JP2000329696A - Method and member for non-inasive observation of living body - Google Patents

Abstract

PROBLEM TO BE SOLVED: To observe a part of a living body which is subjected to a change in a living state by a method wherein a change is given to the physiological state of an animal, a part of the living body is irradiated with light, a region including an irradiated blood-vessel region is imaged, an imaged image is image-processed and a dynamic image is displayed. SOLUTION: A rat 2 is abdominally anesthetized by using an anesthetic (pentobarbital), and it is then put into a fixation device 1. A tail part 3 is taken out from a hole in the fixation device 1, and it is held softly from both sides by a fixation member 48a and a fixation member 48b which are installed on a stage 35. Light from a light source 23 is supplied to an observation device body part 41 by an optical fiber 24. The image of a very small circulation inside the tail part 3 is imaged by a CCD camera 40 which is installed at the observation device body part 41. Information on the imaged image is supplied to an analyzer 20, a video device 21 and a monitor 22. Adrenalin as a vasopressor is hydrodermically injected into a place at a proper distance from the tip of the tail part 3, the place is observed, and an image which is obtained in its observation is retained in the video device 21 so as to be analyzed or displayed on the monitor 22.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for observing a part of a living body after giving a physiological change by administering a drug to an animal or the like.

[0002]

2. Description of the Related Art As a method for confirming a change in a living body by administering a drug to an animal, there is known a method in which a rat is laparotomized, a mesentery is stretched on a stage, and observed with a biological microscope. In that case, surgery is required, which takes time and labor, and furthermore, the animals die after the observation, so that it is difficult to observe the same individual for a long time.

[0003] In the case of administering a drug that forms a thrombus to the tail of a rat, causing a thrombus to occur, and then administering a therapeutic agent for thrombosis and confirming its efficacy, a method of visually observing the necrosis of the tail is known. ing. However, that method required about two weeks. In addition, when preparing a pathological specimen by cutting the tail, it is not possible to observe the same individual animal over time, so there is a problem with reproducibility (modern medicine 18
(II): 1-11, 1986).

As a method for observing the behavior of leukocytes such as stagnation and adhesion to the blood vessel wall by injecting a fluorescent dye into the blood vessel of a rat, a fluorescence microscope or a confocal laser microscope is used to observe the mesentery of an experimental animal. Observing retinal vessels has been performed. In this case, surgery is necessary, and after observation,
There was a problem that animals died. Furthermore, the apparatus using a fluorescence microscope or a confocal laser microscope has a disadvantage that the apparatus becomes large.

Also, without collecting blood from a living body,
A non-invasive blood analyzer that non-invasively images a predetermined volume of blood in a blood vessel with good contrast has been proposed (JP-A-8-206101). However, there is no disclosure of a method of observing microcirculation after giving a physiological change to an animal, such as administering a drug using this device.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and provides a change in the physiological state of an animal, such as administration of a drug, while the animal is alive. Provides a way to observe.

[0007]

SUMMARY OF THE INVENTION The present invention comprises a step of changing a physiological state of an animal, a step of irradiating a part of the living body with light, and a step of imaging an area including an irradiated blood vessel area. An image processing step of performing image processing on a captured image;
Displaying a moving image of a region including a blood vessel region on a display means. Changing the physiological state of an animal means giving a stressful stimulus to the animal due to drug administration, light, sound, smell, or the like, or a stimulus to relax the animal.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In light irradiation, a continuous light source such as a laser or a halogen lamp that continuously emits light can be used as a light source. Further, in order to obtain a still image, an intermittent light source such as a pulse laser, a multi-strobe, or a Xe flash lamp which emits light intermittently can be used.

Furthermore, an optical shutter can be combined with the continuous light source and used as an intermittent light source. As an optical shutter, a known acousto-optic effect element (acoustic to op
ticmodulator or electro-to-optic element
modulator) can be used.

Further, in the light irradiation, a means comprising a plurality of light emitting elements arranged around the objective lens and light guiding means for guiding light emitted from the light emitting elements to a detection area can be used. In this case, the light-emitting elements include LEDs (light-emitting diodes) in that they are small and inexpensive.
It is preferable to use an LD (laser diode) or an SLD (super luminescent diode).

In the light irradiation, a plurality of light emitting elements having different emission wavelengths arranged around the objective lens, light guiding means for guiding light emitted from the light emitting element to a detection region, and light emitting elements having different wavelengths And a control means for selectively emitting light.

In this case, the light emitting elements having different emission wavelengths are, for example, a blue LED (peak emission wavelength: 450 nm), a green LED (peak emission wavelength: 560 nm), and a red LED in accordance with the light absorption characteristics of the object to be imaged. (Peak emission wavelength: 660 nm) or the like is preferably used.

In the image pickup of the present invention, a general CCD image pickup device for visible light, infrared light or ultraviolet light can be used. A CCD image pickup device having an electronic shutter function with a shutter speed of 10,000 / 1 second or more can also be used.

In the image processing, various filters, γ correction, jitter correction, color tone conversion, color balance correction, white balance, shading correction and the like are selectively performed in order to perform pre-processing of the image picked up by the image pickup means. It is preferable to provide analog and / or digital image processing means.

As the display means, an LCD, a CRT or the like can be used.

The dyes to be administered to animals are acridine orange, dye No. 33258
Etc. can be used. The dose of the dye is 0.5-2.0%
(W / v) is preferred. The step of administering the dye to the animal is preferably before or after the step of changing the physiological state of the animal.

In administering a drug to an animal, the drug may be administered from an external organ such as the mouth, nose and eyes of the animal, or may be administered intraperitoneally, subcutaneously,
It can be administered by injection into a vein or the like.

Further, in order to obtain a good image from the living body, it is preferable that a deformable translucent member is closely attached to the living body, and light irradiation and imaging are performed through the translucent member. As the deformable light-transmitting member, a member made of a resin bag in which immersion oil is sealed can be used. As a material of the resin bag, a refractive index of immersion oil of 1.51
General resin close to 5 (refractive index: 1.45 to 1.55)
Can be used. Since such a translucent member is freely deformable, it can be brought into close contact with a living body in a form conforming to the external shape of the living body. As a result, irregular reflection of light from the surface of the living body can be prevented, and even if hair is grown on the living body, it can be colorless and a clear image can be obtained. The optical system side of the light-transmitting member is preferably planarized by a light-transmitting flat plate such as a sheet glass.

[0019]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A first embodiment will be described with reference to a perspective view (FIG. 1) of an apparatus used in the present invention. The rat 2 is anesthetized with the anesthetic pentobarbital intraperitoneally. The tail 3 is taken out of the hole of the fixing device 1, and fixed members 48 a and 48 provided on the stage 35 are fixed.
b lightly holds from both sides.

Immersion oil is applied to the observation site of the tail 3, and a transparent plate 66 is placed. Since the tail 3 is only held lightly, a natural blood flow state can be observed. In addition, the immersion oil prevents irregular reflection of light from the surface of the living body, and even if the living body has hair, it is colorless and a clear image can be obtained.

The main body 41 of the observation device is movable in the vertical direction, and is fixed to a support bar 43 via a movable portion 44.
The movable section 44 is used for fixing the observation apparatus main body section 41 and for focusing a captured image. Light source 23
From the observation device main body 41 through the optical fiber 24.
Supplied to

An image of the microcirculation in the tail 3 is captured by the CCD camera 40 provided in the observation apparatus main body 41. The captured image information is analyzed by an analyzer 2 that performs various image processing.
0, a video 21 for storing the image, and a monitor 22 for visualizing the image in real time. Stage 35
By operating the X-direction actuator 46 and the Y-direction actuator 47 with the XY operation unit 25, the observation region can be moved with respect to the observation device main unit 41.

Adrenaline, a vasoconstrictor, is injected subcutaneously about 5 cm from the tip of the tail 3, and ergotamine tartrate, a blood coagulant, is injected into the back. The observation site was 10 to 13 cm from the tip of the tail. As soon as the subcutaneous injection is over, observation starts, and at the time of observation, 30 seconds or more per visual field,
Observe 5 or more visual fields. The image obtained during observation is video 2
Save with 1. Analyze saved data and monitor 2
2 can be displayed.

FIG. 2 is a block diagram of the first embodiment. FIG.
, The light emitted from the light source 23 is
4, the light is guided into the observation device main body 41 and irradiates the diffuser 26. The light is converted into parallel light by the collimating lens 30 diffused by the diffuser 26.
The central portion of the parallel light is shielded from light by a disk-shaped light shielding plate 67, and the peripheral portion of the parallel light is emitted from the distal end 59 of the observation device main body via the ring-shaped mirrors 34a and 34b. The zonal light emitted from the observation device main body tip 59 illuminates the detection region V in the tail 3 via the transparent plate 66. Light from the region V is transmitted through the transparent plate 66 and the objective lens 38b to the CCD.
The light is received at 40a. The image captured by the CCD 40a is analyzed by the analysis device 20. The analysis device 20 can analyze the image of the captured region and display a blood vessel image on the monitor 22.

FIGS. 3 (a), 3 (b) and 3 (c) show the tail 3 of a rat.
FIG. 4 is a schematic diagram of a captured image of microcirculation of a tail 3 after a thrombotic agent has been administered to a subject. (A) 90 seconds after administration, (b) 18
(C) is an image after 540 seconds after 0 seconds. It can be seen that the flow of the blood flow stops as the time elapses due to the effect of the thrombotic drug and the blood vessels shrink. In this way, it is easy to observe the microcirculation dynamics of blood while the animal is alive,
It becomes possible to obtain the observation result over time with the same individual.

Next, a second embodiment will be described. As in the first embodiment, the rat 2 is anesthetized and lightly held from both sides by fixing members 48a and 48b provided on the stage 35 (FIG. 1). Next, 1.0 vein in the vein of tail 3
Inject saline containing% (w / v) acridine orange. Immersion oil is applied to the observation site, and the transparent plate 66 is placed thereon, and the microcirculation dynamics of the blood in the tail 3 is imaged.

FIG. 5 is a block diagram of the second embodiment. FIG.
The excitation light cut filter 50 is added to the first embodiment of the configuration diagram shown in FIG. The light emitted from the light source 23 is guided into the observation device main body 41 and finally the transparent plate 66
Irradiates the detection region V in the tail 3 via the. Light from the region V is received by the CCD 40a via the transparent plate 66, the objective lens 38b, and the excitation light cut filter 50, and a fluorescent image of blood microcirculation dynamics is obtained.

In the fluorescence image, leukocytes specifically stained with acridine orange can be clearly distinguished from other blood cells and plasma, and the number of leukocytes per time can be counted. In addition, phenomena such as retention and adhesion of leukocytes to the blood vessel wall can be observed.

A third embodiment will be described. FIG. 4 shows a cross-sectional view when a deformable translucent member is arranged at the observation site. Oil 73 on the observation site of tail 3
Immersion oil (refractive index 1.
The objective lens unit 71 is approached via the transparent plate 66 such that the oil container 72, which is a deformable light-transmitting member containing 515), is in close contact. By performing observation in this manner, favorable optical conditions can be created even if the distance between the objective lens and the observation site is large. With this effect, the imaging performance is improved, and a clear image can be obtained. The oil container 72 can be made of a resin. In this embodiment, a polyolefin resin is used. The oil container can be easily deformed compared to the observation in which the observation site is pressed against a glass plate, so that the influence of the compression is very small, and the observation can be performed under conditions close to physiological conditions. Also, if this oil container is used, oil will not adhere to the optical device side,
This eliminates the need for wiping later and is convenient.

[0030]

According to the present invention, since a physiological change is caused by administering a drug to an animal and a moving image of a region including a blood vessel region is captured, the microcirculation can be performed while the animal is alive. Observation can be easily performed and observation results with good reproducibility can be obtained for the same individual over a long period of time. Further, in the present invention, by disposing a deformable translucent member at the observation site, the imaging performance is improved, and a clear image can be obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing a perspective view of a product of the present invention.

FIG. 2 is a diagram showing a configuration diagram of a product of the present invention.

FIG. 3 is a schematic diagram of a captured image captured by the product of the present invention.

FIG. 4 is a diagram showing a cross-sectional view of the product of the present invention.

FIG. 5 is a diagram showing a configuration diagram of a product of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Fixed device 2 Rat 3 Tail 20 Analysis part 21 Video 22 Monitor 23 Light source 24 Optical fiber 40 CCD camera 50 Excitation light cut filter 66 Transparent plate

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Yasunori Maekawa 1-5-1, Wakihama Kaigandori, Chuo-ku, Kobe F-term (reference) in Sysmex Corporation 2G043 AA03 BA16 CA03 DA02 EA01 FA03 GA07 GB01 GB21 HA01 HA05 HA09 HA11 JA03 KA02 KA08 KA09 LA01 LA03

Claims (5)

[Claims] 1. A step of changing the physiological state of an animal, a step of irradiating a part of the living body of the animal with light, a step of imaging a region including a blood vessel region irradiated with light, and a step of A noninvasive living body observation method, comprising: an image processing step of performing image processing; and a step of displaying a moving image of a region including a blood vessel region on a display unit. 2. The non-invasive living body observation method according to claim 1, further comprising the step of administering a dye for staining blood. 3. The non-invasive living body observation method according to claim 1, wherein the step of changing the physiological state of the animal is a step of administering a drug. 4. The non-transparent member according to claim 1, wherein a deformable translucent member is disposed in close contact with a part of a living body, and an image including a blood vessel region is imaged through the translucent member. Invasive living body observation method. 5. A non-invasive living body observation member, which is a deformable light-transmitting member closely attached to a part of a living body, wherein a fluid substance is sealed in a bag body.