JP2009229290A - Sample holding apparatus and biological image acquisition apparatus equipped with the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological image acquisition apparatus capable of performing multi-directional and simultaneous observations and measurements in a short time and to provide a sample holding apparatus for implementing the biological image acquisition apparatus. <P>SOLUTION: A sample holding member 2 is supported by a supporting mechanism 12. The supporting mechanism 12 supports a pair of opposed end parts of the sample holding member 2. Gaps 14 are provided between unsupported end parts of the sample holding member 2 and the supporting mechanism 12. The gaps 14 are light-passing regions for passing reflected light from reflecting mirrors M3 and M4 on the back side of the sample holding member 2. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an optical bioimaging technique for small animals.

  The technique of imaging how molecular species in a living body are distributed is an important research method in medicine and biology. At the cellular level, methods for imaging molecular species using a microscope with a molecular probe attached with a fluorescent dye or a molecular probe using chemiluminescence have been widely used. In the future, there will be a demand for an apparatus for observing the distribution of the molecular species of interest from the cellular level to larger organs and even individual animals. For example, it is a technique in which a fluorescent probe binds to a cancer cell in an individual such as a mouse to image the state of growth of the cancer cell of interest and observe it as a time-dependent change such as daily or weekly. In order to observe the proliferation of cancer cells inside an animal individual with a conventional measuring device for cell level, the animal is killed and a predetermined part is stained or observed with a fluorescent substance. It is not possible to see the growth of cells over time for a single individual. For this reason, it is desired to develop a device that can observe the molecular species of the internal information of a small animal individual while the individual is alive.

  Near-infrared light has a relatively good light transmittance inside the living body, and therefore, a small animal observation apparatus using a wavelength of about 600 nm to 900 nm is used. However, in the conventional observation method, usually, a sample is photographed from the upper side, and simultaneous observation in multiple directions is not possible. For example, when a mouse is observed from a specific direction, cancer is detected when observed from the opposite side even if the cancer is not visible. When using a device that can only observe in one direction, the operator is forced to take an image that rotates little by little around the body axis of the mouse, and handle it in an operation that approximates observation in multiple directions. There is only. However, with this method, reproducible data cannot be obtained, and each direction cannot be detected simultaneously. In particular, since the emission intensity itself is very weak in light emission observation from a living body, it is usually necessary to perform integral exposure on a two-dimensional detector for several tens of seconds to several minutes. On the other hand, since the light emission intensity changes with time, it is not useful to sequentially switch images in different directions to change the image conditions for each direction. It is desirable that images in multiple directions of a living body can be integrated on a detector for a long time in parallel. Fluorescence image measurement can be performed in a relatively short time, but it is still indispensable to obtain quick and accurate data that can capture information in multiple directions at once.

As a method for obtaining multi-directional images, a method is known in which images of a large number of angles are sequentially acquired in a time-sharing manner using a rotating reflector (see Patent Document 1). This method does not rotate the sample nor the two-dimensional detector, but the multi-directional observation is performed by rotating the mirror on the way and changing the position of the sample, although there is parallel movement of the sample itself. .
US Patent Application Publication No. 20050201414

The disadvantage of the method of Patent Document 1 using a rotating reflector is that the measurement in each direction is time-division, so that simultaneous measurement cannot be performed, and in addition to the time required for the measurement, the structure is complicated.
An object of the present invention is to provide a biological image acquisition device that can simultaneously measure multi-directional observations in a short time and a sample holding device for realizing the biological image acquisition device.

  A sample holding device of the present invention is a sample holding device of a biological image acquisition device that acquires images from a plurality of directions of a biological sample using a two-dimensional detector, and includes a sample holding region for holding a sample. In addition, the living body is supported in a state where the sample holding member is supported while securing a substantially transparent sample holding member and a light passage area for allowing the light guided to the two-dimensional detector to pass through the side of the sample holding area. And a support mechanism disposed in the image acquisition apparatus.

  The biological image acquisition device of the present invention provided with the above sample holding device includes the sample holding device and a sample holding member of the sample holding device for imaging the sample placed on the sample holding member of the sample holding device. A two-dimensional detector disposed on the front surface, and a reflecting mirror for guiding light on the opposite side of the sample placed on the sample holding member to the two-dimensional detector. And an optical system that images light in a plurality of directions of the sample on the dimension detector.

In the above-described biological image acquisition device, since it is configured to image light in a plurality of directions of the sample on one two-dimensional detector arranged in front of the sample holding member of the sample holding device, Observation images in a plurality of directions can be acquired simultaneously in a short time.
In such a multi-directional simultaneous observation type biological image acquisition apparatus, since the light on the back side of the sample held by the sample holding member is also guided to the two-dimensional detector, the back side of the sample needs to be visible. Therefore, it is necessary to make the sample holding member on which the biological sample is placed substantially transparent. The light on the back side of the sample (opposite to the two-dimensional detector) is guided to the two-dimensional detector by the optical system and imaged on the two-dimensional detector, but the light guided to the two-dimensional detector. If an opaque member is arranged on the optical path, there is a problem that the member is reflected in the image picked up by the two-dimensional detector, and a portion to be picked up cannot be picked up, or the image becomes confusing.

  Therefore, the support mechanism of the sample holding device is configured to support the sample holding member while securing a light passage region for allowing light guided to the two-dimensional detector through a region different from the sample holding region. ing. “The support mechanism secures a light passage region” means that the support mechanism is provided so as to avoid the optical path of light guided to the two-dimensional detector. Therefore, since the light guided to the two-dimensional detector is not blocked by the support mechanism, the support mechanism does not appear in the image formed on the two-dimensional detector.

  In the claims and specification of the present application, “the sample holding member is substantially transparent” means that the sample can be optically measured through the sample holding member, and the sample holding member is completely transparent. It is not limited to the case. Examples of such a substantially transparent sample holding member include a transparent plate, a transparent film, or a net-like member that is so fine as to be negligible with respect to the size of the measurement site of the sample. An example of the transparent plate is a glass plate, an example of the transparent film is a transparent resin film, and an example of the mesh member is a mesh made of fibers or a wire mesh made of fine wire. Here, the net shape includes a lattice shape, a honeycomb shape, or a shape in which lines are arranged in parallel in the same direction.

Here, as a required property about the material of the sample holding member,
(1) having transparency for multi-directional observation;
(2) Do not emit fluorescence,
(3) The sample holding member is inexpensive because it is desired to replace many sample holding members.
Is mentioned. In general, inexpensive plastics and glass materials tend to emit fluorescence. In order to use these as transparent sample supports, it is necessary to increase the thickness in order to prevent bending, but the fluorescence increases in proportion to the thickness. In order to reduce fluorescence, the thickness may be reduced, but the mechanical support is insufficient.

  Therefore, for example, a thin material or a net-like material can be used as the sample holding member by surrounding the periphery of the sample holding member with a frame-shaped support mechanism and reinforcing the sample holding member. By making the sample holding member a thin material or a net-like structure, the possibility of emission of fluorescence can be reduced and substantial transparency can be maintained.

  The light passage region may be a region in the region where the sample holding member is disposed. Since the sample holding member is substantially transparent, there is no problem in measurement even if the light guided to the two-dimensional detector passes through the region where the sample holding member is disposed. Therefore, the sample holding member can be arranged in a wider area than the sample holding area that covers the light passage area. When placing a biological sample in the sample holding area on the sample holding member, the small animal that is the biological sample is pre-anesthetized so that it does not move, but there is nothing in the light passage area on the side of the sample holding area. There is a risk that the small animal may cramp and fall from the sample holding member. This problem can be solved by disposing a substantially transparent member also in the light passage region on the side of the sample holding region. In this case, by disposing the sample holding member in a wide area so as to cover the light passage region, it is possible to eliminate the gap between the sample holding member and the light passage region and prevent the sample from falling from the gap.

  The embodiment is shown in FIG. In this embodiment, a common member having a common substantially transparent joint is disposed in a region including the sample holding region and the light passage region. In that case, the portion on which the sample is placed can be regarded as the sample holding region, and the surrounding portion can be regarded as the light passage region. In FIG. 5A, a transparent plate is used as the sample holding member, and in FIG. 5B, a net is used as the sample holding member.

  The simplest sample holding member is to place a sample on the upper surface. As such a sample holding member, a glass plate, a wire net, or the like can be used. However, such a simple plate-shaped member may fall from the sample holding member when a small animal causes convulsions. Therefore, a sample holding member that is curved so that the upper surface side is concave may be used. If it does so, even if a small animal raises a cramp, it will become difficult to fall from a sample holding member. Examples of such a sample holding member include a curved glass plate, a loose transparent seal, and a mesh member.

  In addition, even when the sample is loosely anesthetized or when the sample is awakened, it is substantially transparent to clamp the sample with the sample holding member in order to actively prevent the sample from dropping or moving. A sample fixing member can also be provided. By sandwiching the sample between the sample holding member and the sample fixing member, the movement of the sample can be sealed. When the sample holding member is a non-flexible plate member such as a glass plate, the sample fixing member must be held in contact with the sample without being deformed. It is desirable that Conversely, when the sample holding member is a flexible slack net or the like, the sample fixing member may be a flexible member or a non-flexible plate member such as a glass plate. It may be.

The sample holding member may be a container that stores a sample therein. By storing and holding the sample in the container, it is possible to reliably prevent the sample from falling and to supply the anesthetic gas continuously into the container and image the biological sample in an anesthesia state for a long time. Can also be performed.
In that case, the shape of the container is preferably a cylindrical shape having no end face, such as a cylindrical shape. When the observation direction from the side of the biological sample is included, if there is an end surface on the sample holding member, light may be reflected on the end surface of the sample holding member, which may be reflected in the observation image. If the shape of the container is cylindrical and imaging is performed from a plurality of observation directions along the circumferential direction, the sample holding member does not have an end surface, and the above problem is solved.

It is preferable that the sample holding member is detachably supported with respect to the support mechanism. Then, when there are a plurality of biological samples to be measured, each biological sample is placed on the sample holding member, and an image of each biological sample can be taken efficiently while exchanging the sample holding member.
Further, the support mechanism may be detachable from the biological image acquisition apparatus.

  By the way, in the sample holding device of the present invention, the support mechanism may include a reflecting mirror for guiding light from the opposite side of the sample held by the sample holding member to the two-dimensional detector. . By doing so, a multi-directional simultaneous observation type biological image acquisition device can be obtained simply by arranging the sample holding device in a device that includes a two-dimensional detector and an imaging lens and performs observation in only one direction. it can.

  In the above case, it is preferable that the support mechanism further includes an auxiliary lens on each optical path for correcting the optical path length difference between the direct light from the sample and the light from the reflecting mirror. Then, the light of the image from each observation direction can be focused, and the blur of the image due to the optical path length difference of the light from each observation direction can be eliminated.

  By the way, although the biological image acquisition apparatus of this invention is equipped with the sample holding device of this invention, the biological image acquisition apparatus is provided in the circumference | surroundings of a sample holding device, and takes the fluorescence image from a sample. An excitation light source that emits excitation light to excite the sample to generate fluorescence and an optical path of the light that is guided to the two-dimensional detector when taking a fluorescent image from the sample are arranged to block the excitation light. A fluorescence side filter for guiding fluorescence from the sample to the two-dimensional detector may be further provided.

  In addition, the biological image acquisition apparatus may further include a guide that allows the sample holding device to move to different positions while supporting the sample holding device. Parts such as a reflecting mirror are arranged around the sample holding device. In such a state, it is difficult to mount the sample on the sample holding member of the sample holding device, and when the sample is placed, the hand may contact the reflecting mirror and the optical path of the light from the reflecting mirror may be shifted. sell. If the sample holder is configured so that it can be moved to a different position, the sample holder can be moved to an environment where there are no reflectors or other parts around it, and the sample is mounted on the sample holder. Becomes easier.

  Moreover, it is preferable that the biological image acquisition apparatus includes an auxiliary lens on each optical path for correcting the optical path length difference of light from each direction guided to the two-dimensional detector by the optical system. By doing so, it is possible to focus the light from each observation direction, and it is possible to eliminate blurring of the image due to the optical path length difference of the light from each observation direction.

Since the biological image acquisition apparatus of the present invention is configured to image light in a plurality of directions of a sample on one two-dimensional detector disposed in front of the sample holding member of the sample holding apparatus, It is possible to simultaneously acquire observation images in a plurality of directions in a short time.
In the sample holding device of the present invention used in such a multi-directional simultaneous observation type biological image acquisition device, the sample holding member is substantially transparent, and the support mechanism for supporting the sample holding member is the sample holding member. Since it is provided so as to secure a light passage area for passing the light guided to the two-dimensional detector through the side of the sample holding area, the sample holding member and the support are added to the image captured by the two-dimensional detector. The mechanism can be prevented from being reflected.

FIG. 1 shows an embodiment of a multi-directional simultaneous observation type biological image acquisition apparatus.
The living body image acquiring apparatus of this embodiment uses a plurality of reflecting mirrors M2 to M5 arranged around the multidirectional light of the sample 4 placed on the sample holding member 2 as a common imaging lens. By guiding the light to 8, a plurality of lights are imaged on the two-dimensional detector 6.

  A small animal (typically a mouse) 4 as a biological sample is placed in a sample holding region of a substantially transparent sample holding member 2 disposed in the center. An imaging unit including a two-dimensional detector (for example, a CCD imaging device) 6 and an imaging lens 8 is disposed above the sample 4 on the sample holding member 2. In the figure, the fluorescence side filter 10 that shields the excitation light component and transmits the fluorescence component is shown, but the fluorescence side filter 10 is placed on the incident side of the imaging lens 8 when measuring the fluorescence from the sample 4. It is arranged and is not arranged in cases other than fluorescence observation.

  Reflecting mirrors M <b> 2 to M <b> 5 that guide light (images) in the respective observation directions of the sample 4 to the imaging lens 8 are arranged at different positions around the sample holding member 2. The reflecting mirrors M2 to M5 reflect the light of the biological sample in every 72 ° counterclockwise with the direction of the two-dimensional detector 6 as 0 ° with respect to the sample 4 and guide it to the imaging lens 8. is there. That is, the reflecting mirror M2 guides the light of the sample 4 in the direction of 72 °, the reflecting mirror M3 to 144 °, the reflecting mirror M4 to 216 °, and the reflecting mirror M5 to the imaging lens 8 in the direction of 288 °.

  The light in each direction guided to the imaging lens 8 is imaged on the two-dimensional detector 6, and an image as shown in FIG. In the image of FIG. 2, the images are arranged in order of 72 °, 144 °, 0 °, 216 °, and 288 ° from the right end. The central 0 degree image is close to the imaging lens 8 because it does not go through the reflecting mirror, and becomes a large image. The remaining four images that pass through the reflecting mirrors M2 to M5 are smaller in size than the image in the 0 ° direction and the left and right are reversed. Further, since the light guided to the imaging lens 8 via the reflecting mirrors M2 to M5 has different optical path lengths, blurring occurs on the two-dimensional detector 6. This problem is slightly caused by focal length on each optical path. This can be solved by inserting mosaic lenses (auxiliary lenses) L1 to L5 for different fields of view.

  For example, a glass plate can be used as the sample holding member 2. In that case, if the thickness of the glass plate is increased in order to maintain the mechanical strength of the sample holding member 2, there is a disadvantage that light is reflected by the thickness of the glass plate during observation from the side. Such inconvenience can be avoided if the thickness of the glass plate is 1 mm or less. However, the mechanical strength of the sample holding member 2 is insufficient.

  As shown in FIG. 3, the strength of the sample holding member 2 can be reinforced by supporting the sample holding member 2 with a frame-like support mechanism 12. However, if the entire periphery of the sample holding member 2 is simply reinforced with a thick support frame, the support frame blocks the observation field during observation from the side, which becomes an obstacle. Therefore, the support mechanism 12 supports only two portions of a pair of opposite ends of the sample holding member 2 (for example, the head side and the tail side of the sample 4 to be placed). For this reason, the support mechanism 12 is intentionally sized so as to allow a gap 14 between the end of the sample holding member 2 that is not supported, and the gap 14 is formed on the back side of the sample holding member 2. The light passing area for allowing the reflected light from the reflecting mirrors M3 and M4 to pass therethrough. The sample holding member 2 and the support mechanism 12 constitute a sample holding device of the present invention.

4A to 4F are perspective views showing examples of such a sample holding device.
In the example of FIG. 4A, a pair of end portions of a transparent plate 2 a made of, for example, a glass plate having a size capable of placing a sample as the sample holding member 2 is supported at the center portion of the support mechanism 12. A gap (light passage region) 14 is provided between the transparent plate 2 a on the side of the transparent plate 2 a and the support mechanism 12. The point here is that in order to prevent the light behind the sample placed on the transparent plate 2 a from being blocked by the support mechanism 12, a gap 14 is formed between the frame-shaped member as the support mechanism 12 and the sample holding member 2. The light of 144 ° and 216 ° from the back surface side reflected by the reflecting mirrors M3 and M4 is allowed to pass through.

  As the sample holding member 2, in addition to the transparent plate 2a shown in FIG. 4A, see a transparent member 2b such as a glass plate curved so as to be concave on the sample mounting side (see FIG. 4B). ) Or the net-like member 2e (see FIG. 4E) can also be used. Since the sample placement side is concave, it is possible to prevent the placed animal from falling due to convulsion or the like. In addition, a net-like member 2c such as a net or a wire net (see FIG. 4C) that is negligibly small with respect to the measurement site of the sample to be placed can also be used. That is, it is sufficient if the sample can be held at a predetermined position to be observed with a pin or loosely stretched on the support frame 12. This is because the net and the film can be supported using the mechanical strength of the support mechanism as long as the tensile strength is sufficient. By using such a member, it is possible to avoid surface reflection that inevitably occurs when a transparent plate such as a glass plate is used, and to reduce fluorescence.

  FIG. 4D shows an example in which a container that accommodates a sample is used as the sample holding member 2. As shown in this example, by using a cylindrical container 2d such as glass as the sample holding member 2, an anesthetic gas is continuously supplied into the cylinder 2d to hold the animal in an anesthesia state for a long time. Can do. Further, by using the cylindrical container 2d, it is possible to eliminate the inconvenience of end face reflection that occurs when a transparent plate such as a glass plate is used as the sample holding member 2. In addition, as shown in (F), the container 2f can be configured with a fine mesh as the sample holding member 2 that can prevent the sample from dropping.

  In addition, as a method for positively sealing the movement of the sample even when the sample is no longer anesthetized, a substantially transparent member (sample fixing member) 15 is provided as shown in FIG. There is also a method in which the sample 4 is sandwiched between (here, the transparent plate 2a). When the sample holding member 2 is a non-flexible member such as a glass plate, a flexible member such as a net is used as the sample fixing member 15, and conversely, the sample holding member 2 is a flexible member. In some cases, the sample fixing member 15 may be a member that is not a flexible member. By using such a sample fixing member 15, the sample can be captured without applying pressure to the sample. Note that it is important to sandwich the sample, and the sample fixing member and the sample holding member do not matter up, down, left, or right.

  The sample holding members 2a to 2f shown in FIGS. 4A to 4F are preferably detachably attached to the support mechanism 12. Then, a plurality of sample holding members 2 on which the sample 4 is mounted can be prepared, and the replacement of the sample 4 can be replaced together with the sample holding member 2, and the time required for replacing the sample 4 can be shortened.

  Note that the sample holding member 2 in the example shown in FIGS. 4A to 4F has only a sample holding region for holding the sample, and the light passage region 14 on the side of the sample holding region has Nothing is arranged. However, if the light passage region 14 is substantially transparent, light from the back surface side of the sample holding member 2 can pass therethrough, so that the sample holding region and the light passage region 14 are shown in FIG. A transparent plate 2g (see FIG. 5A) having a size covering both of the regions and a fine mesh member 2h (see FIG. 5B) can be disposed as a sample holding member. In this way, by disposing the substantially transparent member identical to the sample holding region also in the light passage region 14, it is possible to prevent the sample from falling, and the boundary between the sample holding member 2 and the other region. There is also an advantage that the end of the sample holding member 2 is not shown in the captured image.

  The support mechanism 12 is preferably movable as shown in FIG. In the example of FIG. 6, the support mechanism 12 is mounted on a guide 16 provided in the apparatus and is movable. Thereby, after mounting the small animal used as a sample in the state pulled out to the front to the sample holding member 2, the support mechanism 12 can be pushed into the original measurement position and set. Therefore, the sample can be easily mounted without being obstructed by components such as a reflecting mirror. In addition, in FIG. 6, although the transparent plate 2a is shown as the sample holding member 2, it is not limited to this.

  By the way, when performing fluorescence measurement of a sample, it is necessary to arrange an excitation light source for fluorescence. Particularly in a multi-directional simultaneous observation type biological image acquisition apparatus such as the present invention, a plurality of excitation light sources are arranged around a sample. The excitation light may be irradiated from multiple directions. FIG. 7 is a configuration diagram schematically showing an example in which a plurality of excitation light sources are arranged, and FIG. 8 is a perspective view showing an example of a specific structure of the support mechanism in the example of FIG. Since the configuration other than the support mechanism 12a for supporting the sample holding member 2 and the excitation light sources S1 to S5 in the configuration illustrated in FIG. 7 is the same as that in FIG. 3, detailed description thereof is omitted here.

  The fluorescence excitation light sources S1 to S5 are arranged so as to irradiate the sample 4 placed on the sample holding member 2 with excitation light through the gaps between the reflecting mirrors M2, M3, M4, and M5. The excitation light sources S1 and S5 irradiate the excitation light from above the sample 4, the excitation light sources S2 and S4 from the side of the sample 4, and the excitation light source S3 from the bottom of the sample 4. The support mechanism 12a that supports the sample holding member 2 with respect to the arrangement of the excitation light sources S1 to S5 is configured as a three-dimensional frame as shown in FIG.

  A gap (light passage region) 14 a for allowing the light reflected by the reflecting mirrors M 3 and M 4 below the sample holding member 2 to pass is provided on the side of the sample holding member 2. By making the support mechanism 12a in such a three-dimensional shape, the frame portion of the support mechanism 12a does not exist between the light sources S1 to S5, particularly the light sources S2 and S4 and the sample 4, and the support mechanism 12a is located on the side. It is possible to prevent the irradiation of excitation light from the direction.

Also in this case, since the sample holding member 2 is detachably attached to the support mechanism 12a, a plurality of samples 4 are prepared in a state where the sample 4 is placed on the sample holding member 2, and the following The whole sample holding member 2 can be replaced at the time of measurement, and the time required for replacing the sample 4 can be shortened.
In FIG. 8, as the sample holding member 2, various members 2a to 2f as shown in FIGS. 4A to 4F can be used.

  In the example of the sample holding device described above, the example in which the sample holding member 2 is supported by the frame-like support mechanism 12 has been shown. However, as shown in FIG. It may be supported by the support mechanism 12b inside the passage region 14. Further, as shown in FIG. 13, one end of the sample holding member 2j may be supported by the support mechanism 12c and the other end may be three-dimensionally supported by a tension member 13 such as a thread.

  In the above embodiment, the sample holding member 2 is provided separately from the support mechanism 12. However, the sample holding member 2 may be integrally formed with the support mechanism 12 using resin or glass. .

  As described above, by using the sample holding member 2 in combination with the support mechanism 12, the bending strength of the sample holding member 2 can be reinforced by the support mechanism 12, and the thickness of the sample holding member 2 can be reduced. Thereby, it is possible to reduce the fluorescence generated from the sample holding member 2 while solving the problem of reflection of the boundary. Further, if a net is used as the sample holding member 2 and a sufficiently thin metal or plastic is used as the material of the net, the light is substantially transparent by passing through the gap of the net, and the wire material is made thin by thinning the wire material. Fluorescence can be reduced regardless of plastic. Moreover, since the net is highly flexible, it can also be used as the sample fixing member 15 that sandwiches the sample with the sample holding member 2 and seals the movement of the sample. That is, the fine net is suitable as a material that transmits light and does not emit fluorescence while restraining the sample not to move.

  Note that, similarly to the example shown in FIG. 6, it is preferable that the support mechanism 12 a can be moved to a position different from the measurement position by providing a guide in the apparatus. In particular, in the biological image acquisition apparatus of this example, since the reflecting mirrors M2 to M5 and the fluorescence excitation light sources S1 to S5 are densely arranged, it is difficult to replace the sample 4 when the support mechanism 12a is arranged at the measurement position. . By making the support mechanism 12a movable and moving it to a position different from the measurement position, both the holding of the sample holding member 2 and the easy exchange of the sample 4 can be satisfied.

  The fluorescence-side filter 10 disposed on the incident side of the imaging lens 8 of such a biological image acquisition device blocks the excitation light component and transmits the fluorescence component emitted from the sample by the excitation light. The two-dimensional detector 6 detects only those that enter the transmission region of the fluorescent filter 10. Of the wavelength components included in the excitation light, the components that are scattered as they are without changing the wavelength become background light that interferes with the measurement, so the wavelength of the excitation light source and the fluorescence side filter should not be transmitted. Ten properties have been selected.

  In the above embodiment, it is described that the reflecting mirrors M2 to M5 for guiding the light of the sample in a plurality of directions to the two-dimensional detector 6 are provided inside the apparatus. It may be arranged so as to be detachable from the apparatus. An example is shown in FIG.

  The sample holding device 18 shown in FIGS. 9A and 9B constitutes a support mechanism for supporting the sample holding member 2 and light on the back side of the sample 4 placed on the sample holding member 2. Reflecting mirrors M6 and M7 for holding the reflected light and guiding it to the two-dimensional detector disposed above the sample holding member 2 are held.

  In the example of (A), a transparent plate such as a glass plate is used as the sample holding member 2 by the same method as in FIG. 4 (A), and a pair of opposite ends are supported by a frame-like member. This is an example. A light passage region for allowing the light reflected by the reflecting mirrors M6 and M7 to pass through is provided as a gap on the side of the sample holding member 2. In addition to the glass plate, the sample placement unit 2 may be a substantially transparent member such as a fine mesh member or a transparent sheet, and may be a transparent cylindrical container.

  In the example of (B), a substantially transparent member constituting the sample holding member 2 is arranged in the light passage region through which the light reflected by the reflecting mirrors M6 and M7 passes, as in the case shown in FIG. Is. In this example, a transparent plate such as a glass plate is used as the sample holding member 2.

  Next, how to use the sample holding device 18 shown in FIG. 9 will be described with reference to FIG. In the same figure, (A) is a biological image acquisition device of a publicly known form which acquires an observation image of a single direction. Here, an example is shown in which three samples 4a, 4b, and 4c are arranged side by side on the sample holder 20 and photographed with the two-dimensional detector 22 and the imaging lens 24 arranged above them. Any number is acceptable. (B) shows a state in which the sample holder 20 of (A) is replaced with the sample holder 18.

  In the configuration of FIG. 10 (A), the front images of the three samples 4a, 4b, and 4c can be acquired at a time, but only the observation image from a single direction can be obtained. In order to acquire, it is necessary to turn each sample 4a, 4b, 4c upside down and perform imaging again. On the other hand, according to the configuration of (B), the light in the front of the sample 4 and the light in the two directions on the back side through the reflecting mirrors M6 and M7 are guided to the two-dimensional detector 22, so that the observation in the three directions is performed simultaneously. Images can be acquired. In addition, when the difference in the focal position between the light on the front surface of the sample 4 and the light on the back surface through the reflecting mirrors M6 and M7 caused by the difference in the optical path length becomes a problem, as shown in FIG. In addition, the auxiliary lenses L1 ′, L6, and L7 for correcting the optical path lengths are also made detachable to solve the problem. When the fluorescence / emission intensity from the sample 4 is strong, the auxiliary lenses L1 ′, L6, and L7 can be omitted by narrowing the imaging lens 24 (increasing the aperture value) to increase the depth of focus. It is.

  In this way, by using the sample holding device 18 including the reflecting mirrors M6 and M7 that reflect the light on the back side of the sample on the sample holding member 2, a known unidirectional observation type biological image acquisition device can be made multi-directional. It can be set as a simultaneous observation type biological image acquisition device. In particular, in the embodiment in which the observation images in the three directions shown in FIGS. 9 and 10 are simultaneously acquired, information on the back side of the sample 4 can be obtained simply by attaching the two reflecting mirrors M6 and M7 to the sample holding device 18. Can be said to be simple and useful. By preparing such a sample holding device 18, it is possible to easily switch between two different measurement methods (unidirectional observation type and multidirectional simultaneous observation type).

It is a schematic block diagram which shows one Example of the multi-directional simultaneous observation type biological image acquisition apparatus. It is a figure which shows an example of the image acquired with the biometric image acquisition apparatus of the Example. It is a figure which shows the relationship between the support mechanism which supports a sample holding member, and the optical path of the light guide | induced to a two-dimensional detector. It is a perspective view which shows a support mechanism with a sample holding member, (A) is an example using a glass plate as a sample holding member, (B) is an example using a transparent plate with a curvature as a sample holding member, (C) is An example using a mesh member as a sample holding member, (D) an example using a cylindrical container as a sample holding member, (E) an example using a mesh member with curvature as a sample holding member, and (F) a sample holding Each example uses a cylindrical container made of a net as a member. It is a perspective view which shows a support mechanism with a sample holding member, (A) is the example which has arrange | positioned the transparent plate as a sample holding member to a light passage area, (B) is a net-like member as a sample holding member in a light passage area. Each example is shown in FIG. It is a perspective view which shows the structure which enables a support mechanism to move. It is a schematic block diagram which shows an example of a structure at the time of providing the excitation light source for irradiating a sample with excitation light. It is a perspective view which shows the other example of a support mechanism with a sample holding member. It is a figure which shows the example of the sample holding apparatus provided with the reflective mirror, (A) supports a sample holding member in the state which provided the clearance gap in the area | region which passes the reflected light from a reflective mirror between sample holding members. (B) is a sectional view showing an example in which a transparent plate as a sample holding member is arranged up to a region through which reflected light from a reflecting mirror is passed. It is a figure for demonstrating the usage of the sample holding | maintenance apparatus provided with the reflecting mirror, (A) replaces with the sample holding | maintenance part of the example of a well-known biological image acquisition apparatus, (B) is the biological image acquisition apparatus of (A). It is a schematic block diagram which shows the example which attached the sample holding device provided with the reflecting mirror. It is a figure which shows the example which provided the sample fixing member on the sample holding member, (A) is the state which has not mounted the sample on the sample holding member, (B) has mounted the sample on the sample holding member It is a perspective view which shows the state fixed by the sample fixing member. It is a figure which shows the modification of a support mechanism. It is a figure which shows the further modification of a support mechanism, (A) is a top view, (B) is a side view.

Explanation of symbols

2, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, 20 Sample holding member 4, 4a, 4b, 4c Biological sample 6, 22 Two-dimensional detector 8, 24 Imaging lens 10 Fluorescence Side filter 12, 12a, 12b, 12c Support mechanism 14, 14a Light passage region 16 Guide 18 Sample holder L1, L1 ′, L2, L3, L4, L5, L6, L7 Auxiliary lens M2, M3, M4, M5, M6 , M7 reflector S1, S2, S3, S4, S5 Excitation light source

Claims (17)

In a sample holding device of a biological image acquisition device that acquires images from a plurality of directions of a biological sample using a two-dimensional detector,
A substantially transparent sample holding member with a sample holding region for holding the sample;
The specimen holding member is disposed in the living body image acquisition device while securing the light passage area for allowing the light guided to the two-dimensional detector to pass through the side of the specimen holding area. And a support mechanism.   The sample holding device according to claim 1, wherein the sample holding member is formed of a transparent plate, a transparent film, or a net-like member that is so fine as to be negligible with respect to the size of the measurement site of the sample.   The sample holding device according to claim 1, wherein the light passage region is a region in a region where the sample holding member is disposed.   The sample holding device according to claim 1, wherein the sample holding member is made of the same material as the support mechanism.   The sample holding device according to any one of claims 1 to 4, wherein the sample holding member mounts a sample on an upper surface.   The sample holding device according to claim 5, wherein the sample holding member is curved so that the upper surface side is concave.   The sample holding device according to any one of claims 1 to 6, further comprising a substantially transparent sample fixing member for fixing the sample between the sample holding member and the sample holding member.   The sample holding device according to any one of claims 1 to 4, wherein the sample holding member is a container that contains a sample therein.   The sample holding device according to claim 8, wherein the container has a cylindrical shape.   The sample holding device according to any one of claims 1 to 9, wherein the sample holding member is detachably supported with respect to the support mechanism.   The sample holding device according to any one of claims 1 to 10, wherein the support mechanism is detachable from the biological image acquisition device.   The said support mechanism is equipped with the reflective mirror for guide | inducing the light from the opposite side to the said two-dimensional detector of the sample hold | maintained at the said sample holding member to the said two-dimensional detector. The sample holding device according to claim 1.   The said support mechanism equips each optical path with the auxiliary | assistant lens for correct | amending the optical path length difference of the direct light from the said sample, and the light from the said reflecting mirror. Sample holding device. A sample holding device according to claim 1;
One two-dimensional detector arranged in front of the sample holding member of the sample holding device to image the sample placed on the sample holding member of the sample holding device;
A plurality of samples on the two-dimensional detector, including a reflecting mirror for guiding at least the light opposite to the two-dimensional detector of the sample placed on the sample holding member to the two-dimensional detector; And a biological image acquisition apparatus. An excitation light source that is provided around the sample holding device and emits excitation light for generating fluorescence by exciting the sample when capturing a fluorescent image from the sample;
Fluorescence side filter disposed on the optical path of light guided to the two-dimensional detector when taking a fluorescent image from the sample, and shielding the excitation light to guide the fluorescence from the sample to the two-dimensional detector The biological image acquisition apparatus according to claim 14, further comprising:   The biological image acquisition apparatus according to claim 14, further comprising a guide that allows the sample holding device to move to a different position while supporting the sample holding device.   The living body according to any one of claims 14 to 16, further comprising an auxiliary lens on each optical path for correcting a difference in optical path length of light from each direction guided to the two-dimensional detector by the optical system. Image acquisition device.

Images