumination means 150 may be omitted.

  The marker applying means is constituted by, for example, a stabilizer 1, and the stabilizer 1 is connected to an elevating mechanism via an arm portion, and the elevating mechanism is connected to the body observation apparatus main body. The stabilizer 1 presses the sample A placed on the stage 101 from above. The stage 101 is installed on the vibration isolation table 103 and can be moved and adjusted by the aiming knob 102.

  The sample A is a living organism that is actually living, for example, various tissues (for example, epithelial tissue, connective tissue, muscle tissue and nerve tissue) of small experimental animals and organs (for example, heart, brain) composed of the above various tissues. , Liver, stomach). The desired observation site is previously labeled with a fluorescent dye or GFP, and is exposed to the outside by a normal surgical technique. The stabilizer 1 includes a movable member that suppresses vibration in the optical axis direction (Z-axis direction) among vibrations of a living body and allows movement in a direction (X, Y-axis direction) perpendicular to the optical axis. Therefore, the sample A pressed from above on the stabilizer 1 is suppressed from vibration in the X and Y axis directions, although vibration in the Z axis direction is suppressed.

  The observation imaging means is constituted by, for example, a high-sensitivity camera 110 and captures a fine observation image of the sample A. The captured image data is transmitted to the PC 111 and displayed on the monitor 112.

  The vibration detection imaging means is constituted by, for example, a high-speed camera 120 and images the marker 9.

  The control means 130 is a position control arithmetic device that calculates the displacement vector of the marker from the image captured by the high-speed camera 120 and drives the objective lens driving means 140 in accordance with the calculated displacement vector.

  The objective lens driving means is constituted by, for example, an XY stage 140 that can be driven according to the displacement vector, and the objective lens 10 is attached to the XY stage 140. Since the stabilizer 1 suppresses the vibration of the sample A in the optical axis direction (Z-axis direction), the calculated displacement vector is a two-dimensional vector represented by (X, Y). Therefore, the objective lens driving unit 140 only needs to be able to be driven in the XY plane direction, and does not need to have a drive system in the Z direction. In addition, the objective lens driving means 140 is attached to the revolver and the lifting mechanism, and the objective lens can be changed, exchanged, and lifted. This lifting mechanism is independent of the lifting mechanism to which the stabilizer 1 is connected.

  A cover glass may be used instead of the stabilizer 1 as the marker applying means. The cover glass is a member that is separated and independent from the living body observation apparatus, and a marker that can generate high-luminance light such as fluorescence or chemiluminescence is provided on the upper surface (non-contact surface of the living body) of the cover glass. When the cover glass is used as the marker applying means, the vibration of the living body is developed also in the Z-axis direction, so the displacement vector calculated by the control means 130 is a three-dimensional vector represented by (X, Y, Z). is there. Therefore, the objective lens driving means 140 needs to be able to be driven to an arbitrary position in the three-dimensional space defined by XYZ.

  In the first embodiment, the optical system includes, for example, a dichroic mirror (hereinafter referred to as DM) and an excitation filter (hereinafter referred to as EX filter).

  For example, the light irradiated by the lamp 150 enters the DM1 170 through the EX filter 160 and is reflected by the DM1 170 toward the DM2 180 side. Thereafter, the light is reflected to the objective lens 10 side by the DM2 180 and irradiated onto the sample A through the objective lens 10.

  Next, the fluorescence / reflected light from the sample A enters the DM2 180 through the objective lens 10, is reflected by the DM2 180 to the DM1 170 side, passes through the DM1 170, and then enters the high sensitivity camera 110.

  On the other hand, the fluorescence from the marker 9 passes through the objective lens 10 and DM2 180 and then enters the high-speed camera 120. In addition, when using the pigment | dye etc. which do not require excitation light like chemiluminescence as a marker, what is necessary is just to use the light of the wavelength suitable for living body observation as illumination light or excitation light exclusively.

  In the first embodiment, the marker 9 is arranged at a distance determined by the numerical aperture of the observation optical system from the observation part of the living body in the optical axis direction. When the sample A is set to the sample A, the marker 9 does not appear on the high sensitivity camera 110. Therefore, the living body observation apparatus according to the first embodiment can continuously capture a clear living body image in which a marker is not reflected on a vibrating living body using a high sensitivity camera (observation camera).

  In general, a marker is very bright as compared with a living body, and the influence of reflection in an image is very large (smearing phenomenon, blooming phenomenon), which is one of serious problems in living body observation. The living body observation apparatus of the present invention that has succeeded in solving the problem of the reflection of a marker on an observation image has extremely high technical significance in real time observation of a vibrating living body.

  Here, in order to prevent the reflection of the marker 9 on the high-sensitivity camera 110, the distance Zm from the observation site of the living body to the marker needs to be separated by the distance indicated by the following inequality.

a: Diameter of the light beam at the marker position / diameter of the marker
NAo: Numerical aperture of observation optics
D: Marker diameter
n: The refractive index of the medium between the observation site of the living body and the marker.

  For example, when a = 4, NAo = 0.5, D = 10 μm, and n = 1.5, if the marker 9 is separated from the observation site of the sample A by 60 μm or more in the optical axis direction, the focus of the high-sensitivity camera 110 is adjusted. The marker 9 does not appear in the image picked up by the high sensitivity camera 110.

  The distance Zm is basically a distance determined by the numerical aperture of the observation optical system, as is clear from Equation 1 (Equation 1). Here, the numerical aperture of the observation optical system is a numerical aperture defined by various means of the observation optical system that can change the numerical aperture of the observation optical system, for example, the imaging means for observation, the illumination means, and the optical system. means. The numerical aperture (hereinafter referred to as NA) is a measure of the resolving power of the objective lens. The refractive index of the medium in front of the objective lens, the outermost ray incident on the objective lens, and the optical axis. It is represented by the product of the sine of the angle formed.

  The distance Zm is provided by the marker applying means. For example, when the marker applying means is constituted by a stabilizer, the thickness of the transparent member attached to the tip of the stabilizer corresponds to this distance. Moreover, when the marker provision means is comprised with the cover glass, the thickness of a cover glass corresponds to this distance.

  In the first embodiment, since the marker 9 is arranged away from the observation site of the living body by a distance determined by the numerical aperture of the observation optical system in the optical axis direction, the focus of the high-speed camera 120 is It is necessary to adjust the position to the marker 9 side. That is, when the high-sensitivity camera 110 is focused on the sample A, the focal point of the high-speed camera 120 whose focal length is generally adjusted in conjunction with the high-sensitivity camera 110 is also focused on the sample A. Needs to be adjusted to the marker 9 again.

<Stabilizer structure>
FIG.
FIG. 2 is a perspective view showing the appearance of the stabilizer 1. The stabilizer 1 includes a cylindrical member 2 constituted by an upper member 3 and a lower member 4, a movable member 5, and a transparent member 6. The upper member 3 has an arm portion 7, and the arm portion 7 is attached to an elevating mechanism 8 so as to be movable up and down. The lifting mechanism 8 is attached to the body of the ecology observation apparatus 100. The arm unit 7 may be an articulated arm that can move in all directions, and the biological sample can be pressed down accurately by providing the lifting mechanism 8 with a mechanism that can move in the plane direction.

  The transparent member 6 is provided with a marker 9. The marker 9 only needs to be provided within the observation range B of the objective unit 9 in the transparent member 6. Moreover, the marker 9 is provided on the living body non-contact surface 6b side of the transparent member 6, and the thickness of the transparent member 6 is a distance determined by the numerical aperture of the observation optical system from the observation part of the living body toward the optical axis direction. .

  When observing the sample A, first, the outer skin D of an experimental small animal or the like is cut open to expose the sample A such as an organ. And the raising / lowering mechanism 8 is operated, the stabilizer 1 is lowered | hung, and the contact surface 6a of the transparent member 6 is made to contact the upper surface of the sample A. FIG.

  At this time, the stabilizer 1 is lowered until the biological contact surface 6a presses the sample A with a predetermined pressure, and the stabilizer 1 is fixed at that position. In the sample A, vibration in the direction of the optical axis C (Z-axis direction) is suppressed by the pressing force from above by the contact surface 6a. On the other hand, since the movable member 5 is allowed to move in the direction perpendicular to the optical axis C (X and Y axis directions), vibration of the living body in the X and Y axis directions is not suppressed. When the sample A vibrates in the direction perpendicular to the optical axis C (X and Y axis directions) with the contact surface 6a pressing the sample A, the movable member 5 moves in conjunction with the vibration of the sample A. Is not deviated from the initial position of the sample A. Therefore, the marker 9 can always indicate a specific position of the sample A regardless of the vibration of the sample A.

  In this state, the objective lens 10 is lowered to the inner space of the cylindrical member 2 to focus on the sample A. The light of the marker 9 captured by the objective lens 10 is picked up by the vibration detecting image pickup means (high speed camera 120), and the displacement vector of the marker is calculated by the control means 130 from the picked up image. Then, the control means 130 drives the objective lens 10 according to this displacement vector. Therefore, since the objective lens 10 also moves in accordance with the movement of the marker 9 that moves following the vibration of the living body, image blurring does not occur and a clear image can always be obtained.

FIG.
3 is a perspective view of a cross section taken along line BB in FIG. The lower member 4 is joined to the concave portion of the upper member 3, and the cylindrical member 2 having the holding portion 12 is formed. On the other hand, the movable member 5 includes a protrusion (flange 11). Then, the movable member 5 is carried on the cylindrical member 2 by the flange 11 of the movable member 5 being held between the holding portions 12. The cylindrical member 2 only needs to have an inner space for inserting the objective lens 10, and the shape thereof is not particularly limited. Moreover, there is no restriction | limiting in particular in the material of the upper member 3 and the lower member 4 which comprise the cylinder member 2, For example, it selects from the resin material and a metal material suitably.

  The movable member 5 is made of an elastic member having slidability, for example, a resin material such as a plastic film, and its trunk can be deformed by finger pressure. The movable member 5 is deformed so as to be crushed by finger pressure, and the movable member 5 is inserted from below in the cylindrical member 2 in this state. At this time, it is advisable to insert the movable member 5 so that the flanges 11 of the movable member 5 are inserted into the holding portion 12 in the same rotational direction. Here, when the finger is released, the movable member 5 returns to its original shape due to its own elasticity, the flange 11 enters the holding portion 12, and the movable member 5 is carried on the cylindrical member 2. It is easy to put a mark indicating the direction of the holding portion 12 on the outer lower surface of the lower member 4.

  The movable member 5 may be provided with a slit 13 in the upper part of the movable member 5. By providing the slit 13, the deformation of the body portion of the movable member 5 can be made easier.

  Moreover, since the movable member 5 can be easily removed, contamination between biological samples can be prevented by exchanging or washing the movable member 5. Furthermore, it is possible to prevent the observation image from being deteriorated due to dirt on the cover glass.

  The sandwiching portion 12 needs to be provided at a corresponding portion of the flange 11 of the movable member 5, but more sandwiching portions 12 may be provided in the cylindrical member 2 as necessary. For example, with respect to the movable member 5 including the pair of flanges 11, by providing the four holding portions 12 in the cylindrical member 2, the direction in which the flange 11 of the movable member 5 is oriented when the movable member 5 is inserted. Even if it is, it is possible to easily insert the flange 11 into the holding portion 12.

  Holding members 12a and 12b are attached to the upper and lower surfaces of the holding portion 12, respectively, and allow the flange 11 to move in a direction perpendicular to the optical axis C (X and Y axis directions). The holding member includes all the holding members known at the time of filing of the present application that allow the flange 11 to move in the X and Y axis directions, and particularly preferably a group consisting of a sliding member, a ball and a leaf spring. It is comprised from the arbitrary combinations selected more. In FIG. 4, each of the holding members 12a and 12b is illustratively constituted by a sliding member. As the sliding member, a resin material such as a fluororesin is preferable. Note that the upper member 3 and the lower member 4 themselves may be formed of sliding members.

  The flange 11 is allowed to move in the X and Y axis directions between the sliding members 12a and 12b. The movement of the movable member 5 following the vibration of the living body in the X and Y axis directions is brought about by the sliding action of the flange 11.

FIG.
FIG. 4 is a cross-sectional view taken along line BB in FIG. The flange 11 has a dimension such that the movable member 5 does not come off the holding portion 12. That is, the flange 11b at the other end needs to be carried on the holding portion 12 when the flange 11a at the one end swings to the extreme end. Specifically, when observing a living body in which vibration of 400 μm is estimated, for example, the inner diameter of the cylindrical member 2 is designed to be 1 mm larger than the outer diameter of the movable member 5 and the inner diameter of the holding portion 12 is set to the flange 11. By designing it to be 1 mm larger than the outer diameter as described above, the movable member 5 installed on the central axis is provided with a movable space of 500 μm on the front, rear, left and right. At this time, if the outer diameter of the flange 11 is designed to be 4 mm larger than the outer diameter of the body portion of the movable member 5 (that is, a protrusion having a total length of 4 mm), the movable member 5 will not come off the holding portion 12. Furthermore, the slidability is not impaired by being caught by the upper member 3 or the lower member 4.

  The marker 9 may be disposed at any position as long as it enters the observation range B of the objective lens 10 (more specifically, within the field of view of the high-speed camera). For example, the marker may be arranged at the center of the visual field, or may be arranged at a position outside the observation visual field. Further, a plurality of markers 9 may be arranged so that they always enter the field of view of the high-speed camera regardless of the movement of the living body or the initial position of the movable member. Furthermore, the marker 9 is provided on the living body non-contact surface 6 b side of the transparent member 6. There is no restriction | limiting in the kind, shape, etc. of the marker 9, All the markers well-known at the time of this-application application are included. For example, the marker 9 is a fluorescent paint printed in a circle on the transparent member 6 or a reflective material plated in a cross shape. Since the marker 9 is provided on the inner surface 6b side, the marker 9 does not contact the living body, and the material of the marker can be freely selected without considering biocompatibility.

FIG.
FIG. 5 is a perspective view of the movable member 5. A pair of flanges 11 a and 11 b are provided on the cylindrical main body of the movable member 5. The shape and number of the flanges 11 are not particularly limited, but as shown in the figure, the pair of flanges 11a and 11b are connected to each other on the cylinder circumference so that they can be easily crushed and inserted into the cylindrical member 2. It is preferable to provide it so as to face each other. Optionally, a slit 13 may be provided on the cylindrical circumferential frame so that the movable member 5 can be crushed more easily. The shape and number of the slits 13 are not particularly limited, but a pair of slits are preferably provided so as to face each other. If the slit 13 is provided, the cylindrical body of the movable member 5 can be thickened to some extent by the amount of deformation. If the thickness of the cylindrical body portion of the movable member 5 is reduced, it can naturally be easily deformed, but irreversible deformation is easily caused accordingly. The slit 13 is provided so that the cylindrical body of the movable member 5 has a certain thickness, and the irreversible deformation of the movable member 5 is prevented, and the ease of deformation can be realized simultaneously by the effect of the slit 13.

  A transparent member 6 is attached to the tip of the movable member 5. The transparent member 6 should just be an optically transparent member, for example, is comprised with a cover glass etc. Glass has good adhesion to a living body and enables translational movement of the movable member 5 following the vibration of the living body. On the surface of the transparent member 6, at least one marker 9 is provided. The transparent member 6 can be fitted and fixed so as to be inscribed in the inner space of the movable member 5. At this time, for example, a protrusion (not shown) may be provided in the inner space of the movable member 5 and the transparent member 6 fitted in the inner space of the movable portion 5 may be fixed. Further, the transparent member 6 may be stuck and fixed on the cylindrical circumferential frame of the movable member 5. In addition, the transparent member 6 in which the distal end portion of the movable member 5 is constituted by a separable member and is integrated with the distal end portion may be combined with the movable member 5.

<Composition of cover glass>
FIG.
FIG. 6 is a perspective view showing the appearance of the cover glass 20. The cover glass 20 is placed on the observation site of the sample A. Unlike the stabilizer 1, the cover glass 20 allows the living body to vibrate in the optical axis direction (Z-axis direction) of the living body, and thus the living body vibrates on the three-dimensional space coordinates (X, Y, Z). Therefore, the displacement vector calculated by the control means 130 is a three-dimensional vector represented by (X, Y, Z), and the objective lens driving means needs to be composed of an XYZ stage. The XYZ stage means a stage that can drive the objective lens to a desired position in a space represented by three-dimensional space coordinates (X, Y, Z).

  The cover glass 20 is provided with a marker 9 on the living body non-contact surface side. The distance Zm between the marker 9 and the observation part E of the living body is a distance determined by the numerical aperture of the observation optical system, and the distance is controlled by the thickness of the cover glass.

<Second Embodiment>
In addition to the living body observation apparatus 100 according to the first embodiment shown in FIG. 1, the living body observation apparatus 100 according to the second embodiment further includes a first adjustment unit that adjusts the numerical aperture of the observation optical system, and a detection. Second adjusting means for adjusting the numerical aperture of the optical system.

  Here, the numerical aperture (NA) of the detection optical system is defined by various means of the detection optical system that can change the numerical aperture of the detection optical system, for example, imaging means for vibration detection, illumination means, and the optical system. Numerical aperture. On the other hand, the numerical aperture (NA) of the observation optical system is, as described above, various means of the observation optical system that can change the numerical aperture of the observation optical system, for example, the imaging means for observation, the illumination means, and the optical system. Means the numerical aperture defined by.

  In the living body observation apparatus 100 according to the second embodiment, the first adjustment unit includes a first diaphragm member for the observation imaging unit (high-sensitivity camera 110), and the second adjustment unit includes vibration detection. It comprises a second diaphragm member for the image pickup means (high speed camera 120). The diaphragm member (diaphragm; hereinafter referred to as DP) can reduce the NA by narrowing the light width of the incident light to the objective lens or by narrowing the light width of the reflected light from the living body.

  The first aperture member DP1 183 provided close to the high-sensitivity camera 110 serves to adjust the NA of the imaging means for observation, that is, the high-sensitivity camera 110. On the other hand, the second diaphragm member DP2 184 provided close to the high-speed camera 120 plays a role of adjusting the vibration detection imaging means, that is, the NA of the high-speed camera 120. Instead of using the diaphragm member, the NA of each camera may be adjusted by adjusting the curvature of the imaging lens of each camera.

  The biological observation apparatus 100 according to the second embodiment is configured so that the aperture members DP1 183 and DP2 184 set the numerical aperture of the detection optical system to be lower than the numerical aperture of the observation optical system. Therefore, when the high-sensitivity camera 110 is focused on the sample A, the high-speed camera 120 is simultaneously focused on the marker 9 as well as the sample A. At this time, the dark biological sample A is hardly imaged by the high-speed camera 120 with a short exposure time, and only the marker 9 is imaged. Therefore, just by focusing the high sensitivity camera 110 on the sample A, the high speed camera 120 automatically focuses on the marker 9 and only the marker 9 can be imaged. For this reason, the work of individually refocusing the high-speed camera 120 on the marker 9 becomes unnecessary, and the objective lens is immediately followed by the vibrating living body by simply focusing the high-sensitivity camera 110 on the biological sample, and image blurring is performed. It is possible to obtain a clear living body image without any problem.

  Here, the relationship between the numerical aperture (NAm) of the detection optical system and the numerical aperture (NAo) of the observation optical system is expressed by the following equation.

  (I) For a CCD camera with a small pixel size

NAm: Numerical aperture of detection optical system
NAo: Numerical aperture of observation optical system λ: Marker illumination wavelength
a: Light flux diameter at marker position / marker diameter
D: Marker diameter For example, when NAo = 0.5, λ = 1 μm, a = 4, D = 10 μm, NAm ≦ 0.0125.

  (Ii) For a CCD camera with a rough pixel size

NAm: Numerical aperture of detection optical system
NAo: Numerical aperture of observation optics
M: Horizontal magnification of the entire detection-side optical system
p: Pixel pitch of the image sensor on the detection side
a: Light flux diameter at marker position / marker diameter
D: Diameter of marker For example, when NAo = 0.5, M = × 20, p = 10 μm, a = 4, D = 10 μm, NAm ≦ 0.025.

  The looser one of (i) and (ii) is adopted depending on the pixel pitch of the CCD camera and the conditions of the optical system. Therefore, here, (ii) is applied, and the numerical aperture (NAm) of the detection optical system is 0.025 or less.

<Third Embodiment>
FIG.
In FIG. 1, the microscope apparatus is illustrated and described as an example, but the living body observation apparatus of the present invention is not limited to this, and vibrates continuously due to an actually living living body, that is, an external stimulus or a physiological phenomenon. All living body observation devices for observing living bodies are included. As an example, an endoscope 150 to which the configuration of the living body observation apparatus of the present invention is applied is illustrated.

  FIG. 7 is a schematic diagram of the distal end portion of the endoscope 400 according to the third embodiment. An endoscope lens (objective lens 10) is fixed to an XY stage 30 that can be driven in the X and Y axis directions. The XY stage 30 is attached to an elevating mechanism 35 and can raise and lower the objective lens 10 in the Z-axis direction. The objective lens 10 is connected to an optical fiber 40, and the optical fiber 40 transmits reflected light of an object captured by the objective lens 10 to an endoscope main body (not shown) through the optical fiber 40. The control mechanism in the endoscope main body is the same as that of the above-described microscope apparatus, and can make the objective lens 10 follow in accordance with the movement of the marker 9.

  The stabilizer 1 of the present invention is attached to the distal end portion of the endoscope 150. The stabilizer 1 includes a cylindrical member 2 and a movable member 5, and a transparent member 6 is attached to the distal end portion of the movable member 5.

  When the transparent member 6 is pressed against the observation site of the living body, the movable member 5 moves on the X and Y planes according to the vibration of the living body. Since the transparent member 6 is provided with the marker 9, the marker 9 moves in accordance with the vibration of the living body and can keep contact with a certain point of the living body.

  The marker 9 is provided on the living body non-contact surface (upper surface) side of the transparent member 6, and the thickness of the transparent member is determined by the numerical aperture of the observation optical system from the observation part of the living body toward the optical axis direction. Just placed apart. Therefore, when the focus of the observation camera is focused on the living body, the marker 9 is not reflected in the observation image, and a precise observation image can be obtained.

<Fourth Embodiment>
FIG.
FIG. 8 is a schematic diagram of a living body observation apparatus 200 according to the fourth embodiment. The basic configuration is the same as that of the first embodiment, but the illumination means includes a first lamp 201 for the high-sensitivity camera 110 and a second lamp 202 for the high-speed camera 120. Yes.

  The light emitted by the first lamp 201 passes through the DP3 211, enters the DM1 170 via the EX1 161, and is reflected by the DM1 170 toward the DM2 180 side. Thereafter, the light is reflected by the DM2 180 toward the objective lens 10 and irradiated onto the sample A through the objective lens 10.

  On the other hand, the light irradiated by the second lamp 202 passes through the DP4 212, enters the DM3 171 via the EX2 162, and is reflected by the DM3 171 to the DM2 180 side. Thereafter, the light is reflected to the objective lens 10 side by DM2 180, and the sample A is irradiated by the objective lens 10.

  Next, the fluorescence / reflected light from the sample A enters the DM2 180 through the objective lens 10, is reflected to the DM1 170 side by the DM2 180, passes through the DM3 171 and DM1 170, and then passes through the DP1 183. Is incident on the high-sensitivity camera 110.

  On the other hand, the fluorescence from the marker 9 passes through the objective lens 10 and the DM2 180 and then passes through the DP2 184 and enters the high-speed camera 120.

  By dividing the illumination means into two, the NA can be adjusted by the illumination means. That is, by narrowing the slit of the diaphragm member DP4 212 of the second lamp 202, the NA of the detection optical system is lowered, and as a result, the depth of focus of the high-speed camera 120 is increased. On the other hand, by opening the slit of the diaphragm member DP3 211 of the first lamp 201, the NA of the observation optical system is increased. As a result, the high-sensitivity camera 110 has a shallow focal depth, and a more precise observation image can be obtained. it can.

  In the fourth embodiment, the NA of the observation optical system is defined by the first diaphragm member DP1 183 for the high sensitivity camera 110 and / or the third diaphragm member DP3 211 for the first lamp 201, On the other hand, the NA of the detection optical system is defined by the second diaphragm member DP2 184 for the high-speed camera 120 and / or the fourth diaphragm member DP4 212 for the second lamp 202. Since NA can be defined using both the camera side and the illumination side, or one of the diaphragm members, the degree of freedom of NA definition is increased. Of course, the NA may be defined by changing the curvature of the imaging lens of the camera instead of the diaphragm member.

  In the fourth embodiment, as in the first embodiment, the stabilizer 1 is used as the marker applying means, and the marker 9 is determined by the numerical aperture of the observation optical system from the observation site of the living body toward the optical axis direction. They are spaced apart by a distance. In addition, you may use a cover glass instead of the stabilizer 1 similarly to 1st Embodiment.

<Fifth Embodiment>
FIG.
FIG. 9 is a schematic diagram of a living body observation apparatus 300 according to the fifth embodiment. The basic configuration is the same as that of the first embodiment, but the light emitted from the lamp 150 is branched into the first light for the high-sensitivity camera 110 and the second light for the high-speed camera 120. An optical system is provided.

  The light emitted from the lamp 150 is branched into first light and second light by the DM 1 170. The first light passes through DM1 170, passes through DP5 301, and is irradiated to the half mirror (HM) 301 side. Thereafter, the first light is reflected to the DM3 172 side by the HM 310, passes through the DM3 172, and is irradiated onto the sample A through the objective lens 10. On the other hand, the second light is reflected by the DM1 170 to the DM2 171 side. Thereafter, the second light is irradiated to the DM4 173 side by the DM2 171. The second light passes through DP6 302, passes through DM4 173, reaches DM3 172, is reflected by DM3 172, and is applied to sample A through objective lens 10.

  Next, the fluorescence / reflected light from the sample A enters the DM3 172 via the objective lens 10, is reflected to the DM4 173 side by the DM3 172, is subsequently reflected by the DM4 173, passes through the DP1 183, and has high sensitivity. The light enters the camera 110.

  On the other hand, the fluorescence from the marker 9 passes through the objective lens 10 and DM3 172, then passes through the HM 310, passes through the DP2 184, and enters the high-speed camera 120.

  By branching the light emitted from one illumination means into two lights, the same NA control as in the fourth embodiment having two illumination means is possible. That is, by narrowing the slit of the sixth aperture member DP6 302 capable of adjusting the second light width, the NA of the detection optical system is lowered, and as a result, the depth of focus of the high-speed camera 120 is increased. On the contrary, the NA of the observation optical system is increased by opening the slit of the fifth aperture member DP5 301 capable of adjusting the first light width, and as a result, the depth of focus of the high-sensitivity camera 110 becomes shallower and more precise. An observation image can be obtained.

  In the fifth embodiment, as in the fourth embodiment, the NA can be defined using both the camera side and the illumination side, or one of the diaphragm members, so the degree of freedom of the NA definition is increased. Of course, the NA may be defined by changing the curvature of the imaging lens of the camera instead of the diaphragm member.

  In the fifth embodiment, as in the first embodiment, the stabilizer 1 is used as the marker applying means, and the marker 9 is determined by the numerical aperture of the observation optical system from the observation site of the living body toward the optical axis direction. They are spaced apart by a distance. In addition, you may use a cover glass instead of the stabilizer 1 similarly to 1st Embodiment. In addition, since the present invention can be applied to bright field observation (transmitted light, reflected light, interference light, etc.) observation and dark field observation (fluorescence, light emission), it does not matter whether there is light illumination.

Schematic diagram of the biological observation apparatus 100 in the first embodiment The perspective view which shows the external appearance of the stabilizer 1 FIG. 2 is a perspective view of a cross section taken along line BB in FIG. Sectional view along line BB in FIG. Perspective view of movable member 5 A perspective view showing the appearance of the cover glass 20 The schematic diagram of the front-end | tip part of the endoscope 400 in 3rd Embodiment The schematic diagram of the biological observation apparatus 200 in 3rd Embodiment Schematic diagram of the biological observation apparatus 300 in the fifth embodiment

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Stabilizer, 2 ... Cylindrical member, 3 ... Upper member, 4 ... Lower member, 5 ... Movable member, 6 ... Transparent member, 6a ... Living body contact surface, 6b ... non-biological contact surface, 7 ... arm part, 8 ... elevating means, 9 ... marker part, 10 ... objective lens, 11 ... projection part, 11a ... flange at one end 11b: flange at the other end, 12: clamping part, 12a, 12b: clamping member, 13 ... slit, 20 ... cover glass, 30 ... XY stage, 35. .. Lifting mechanism, 40 ... optical fiber, 100, 200, 300 ... living body observation apparatus of the present invention, 101 ... stage, 102 ... adjustment knob, 103 ... slow shaking table, 110 ... High sensitivity camera, 111 ... PC, 112 ... Monitor, 120 ... High speed camera, 130 ... Control device, 140 ... XY stage, 150, 201, 202 ..Lamp, 160, 161, 162 ... Excitation filter, 170,171,173,180 ... Dichroic mirror, 183,184, 211,212,301,302 ... Aperture member, half mirror・ 310.

Claims (15)

An imaging means for observation that forms an observation image of a living body;
Marker providing means capable of arranging the marker by a distance determined by the numerical aperture of the observation optical system from the observation site of the living body toward the optical axis direction;
Vibration detecting imaging means for imaging light from the marker;
Control means for calculating a displacement vector of the marker from the image picked up by the vibration detecting image pickup means;
A biological observation apparatus comprising: an objective lens driving unit that can be driven according to the displacement vector. An imaging means for observation that forms an observation image of a living body;
Marker providing means capable of arranging the marker by a distance determined by the numerical aperture of the observation optical system from the observation site of the living body toward the optical axis direction;
Vibration detecting imaging means for imaging light from the marker;
Control means for calculating a displacement vector of the marker from the image picked up by the vibration detecting image pickup means;
An objective lens driving means that can be driven according to the displacement vector;
Since the numerical aperture of the detection optical system is set lower than the numerical aperture of the observation optical system, the depth of focus of the imaging means for vibration detection becomes deep, and the observation imaging means is focused on the living body. A living body observation apparatus comprising adjustment means for allowing light from a marker to be imaged on the vibration detection imaging means.   The said adjustment means is comprised from the 1st adjustment means which can adjust the numerical aperture of an observation optical system, and the 2nd adjustment means which can adjust the numerical aperture of a detection optical system. Living body observation device.   The first adjusting means is constituted by a first diaphragm member for the observation imaging means, and the second adjusting means is constituted by a second diaphragm member for the vibration detecting imaging means. The living body observation apparatus according to claim 3. An imaging means for observation that forms an observation image of the living body;
Marker providing means capable of disposing the marker by a distance determined by the numerical aperture of the observation optical system from the observation site of the living body toward the optical axis direction;
Vibration detecting imaging means for imaging light from the marker;
Control means for calculating a displacement vector of the marker from the image picked up by the vibration detecting image pickup means;
An objective lens driving means that can be driven according to the displacement vector;
First illuminating means for the imaging means for observation, which irradiates light to an observation site and a marker of the living body;
A second illuminating means for the vibration detecting imaging means for irradiating the observation site and marker of the living body with light;
Since the numerical aperture of the detection optical system is set lower than the numerical aperture of the observation optical system, the depth of focus of the imaging means for vibration detection becomes deep, and the observation imaging means is focused on the living body. A living body observation apparatus comprising adjustment means for allowing light from a marker to be imaged on the vibration detection imaging means.   The said adjustment means is comprised from the 1st adjustment means which can adjust the numerical aperture of an observation optical system, and the 2nd adjustment means which can adjust the numerical aperture of a detection optical system. Living body observation device. The first adjusting means comprises a first diaphragm member for the observation imaging means and / or a third diaphragm member for the first illumination means;
The said 2nd adjustment means is comprised by the 4th aperture member for the 2nd aperture member for the said imaging means for a vibration detection, and / or the said 2nd illumination means, The Claim 6 is comprised. Living body observation device. Illuminating means for irradiating light to the observation site and marker of the living body
An imaging means for observation that forms an observation image of the living body;
Marker providing means capable of disposing the marker by a distance determined by the numerical aperture of the observation optical system from the observation site of the living body toward the optical axis direction;
Vibration detecting imaging means for imaging light from the marker;
Control means for calculating a displacement vector of the marker from the image picked up by the vibration detecting image pickup means;
An objective lens driving means that can be driven according to the displacement vector;
An optical system for branching the light emitted from the illumination unit into a first light for the observation imaging unit and a second light for the vibration detection imaging unit;
Since the numerical aperture of the detection optical system is set lower than the numerical aperture of the observation optical system, the depth of focus of the imaging means for vibration detection becomes deep, and the observation imaging means is focused on the living body. A living body observation apparatus comprising adjustment means for allowing light from a marker to be imaged on the vibration detection imaging means.   The said adjustment means is comprised by the 1st adjustment means which can adjust the numerical aperture of an observation optical system, and the 2nd adjustment means which can adjust the numerical aperture of a detection optical system. Living body observation device. The first adjusting means comprises a first diaphragm member for the observation imaging means and / or a fifth diaphragm member for the first light;
The said 2nd adjustment means is comprised by the 2nd aperture member for the said imaging means for a vibration detection, and / or the 6th aperture member for the said 2nd light. Living body observation device. The marker applying means is composed of a stabilizer,
The stabilizer is configured to be separable into a cylindrical member having an objective lens insertion port and the cylindrical member, and suppresses vibration in the optical axis direction among vibrations of the living body and in a direction perpendicular to the optical axis. A movable member that allows movement; and a living body contact surface that is located at a distal end of the movable member, and is separated from the living body contact surface by a distance determined by the numerical aperture of the observation optical system in the optical axis direction. The living body observation apparatus according to any one of claims 1 to 10, further comprising a transparent member provided with one or more of the markers. The marker applying means is composed of a cover glass,
The said cover glass is comprised by the transparent member to which the one or more said marker was provided | separated by the distance determined by the numerical aperture of an observation optical system toward the optical axis direction from a biological contact surface. The biological observation apparatus according to any one of 10. The distance Zm from the observation site of the living body to the marker is

a: Diameter of the light beam at the marker position / diameter of the marker
NAo: Numerical aperture of observation optics
D: Marker diameter
n: The living body observation apparatus according to any one of claims 1 to 10, which is determined by a refractive index of a medium between a living body observation site and a marker. An imaging means for observation that forms an observation image of a living body;
Marker providing means capable of arranging the marker by a distance determined by the numerical aperture of the observation optical system from the observation site of the living body toward the optical axis direction;
Vibration detecting imaging means for imaging light from the marker;
Control means for calculating a displacement vector of the marker from the image picked up by the vibration detecting image pickup means;
An objective lens driving means that can be driven according to the displacement vector;
Since the numerical aperture of the detection optical system is set lower than the numerical aperture of the observation optical system, the depth of focus of the imaging means for vibration detection becomes deep, and the observation imaging means is focused on the living body. A control method for a biological observation apparatus, comprising: adjusting means for allowing light from a marker to be imaged on the vibration detecting imaging means.
With the numerical aperture of the detection optical system set to be lower than the numerical aperture of the observation optical system, the light from the marker is imaged on the vibration detection imaging means when the observation imaging means is focused on the living body. A process of
Converting the movement of the marker that is arranged at a predetermined position of the living body and that follows the vibration of the living body into the displacement vector of the marker by the vibration detecting imaging means and the control means;
Translating the objective lens driving means according to the displacement vector;
A method for controlling a living body observation apparatus, including a step of displaying an observation image of a living body imaged by the imaging means for observation. An imaging means for observation that forms an observation image of a living body;
Marker providing means capable of arranging the marker by a distance determined by the numerical aperture of the observation optical system from the observation site of the living body toward the optical axis direction;
Vibration detecting imaging means for imaging light from the marker;
Control means for calculating a displacement vector of the marker from the image picked up by the vibration detecting image pickup means;
An objective lens driving means that can be driven according to the displacement vector;
Since the numerical aperture of the detection optical system is set lower than the numerical aperture of the observation optical system, the depth of focus of the imaging means for vibration detection becomes deep, and the observation imaging means is focused on the living body. A biological observation method using a biological observation apparatus, comprising: adjustment means that allows light from a marker to be imaged on the vibration detection imaging means.
A step of setting the numerical aperture of the detection optical system to be lower than the numerical aperture of the observation optical system;
Bringing the marker applying means into contact with a living body (excluding humans);
Focusing the imaging means for observation on the living body;
Converting the movement of the marker that is arranged at a predetermined position of the living body and that follows the vibration of the living body into the displacement vector of the marker by the vibration detecting imaging means and the control means;
Translating the objective lens driving means according to the displacement vector;
And a step of obtaining an observation image of the living body by the imaging means for observation.

Images