JP2004325461A - Specific region detecting method and physiologic measurement method of non-human derived bio-specimen, specific region detector on bio-specimen, and optical fiber holder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device and a method for accurately and easily finding out a specific region of which an activity record is to be kept when in-vivo recording an activity of a bio-specimen. <P>SOLUTION: The bio-specimen 3 with a fluorescent material revealed in the specific region 3a is prepared, optical fiber 7 is stuck into the bio-specimen 3, excitation light 4 is irradiated through the optical fiber 7 to the interior of the bio-specimen 3 by means of a first optical system 9 of a fluorescence microscope 6, and fluorescence 5 excited by the excitation light 4 and emitted is observed through the optical fiber 7 by means of a second optical system 10 of the microscope 6. When the optical fiber 7 is stuck into the specific region 3a or its vicinity, the fluorescence from the specific region 3a is guided through the optical fiber 7 to the microscope 6 and enters an ocular lens 16 of the optical system 10. Only by looking through the ocular lens 16 to check whether the fluorescence 5 is observable, it is determined whether a region then stuck with the optical fiber 7 is the specific region 3a. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a technique for observing and recording the activity of a tissue of a biological sample in vivo, that is, in a living state by a physiological method. The present invention relates to a technique for enabling detection.

  Conventionally, a probe such as a tungsten electrode is inserted into a specific portion of a biological sample as a method of recording changes in the activity of a biological tissue due to external stimuli or the like, that is, changes in a physiological action in vivo, using the living body as an individual sample. An electrophysiological method for observing action potentials of a group of nerve cells in the vicinity thereof is known.

However, when recording the activity of living tissue in vivo, it is only possible to roughly specify the site where the nerve cell group to be observed is present. Therefore, after inserting the probe into the biological sample and recording the activity, a sample section is created. However, a complicated operation such as collating the obtained recording result with the site of the sample section is required. For example, when a mouse is used as a sample and the action potential of a group of nerve cells at a specific site in the brain is recorded in vivo, the site to be measured is roughly specified based on empirically obtained region information of the mouse brain. Then, after inserting an electrode into the site and recording the action potential of the nerve cell group, the brain is taken out, a slice is created, and the obtained recorded result is compared with the site of the brain slice. .
As described above, conventionally, when recording the activity of a specific portion of a biological sample in vivo, it was only possible to roughly specify that portion, so a section of the biological sample was created after recording the activity, and the recorded result was compared with the observed portion. Had to be collated. In addition, since a record of a part to be observed is not always obtained, it is necessary to repeat the above operation until a satisfactory recording result is obtained.
Accordingly, an object of the present invention is to provide a specific site detection device, a specific site detection method, and an activity record that can accurately and easily find a specific site for which an activity record is taken when recording the activity of a biological sample in vivo. It is an object of the present invention to provide a physiological measurement method or the like that can accurately and easily find a specific site for measuring the activity of a biological sample.

In order to solve the above-mentioned problem, the method for detecting a specific site according to the first aspect of the present invention is to express a fluorescent substance (e.g., Green Fluorescent Protein (GFP)) at a specific site of a non-human biological sample. An optical fiber is inserted into the non-human biological sample, and the excitation light emitted from the fluorescence light is emitted while irradiating the inside of the non-human biological sample with the excitation light from the fluorescence microscope through the optical fiber. Is observed by the fluorescence microscope through the optical fiber to detect the specific portion.
Further, the physiological measurement method according to the invention according to claim 2 is a method for expressing a fluorescent substance in a specific portion of a non-human-derived biological sample, inserting an optical fiber into the non-human-derived biological sample, and using a fluorescence microscope. While irradiating the excitation light of the non-human-derived biological sample through the optical fiber, the specific site is detected by observing the fluorescence excited and emitted by the excitation light through the optical fiber with the fluorescence microscope. A probe is inserted into the specific site or its vicinity, and the activity of the specific site or its vicinity is measured via the probe.
The specific site detecting apparatus according to the third aspect of the present invention is configured to irradiate a biological sample with excitation light through an objective lens and to capture fluorescence emitted and excited by the excitation light with the objective lens. A microscope, an optical fiber having one end inserted into a biological sample, and an optical fiber holding device for holding the other end of the optical fiber facing the objective lens are provided.
The specific site detecting device according to the invention of claim 4 is characterized in that the optical fiber holding device is detachably fixed to the stage of the fluorescence microscope. Here, the stage is a stage which is located below the objective lens of the fluorescence microscope, and on which a sample or the like is placed and which moves horizontally or further up and down.
This specific site detection device can be used as the specific site detection device according to claim 3 if an optical fiber holding device is fixed to a stage of the fluorescence microscope and the optical fiber is held by the optical fiber holding device. If the optical fiber holding device is removed from the device, it can be used as a normal fluorescence microscope.

Further, in the specific site detecting device according to the invention of claim 5, the optical fiber holding device includes a flat pedestal detachably fixed to a stage of the fluorescence microscope, and an end of the optical fiber attached to the stage. And a gripping device fixed to the pedestal so as to be held vertically with respect to the pedestal.
The specific site detection device can be used as the specific site detection device according to claim 3 by fixing a pedestal of an optical fiber holding device to a stage of the fluorescence microscope and holding the optical fiber by a holding device. If the optical fiber holding device is removed from the stage, it can be used as a normal fluorescence microscope.
An optical fiber holding device according to the invention according to claim 6, further comprising: a plate-shaped pedestal detachably fixed to a stage of the fluorescence microscope; and the pedestal for holding an end of the optical fiber perpendicular to the stage. And a gripping device fixed to the device.
According to this optical fiber holding device, if the pedestal is fixed to the stage of the existing fluorescent microscope and the optical fiber is held by the holding device, it can be used as the specific site detecting device according to claim 5, so that the existing fluorescent microscope can be used. By using the method, a specific site of a biological sample can be easily found.
In the fifth and sixth aspects, the pedestal is provided with a sample provided on a stage such as a glass plate used for a slide glass or a slide, a metal plate or a synthetic resin plate having the same dimensions as the slide or the like. It is desirable to use one having a size and shape that can be fixed by a presser or the like.

The present invention has the following excellent effects.
According to the specific site detection method of claim 1, when recording the activity of a non-human biological sample in vivo, an operation of inserting an optical fiber into the non-human biological sample and observing fluorescence through the optical fiber is performed. Can be efficiently performed using a fluorescence microscope, so that it is possible to easily find a specific site of a biological sample derived from a non-human whose activity is to be recorded.
According to the physiological measurement method of the second aspect, when recording the activity of a non-human-derived biological sample in vivo, an optical fiber is inserted into the non-human-derived biological sample, and fluorescence is observed through the optical fiber. Work can be performed efficiently using existing fluorescence microscopes, and the specific site can be easily found.Therefore, by inserting a probe into the specific site or in the vicinity thereof and measuring the non-human source, The activity recording operation can be easily performed in vivo on a biological sample.
According to the specific site detecting device of the third aspect, when recording the activity of a biological sample in vivo, an operation of inserting an optical fiber into the biological sample and observing fluorescence through the optical fiber uses a fluorescence microscope. As a result, it is possible to easily find a specific portion of the biological sample for which an activity record is to be taken.
According to the specific site detecting device of the fourth aspect, if the optical fiber holding device is fixed to the stage of the fluorescence microscope and the optical fiber is held on the optical fiber holding device, it can be used as the specific site detecting device of the third aspect, If the optical fiber holding device is removed from the stage of the fluorescence microscope, it can be used as a normal fluorescence microscope, so that it can be used in various ways according to the observation object and purpose.
According to the specific site detecting device of the fifth aspect, if the pedestal of the optical fiber holding device is fixed to the stage of the fluorescence microscope and the optical fiber is held by the holding device, it can be used as the specific site detecting device of the fourth aspect. If the optical fiber holding device is removed from the stage of the fluorescence microscope, it can be used as a normal fluorescence microscope, so that it can be used in various ways according to the observation object and purpose.
According to the optical fiber holding device of the sixth aspect, the pedestal of the optical fiber holding device is fixed to the existing stage of the fluorescence microscope, and the optical fiber is held by the gripping device. Since it can be used, a specific site of a biological sample can be easily found using an existing fluorescence microscope.

Hereinafter, the present invention will be described in detail based on embodiments.
FIG. 1 is a schematic configuration diagram showing an example of an embodiment of a specific site detection device according to the present invention. The specific site detecting apparatus 1 irradiates a biological sample 3 with excitation light 4 through an objective lens 2 having a high numerical aperture n (for example, n = 0.7), and emits fluorescence 5 excited and emitted by the excitation light 4 to the objective lens 2. A fluorescence microscope 6, which is configured to capture one end 7a into the biological sample 3, and a light which holds the other end surface 7b of the optical fiber 7 facing the objective lens 2. And a fiber holding device 8.
The fluorescence microscope 6 includes a first optical system 9 for irradiating the inside of the biological sample 3 with the excitation light 4 through the optical fiber 7 and a fluorescent light 5 excited and emitted by the excitation light 4 for observation through the optical fiber 7. And a second optical system 10.
The first optical system 9 includes an excitation light source 11, a relay lens 12 that collects and collimates the excitation light 4 emitted from the excitation light source 11, and reflects the excitation light 4 from the relay lens 12 to the objective lens 2 side. It has a dichroic mirror (dichroic mirror) 13 for guiding, and a relay lens 14 for relaying the excitation light 4 from the dichroic mirror 13 to make it enter almost the entire surface of the objective lens 2. The excitation light source 11 emits an excitation light 4 having a specific wavelength for exciting a fluorescent substance (such as GFP) specifically expressed in the biological sample 3 by coloring or genetic recombination. As the excitation light source 11, a lamp or a laser light source is used. The dichroic mirror 13 has a characteristic of reflecting only the excitation light 4 and transmitting other wavelength components.
The second optical system 10 includes a relay lens 14 for condensing and collimating light from the objective lens 2, a dichroic mirror 13 for transmitting light from the relay lens 14, and a biological sample among transmitted light from the dichroic mirror 13. A filter 15 that transmits only the fluorescent light 5 emitted from 3 and absorbs light in the other wavelength region, and transmits the transmitted light (fluorescent light 5) from the filter 15 in two directions: the eyepiece lens 16 side and the imaging device 17 side. And a half mirror 18 for branching. In this example, the transmitted light from the filter 15 is partially reflected by the half mirror 18 and enters the eyepiece 16. On the other hand, the light transmitted through the half mirror 18 is condensed by the relay lens 19 and forms an image on the light receiving surface 17a of the imaging device 17 such as a CCD.

The optical fiber holding device 8 is fixed on a stage 20 of the fluorescence microscope 6. As shown in FIG. 2A, the optical fiber holding device 8 includes a pedestal 21 formed of a rectangular flat plate, and a holding device 22 for holding an end of the optical fiber 7. For the pedestal 21, a metal plate or a synthetic resin plate having the same dimensions as a commercially available preparation is used. The gripping device 22 is fixed to the center of the upper surface of the pedestal 21 by a method such as bonding or screwing.
The gripping device 22 includes a main body 22a having a cubic structure made of metal or synthetic resin, and a gripping mechanism 23b provided on one side surface of the main body 22a. The gripping mechanism 23b is rotated in the longitudinal direction of the pedestal 21 (in the direction indicated by arrow A) with respect to the substrate member 24 via a hinge 25 with respect to the substrate member 24 fixed to the main body 22a by a method such as bonding or screwing. And a gripping plate 26 which is connected as possible.
As shown in FIGS. 2 (A) and 2 (B), on one side of the substrate member 24 of the gripping mechanism 23b, a support portion 27 having a concave portion 27a for receiving the optical fiber 7 is provided at both upper and lower ends thereof. (In the state of FIG. 2A, the lower supporting portion 27 is hidden by the holding plate 26). Then, with the end portion 7c of the optical fiber 7 (the end opposite to the side into which the biological sample 3 is inserted) 7c engaged with the concave portions 27a of the upper and lower support portions 27, the grip plate 26 is moved to the support portion 27 side. When the optical fiber 7 is rotated to the limit, the optical fiber 7 is sandwiched and fixed between the holding plate 26 and the substrate member 24. At this time, a lock mechanism (not shown) is operated, and the holding plate 26 is fixed while holding the optical fiber 7.

The optical fiber holding device 8 is mounted on the stage 20 with the pedestal 21 down, and a sample holder (generally, a holding member for holding a slide or the like from above, a screw for adjusting the holding force) provided on the stage 20 is provided. Is fixed on the stage 20 by fixing the pedestal 21. By fixing the optical fiber holding device 8 on the stage 20, the end 7c of the optical fiber 7 is held perpendicular to the stage 20. Therefore, at this time, the end face 7b of the optical fiber 7 is exposed upward.
The position of the stage 20 can be adjusted by operating a moving mechanism (not shown) in the front-rear and left-right directions (x-axis and y-axis directions) and up and down (z-axis direction). Therefore, the optical fiber holding device 8 is set on the stage 20, and after holding the optical fiber 7 on the stage 20, the stage 20 is moved to adjust the position, so that the end face 7 b of the optical fiber 7 is located immediately below the objective lens 2. And the optical axes of the two can be aligned. Then, in this state, by adjusting the height and the like of the stage 20, the focus of the objective lens 2 can be adjusted to the end face 7b of the optical fiber 7.
On the other hand, the end 7 a of the optical fiber 7 on the side inserted into the biological sample 3 is held by the optical fiber moving mechanism 28. The optical fiber moving mechanism 28 is extremely densely moved in the front-rear and left-right directions (x-axis and y-axis directions) and up and down (z-axis direction) while holding the end portion 7a of the optical fiber 7 in a vertically downward direction. In order to support the moving body 29 and the end 7a of the optical fiber 7 so that the end 7a of the optical fiber 7 does not bend when the end 7a of the optical fiber 7 is inserted into the biological sample 3, a metal ( (For example, stainless steel).

  According to the specific site detection device 1 configured as described above, the biological sample 3 in which the fluorescent substance is expressed in the specific site 3a for which the activity is recorded is prepared, and the optical fiber 7 is inserted into the biological sample 3, The first optical system 9 of the fluorescence microscope 6 irradiates the inside of the biological sample 3 with the excitation light 4 through the optical fiber 7, and the fluorescence 5 excited and emitted by the excitation light 4 is transmitted through the optical fiber 7 to the second of the fluorescence microscope 6. By observing with the optical system 10, the specific site 3a of the biological sample 3 can be easily found. That is, when the optical fiber 7 is inserted into the specific portion 3a of the biological sample 3 or in the vicinity thereof, the fluorescence from the specific portion 3a is guided to the fluorescence microscope 6 through the optical fiber 7 and the second optical system 10 Since the light enters the eyepiece 16, it is only necessary to look into the eyepiece 16 to see if the fluorescence 5 is observed, and to judge whether the part where the optical fiber 7 is inserted at that time is the specific part 3 a. be able to. Further, if the fluorescence 5 is received by the imaging device 17 and the data of the received light signal and the received light image are displayed on a monitor, even weak light can be amplified and detected, so that the specific portion 3a can be more reliably located. be able to. At this time, the insertion position of the optical fiber 7 is slightly changed by moving the end 7a of the optical fiber 7 on the side to be inserted into the biological sample 3 by the optical fiber moving mechanism 28. Therefore, even the minute specific portion 3a can be reliably found.

Further, according to the specific site detecting device 1, an operation of irradiating the inside of the biological sample 3 with the excitation light 4 through the optical fiber 7 and observing the fluorescence 5 excited and emitted by the excitation light 4 through the optical fiber 7. Can be performed using the fluorescence microscope 6, which is an experimental tool familiar to researchers in the field of physiology, so that the operation of finding the specific site 3a can be performed extremely efficiently.
As a result, it is possible to accurately and easily perform the action of measuring the action potential in vivo by inserting a probe or the like into the specific site 3a of the biological sample 3 or its vicinity.
Therefore, it eliminates the conventional complicated work of inserting a probe into a biological sample, recording the action potential, etc., into a biological sample, creating a sample section, and comparing the obtained recording result with the site of the sample section. Therefore, it is expected that in vivo studies of biological samples will be drastically promoted. For example, using a transgenic animal in which a specific region of brain tissue is labeled with GFP, for example, using a transgenic mouse in which GFP is expressed in specific brain nerve cells and recording the activity of the brain tissue in vivo, compared with the conventional method. Since it can be done so easily, it can greatly contribute to elucidation of brain function.

In addition, if the optical fiber holding device 8 is detached from the stage 20 of the fluorescence microscope 6, the specific site detection device 1 can be used as a normal fluorescence microscope, and can be used properly according to various observation targets and applications.
Further, since the optical fiber holding device 8 can be easily attached to an existing fluorescence microscope widely used in this type of research field, by attaching the optical fiber holding device 8 to an existing fluorescence microscope, The specific site detecting device 1 described above can be realized.
In the above-described embodiment, the device configuration including the eyepiece 16 and the imaging device 17 is illustrated. However, a configuration including only one of the eyepiece 16 and the imaging device 17 may be employed.
Further, the optical fiber moving mechanism 28 does not necessarily need to be provided, and the optical fiber 7 may be manually inserted into the biological sample 3.
Also, as shown in FIG. 3, for example, a probe 31 for detecting an action potential is arranged in parallel at the insertion end 7a of the optical fiber 7, and the probe 31 is inserted into the biological sample 3 at the same time as the optical fiber 7. You may make it. Further, as shown in FIG. 4, for example, the optical fiber 7 whose outer peripheral surface at the insertion side end 7 a is coated with the probe electrode layer 32 may be inserted into the biological sample 3. The probe 31 and the probe electrode layer 31 are connected to a recording system via a signal line 33. In the case of the configuration shown in FIG. 4, the detection of the specific portion 3a and the measurement of the action potential can be performed simultaneously and simultaneously, and the strength of the optical fiber 7 is reinforced by the probe electrode layer 32. Is prevented. Therefore, the detection of the specific site 3a and the measurement of the action potential can be performed extremely efficiently and accurately.

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic block diagram of the specific site | part detection apparatus which shows an example of Embodiment of this invention. 2A is an enlarged perspective view of the optical fiber holding device shown in FIG. 1, and FIG. 2B is an enlarged view of a main part of FIG. It is a principal part schematic block diagram of the specific site | part detection apparatus which shows another embodiment of this invention. It is a principal part schematic block diagram of the specific part detection apparatus which shows another embodiment of this invention.

Explanation of reference numerals

  Reference Signs List 1 specific site detection device, 2 objective lens, 3 biological sample, 4 excitation light, 5 fluorescence, 6 fluorescence microscope, 7 optical fiber, 7a end (insertion end), 7b end surface (other end surface), 7c end , 8 optical fiber holding device, 9 first optical system, 10 second optical system, 11 excitation light source, 12 relay lens, 13 dichroic mirror, 14 relay lens, 15 filter, 16 eyepiece, 17 imaging device, 17a light receiving surface, 18 Half mirror, 20 stage, 21 pedestal, 22 gripping device, 23b gripping mechanism, 25 hinge part, 26 gripping plate, 27 support, 28 optical fiber moving mechanism, 29 moving body, 30 support tube, 31 probe, 32 probe electrode layer.

Claims (6)

  A fluorescent substance is expressed in a specific portion of the non-human-derived biological sample, an optical fiber is inserted into the non-human-derived biological sample, and excitation light from a fluorescence microscope is passed through the optical fiber into the non-human-derived biological sample. A method for detecting a specific part of a non-human biological sample, wherein the specific part is detected by observing fluorescence emitted and excited by the excitation light through the optical fiber with the fluorescence microscope while irradiating the specimen. .   A fluorescent substance is expressed in a specific portion of the non-human-derived biological sample, an optical fiber is inserted into the non-human-derived biological sample, and excitation light from a fluorescence microscope is passed through the optical fiber into the non-human-derived biological sample. While irradiating the probe, the specific site is detected by observing the fluorescence excited and emitted by the excitation light through the optical fiber with the fluorescent microscope, and the probe is inserted into the specific site or its vicinity, and the probe is inserted. A physiological measurement method for a biological sample derived from a non-human, wherein the activity of the specific site or the vicinity thereof is measured via a computer.   A fluorescence microscope configured to capture fluorescence emitted and excited by the excitation light by the objective lens while irradiating the biological sample with the excitation light through the objective lens, and an optical fiber having one end inserted into the biological sample. An optical fiber holding device for holding the other end surface of the optical fiber so as to face the objective lens.   The device according to claim 3, wherein the optical fiber holding device is detachably fixed to a stage of the fluorescence microscope.   The optical fiber holding device is a plate-shaped pedestal detachably fixed to the stage of the fluorescence microscope, and a gripping device fixed to the pedestal to hold an end of the optical fiber perpendicular to the stage. 5. The device for detecting a specific portion of a biological sample according to claim 4, comprising:   A flat-shaped pedestal detachably fixed to the stage of the fluorescence microscope, and a gripping device fixed to the pedestal to hold the end of the optical fiber perpendicular to the stage. Optical fiber holding device.

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