JP2010017396A - Method and device for observing living body - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To easily measure the velocity of a moving object in vivo, especially the velocity of the blood flow in vivo. <P>SOLUTION: The method for observing the living body comprises an image acquisition step S2 for acquiring a plurality of images of the moving object in vivo at different times in the same visual field; an image composing step S4 for composing a projection image by superimposing the plurality of images acquired in the image acquisition step S2; a detection step S5 for detecting the moving object from the projection image composed in the image composing step S4; a measuring step S6 for measuring the distance between the moving objects detected in the detection step S5; and a computing step S7 for computing the velocity of the moving object based on the distance measured in the measuring step S6 and the time when the image is acquired. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a living body observation method and apparatus.

  In recent years, in research fields such as cell biology and molecular biology, green fluorescent protein (GFP) and luciferase gene, which is a bioluminescent enzyme, are used as expression reporters, and specific sites and functional proteins in cells are fluorescently labeled or luminescent. There is an increasing need to label and observe biological cells. In addition, a technique for performing in vitro measurement using light in an in vivo state of a living body such as a small animal is important for medical research and the like. In particular, for pharmacokinetic analysis and the like, it is an important technique to know the shape and speed of a moving object such as blood flow velocity measurement by image analysis.

  As an analysis method of this kind, the time change of the luminance value caused by the passage of red blood cells is measured at two locations of the blood vessel while irradiating the blood vessel intermittently using pulsed light or a shutter, and the obtained time and luminance A measuring device and a method for calculating a blood flow velocity from a correlation between two graphs of values are known (for example, see Patent Document 1).

JP-A-2-257931

  However, the apparatus and method of Patent Document 1 have a problem that the apparatus becomes complicated and expensive because it is necessary to acquire an image by interlocking pulsed light or a shutter with a camera.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a living body observation method and apparatus capable of easily measuring the speed of a moving object in a living body, particularly the speed of blood flow. .

In order to achieve the above object, the present invention provides the following means.
The present invention provides an image acquisition step of acquiring a plurality of images of a moving object in a living body at different times in the same visual field, and an image in which a plurality of images acquired in the image acquisition step are superimposed to synthesize a projection image A combining step; a detecting step for detecting the moving object from the projection image combined in the image combining step; a measuring step for measuring a distance between the moving objects detected in the detecting step; There is provided a living body observation method including a calculation step of calculating a speed of the moving object based on a measured distance and an acquisition time of the image.

  According to the present invention, an image of a moving object is acquired in the image acquisition step, and a projected image is generated by combining a plurality of images among the acquired images in the image combination step. Then, the moving object in the projection image is detected in the detecting step, and the distance between the detected moving objects is measured in the measuring step. When the speed is calculated in the calculation step from the measured distance and the interval between the acquisition times of the images used for the synthesis, the moving object in the living body can be observed and the speed can be measured.

  In this case, by synthesizing images with the same field of view and different acquisition times, the images of the moving objects in the images with different acquisition times are displayed in one projection image at the same position in each image. The That is, a projection image is obtained in which moving objects are arranged at intervals of acquisition time along a trajectory that has moved. Thereby, the moving distance of the moving object can be easily measured.

  The image for creating the projection image may be an optical image taken by a conventional method. Therefore, a microscope or a camera with a simple configuration can be used, and at the same time, the speed of the moving object in the living body can be measured in vivo.

In the above invention, a luminance value inversion step for inverting the luminance value of the image or the projection image may be provided before or after the image synthesis step.
By inverting the luminance value according to the luminance of the moving object, the moving object can be displayed more clearly and easily detected.

Moreover, in the said invention, you may provide the administration step which administers a labeled | labeling substance to the said biological body before the said image acquisition step.
In this way, the moving object or the observation area other than the moving object can be labeled and the moving object can be easily detected.

Further, in the above invention, the moving object is a blood cell flowing in a blood vessel of a living body, and before the image acquisition step, a collecting step for collecting blood from the living body, and a labeling substance on the blood collected in the collecting step And an addition step of adding a reducing step for returning the blood, to which the labeling substance has been added in the addition step, to the living body.
By doing so, blood cells in the blood vessel of the living body or an observation region other than blood cells can be labeled and blood cells can be easily detected.

In the above invention, the labeling substance may be a fluorescent dye.
In this way, a moving object or an observation region other than the moving object can be labeled with a fluorescent dye to obtain a fluorescent image, and the moving object can be detected more easily and accurately as a bright part or a dark part.

In the above invention, the labeling substance may be a labeling substance recognition unit that binds to a fluorescent dye and a specific substance existing in a moving object or a region outside the moving object by binding the fluorescent dye.
Moreover, in the said invention, the said to-be-labeled object recognition part may be an antibody.
In this way, a moving object or an observation area outside the moving object can be selectively labeled with a fluorescent dye to obtain a fluorescent image, and the moving object can be easily detected as a bright part or a dark part.

In the above invention, the labeling substance may be a fluorescent dye and a non-binding molecular compound to which the fluorescent dye is bound and does not bind to the substance in the living body.
In the above invention, the non-binding molecular compound may be a polysaccharide or a polypeptide.

  In this way, the fluorescent dye can be dispersed in the observation area outside the moving object, and the moving object can be easily detected as a dark part, and at the same time, the influence on the living body can be reduced by using a compound having high biocompatibility. Can be reduced.

  Further, the present invention synthesizes a projection image by superimposing a plurality of images acquired by the image acquisition unit and an image acquisition unit that acquires a plurality of images of moving objects in a living body at different times in the same field of view. An image synthesizing unit, a detecting unit for detecting the moving object in the projection image synthesized by the image synthesizing unit, a measuring unit for measuring a distance between the moving objects detected by the detecting unit, and the measurement There is provided a living body observation apparatus including a calculation unit that calculates a speed of the moving object based on a distance measured by the unit and an acquisition time of the image.

  According to the present invention, a plurality of moving object images are acquired in the image acquisition unit, and a projection image is generated in the image composition unit using a plurality of these images. Then, the moving object in the projection image is detected by the detection unit, and the distance between the moving objects is measured by the measurement unit. By calculating the speed of the moving object from the distance and the interval between the acquisition times of the images, the moving object can be observed and measured in the living body.

  In this way, the moving distance of the moving object can be easily measured from within the projected image. Further, since the image may be a conventional optical image, the acquisition and measurement of the image of the moving object can be performed in vivo using a microscope or camera with a simple configuration for acquiring the image of the moving object.

  According to the present invention, it is possible to easily measure the speed of a moving object in a living body, in particular, the speed of blood flow in the living body.

A living body observation apparatus 1 and a living body observation method according to a first embodiment of the present invention will be described below with reference to FIGS. In the present embodiment, the velocity v of red blood cells B flowing in the blood vessels of a living mouse A is measured, and the velocity of blood flow is analyzed based on the measured velocity v.
As shown in FIG. 1, the living body observation apparatus 1 according to the present embodiment includes a general upright optical microscope 2, a light source 3, an image acquisition unit 4, and a computer 5.

  The upright optical microscope 2 includes a stage 6 on which the mouse A is placed, an objective optical system 7, a dichroic mirror 8 disposed between the objective optical system 7, the image acquisition unit 4, and the light source 3, The second wavelength filters 9 and 10 are used.

The light from the light source 3 transmits only the light having a predetermined wavelength through the first wavelength filter 9, is reflected by the dichroic mirror 8, and irradiates the mouse A through the objective optical system 7.
Light from the mouse A is collected by the objective optical system 7 and transmitted through the dichroic mirror 8, and light having an unnecessary wavelength is cut by the second wavelength filter 10 and detected by the image acquisition unit 4.

The image acquisition unit 4 is, for example, a CCD camera.
The computer 5 is connected to the light source 3 and the CCD camera, and controls the conditions for acquiring the image of the CCD camera, displays the acquired image, and adjusts the light amount of the light source 3.
Further, the computer 5 combines the projected images, detects the red blood cells B in the projected images, measures the distance L between the red blood cells B, and calculates the velocity v.

A living body observation method using the living body observation apparatus 1 configured as described above will be described below.
As shown in FIG. 2, the living body observation method according to the present embodiment includes an administration step S11 for administering a fluorescent dye to mouse A, and an image acquisition step S2 for obtaining a fluorescent image by time-lapse observation of blood vessels of mouse A. A luminance value inversion step S3 for inverting the luminance value of the acquired fluorescent image, an image synthesis step S4 for synthesizing the projection image from the image with the luminance value inverted, and a detection step S5 for detecting red blood cells B from the projection image. And a measurement step S6 for measuring the distance L between the detected red blood B spheres, and a calculation step S7 for calculating the velocity v from the measured distance L and the acquisition time of the fluorescence image.

Although not shown, mouse A is anesthetized in advance.
In the administration step S11, for example, a near-infrared fluorescent dye is administered into the blood vessel of mouse A using a syringe, and the near-infrared fluorescent dye is circulated in the body.
In the image acquisition step S2, the mouse A is illuminated by the light source 3, and the blood vessels of the mouse A are observed in a time-lapse manner at predetermined time intervals. In the whole fluorescence image acquired by the CCD camera, the luminance value of each pixel is inverted in the luminance value inversion step S3, and the bright part is displayed as the dark part and the dark part is displayed as the bright part.

  In the image composition step S4, for example, as shown in FIGS. 3 (a) and 3 (c), two inverted images of luminance values acquired at times t1 and t2 are selected, and FIG. , (D) is synthesized. Specifically, the brightness values of each pixel in two images are compared, and one projection image is created using the lower or higher brightness value or the average of the two brightness values as the brightness value of that pixel. To do. In the present embodiment, the higher of the two luminance values is selected as the luminance value of the pixel.

Subsequently, when a round shape is detected as the red blood cell B from the projection image in the detection step S5, the distance L of the center of gravity position of the detected red blood cell B is measured in the measurement step S6.
In the calculation step S7, the velocity v = L / t of the red blood cell B is calculated from the distance L and the interval t = t2−t1 between the acquisition times of the images.
By performing the processing from the image synthesis step S4 to the calculation step S7 on the acquired all fluorescent images and statistically processing the calculated velocity, the blood flow velocity can be analyzed.

  In this embodiment, by doing so, the near-infrared fluorescent dye is dispersed in the plasma in the blood vessel to label the region other than the red blood cell B in the blood vessel, and the red blood cell B is observed as a dark part in the fluorescent image. can do. Then, by inverting the luminance value of the fluorescent image, the red blood cell B can be displayed as a bright portion, and the red blood cell B can be identified more easily.

  Further, when the projected images are synthesized, the higher luminance value is selected for each pixel in the two images whose luminance values are inverted. By doing so, a portion having a high luminance value on which red blood cells B are displayed is selected from each image, so that an image of red blood cells B is present at each position existing at times t1 and t2 in the projection image. The distance L displayed and the red blood cell B moved from time t1 to time t2 can be easily measured on one image.

  In addition, since a general fluorescent image is used, an upright optical microscope 2, a light source 3 and a CCD camera having a simple configuration can be used, and at the same time, an in vivo erythrocyte can be easily observed by observing a blood vessel of a living mouse A. The velocity v of B can be measured.

In this embodiment, when the image of red blood cells B in the projected image is unclear, red blood cells B are manually marked with dots, circles, ellipses, etc. on the image displayed on the computer 5 using a mouse device or the like. May be detected.
By doing in this way, the precision of detection of red blood cells B can be improved.

Moreover, in the said embodiment, although the near-infrared fluorescent dye was used as a labeling substance, it may replace with this and may use the polysaccharide or polypeptide with which the fluorescent dye was couple | bonded.
By using a compound present in the living body, the influence on mouse A can be reduced.

In the above embodiment, an antibody to which a fluorescent dye is bound may be used as a labeling substance.
In this way, biomolecules such as proteins present in plasma or on the surface of red blood cells B can be selectively labeled with a fluorescent dye.

In the above embodiment, the mouse is irradiated with the light from the light source via the objective optical system. Instead, as shown in FIG. 4, the mouse is guided with the light from the light source through a fiber. May be irradiated.
In this case, the living body observation apparatus 1 includes the fiber 11 in the light source 3 and does not require the dichroic mirror 8.

As for the light from the light source 3, only the light having a predetermined wavelength that has passed through the first wavelength filter 9 is guided through the fiber 11 and irradiated to the mouse A.
The light emitted from the mouse A is collected by the objective optical system 7, and light having an unnecessary wavelength is cut by the second wavelength filter 10 and detected by the CCD camera.

A second embodiment of the present invention will be described below with reference to FIGS.
In the description of the present embodiment, portions having the same configuration as those of the biological observation apparatus 1 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted. Further, as in the first embodiment, the red blood cells B in the blood vessels of a living mouse A are set as the measurement target.
As shown in FIG. 5, the biological observation apparatus 1 according to the second embodiment of the present invention includes a scanning optical microscope 12, an image acquisition unit 4, a computer 5, an analog processing unit 13, and a storage unit 14. And an input unit 15 and a display unit 16.

  The scanning optical microscope 12 includes a stage 6, a light source 3, an objective optical system 7, a two-dimensional scanner 17 disposed between the objective optical system 7 and the light source 3, a dichroic mirror 8, and a wavelength filter 10. And.

The light source 3 is a laser light source, and the laser light output from the laser light source is reflected by the dichroic mirror 8 and is shaken by a two-dimensional scanner 17 on a plane (XY plane) perpendicular to the optical axis direction. Mouse A is scanned through system 7.
Light emitted from the mouse A is condensed by the objective optical system 7, passes through the two-dimensional scanner 17 and the dichroic mirror 8, and light having an unnecessary wavelength is cut by the wavelength filter 10. Detected.

The image acquisition unit 4 is, for example, a light receiving element, and converts detected light information into an analog electric signal and outputs the analog electric signal.
The analog processing unit 13 includes an offset calculation unit 13a, an analog amplifier 13b, and an A / D converter 13c.

  The analog signal output from the light receiving element is offset by the offset calculation unit 13a, amplified by the analog amplifier 13b, converted into a digital signal by the A / D converter 13c, and then output to the computer 5.

The storage unit 14 is, for example, a storage medium such as a CD-ROM or a hard disk, and stores a processing program for processing an image. The processing program is called by the computer 5, and the computer 5 synthesizes the projection image, detects the red blood cells B, measures the distance L between the red blood cells B, and calculates the velocity v.
The input unit 15 is, for example, a mouse device connected to the computer 5, and the display unit 16 displays a light emission image detected by the light receiving element and imaged by the computer 5.

A living body observation method using the living body observation apparatus 1 configured as described above will be described below.
As shown in FIG. 6, the living body observation method according to this embodiment includes a collection step S12 for collecting blood from mouse A, an addition step S13 for adding a fluorescent dye to the collected blood, and a blood to which a fluorescent dye is added. Is returned to the body of the mouse A, and includes a reduction step S14, an image acquisition step S2, an image synthesis step S4, a detection step S5, a measurement step S6, and a calculation step S7.

  Fluorescent dyes that label red blood cells B, for example, DiI or DiO (both manufactured by Invitrogen) are added to blood collected from mouse A in collection step S12. In the reduction step S14, the labeled red blood cells B are returned to the body of the mouse A and circulated in the body of the mouse A.

  In the image acquisition step S2, laser light is output from the laser light source, the mouse A is scanned, time-lapse observation is performed at predetermined time intervals, and light emission from the red blood cells B is detected by the light receiving element. The detected light signal is processed by the analog processing unit 13 and sent to the computer 5. And the processing program memorize | stored in the memory | storage part 14 is read on the computer 5, and the process from image composition step S4 to calculation step S7 is performed.

  In the image composition step S4, for example, as shown in FIGS. 7A and 7C, the higher one of the luminance values of the pixels in the fluorescent image at the times t1 and t2 is selected, and FIG. , (D), a projection image is created. The detection step S5 to the calculation step S7 are performed in the same manner as in the first embodiment. By performing the image synthesis step S4 to the calculation step S7 for the acquired all fluorescence images and statistically processing the calculated velocity v, the blood flow velocity can be analyzed.

In this way, red blood cells B can be easily identified by labeling with fluorescent dyes. In the projected image, a fluorescent image of red blood cell B is displayed at each position at times t1 and t2 on the locus where red blood cell B has moved.
Accordingly, the distance L traveled by the red blood cells B can be easily measured, and at the same time, the speed of the red blood cells B moving in the body of the mouse A and the blood flow velocity can be measured in vivo using the scanning optical microscope and the light receiving element having the conventional configuration. Can be measured easily.

In the detection step S5, the red blood cells B on the image displayed on the display unit 16 may be detected by marking them with dots or circles using the input unit 15.
By doing in this way, the precision of detection of red blood cells B can be improved.

Moreover, in the said embodiment, although the light source and the light receiving element are each provided one each, you may provide two or more, respectively.
In this way, for example, erythrocytes B and other biomolecules can be labeled with a plurality of types of fluorescent dyes having different excitation wavelengths, and the velocity of both can be measured simultaneously.

In the first and second embodiments, the measurement is performed using the red blood cell B as a moving object, but the present invention is not limited to the red blood cell B, and can be applied to other moving objects.
In the first and second embodiments, an upright optical microscope is used, but it can also be applied to an inverted optical microscope and a general biological observation apparatus.

1 is an overall view showing a living body observation apparatus according to a first embodiment of the present invention. It is a flowchart which shows the biological observation method which concerns on the 1st Embodiment of this invention. (A) An image obtained by inverting the luminance of a fluorescent image of blood vessels at times t1 and t2, (b) a projected image, (c) a schematic diagram of FIG. 3 (a), and (d) a schematic diagram of FIG. 3 (b). . It is a modification of the biological observation apparatus which concerns on the 1st Embodiment of this invention. It is a general view which shows the biological observation apparatus which concerns on the 2nd Embodiment of this invention. It is a flowchart which shows the biological observation method which concerns on the 2nd Embodiment of this invention. (A) Fluorescence image of blood vessel at time t1, t2, (b) Projected image, (c) Schematic diagram of FIG. 7 (a), (d) Schematic diagram of FIG. 7 (b).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Living body observation apparatus 2 Upright type optical microscope 3 Light source 4 Image acquisition part 5 Computer (Image composition part, a detection part, a measurement part, a calculation part)
6 Stage 7 Objective Optical System 8 Dichroic Mirror 9 First Wavelength Filter 10 Second Wavelength Filter 11 Fiber 12 Scanning Optical Microscope 13 Analog Processing Unit 13a Offset Operation Unit 13b Analog Amplifier 13c A / D Converter 14 Storage Unit 15 Input Unit 16 display unit 17 two-dimensional scanner S11 administration step S12 collection step S13 addition step S14 reduction step S2 image acquisition step S3 luminance value inversion step S4 image synthesis step S5 detection step S6 measurement step S7 calculation step A mouse B red blood cell L distance

Claims (11)

An image acquisition step of acquiring a plurality of images of moving objects in a living body at different times in the same field of view;
An image combining step of combining a plurality of images acquired in the image acquisition step and combining the projection images;
A detection step of detecting the moving object from within the projection image synthesized in the image synthesis step;
A measuring step for measuring a distance between the moving objects detected in the detecting step;
A living body observation method comprising: a calculation step of calculating a velocity of the moving object based on the distance measured in the measurement step and the acquisition time of the image.   The living body observation method according to claim 1, further comprising a luminance value inversion step of inverting the luminance value of the image or the projection image before or after the image synthesis step.   The living body observation method according to claim 1, further comprising an administration step of administering a labeling substance to the living body before the image acquiring step. The moving object is a blood cell flowing in a blood vessel of a living body;
Before the image acquisition step,
A collecting step of collecting blood from the living body;
An addition step of adding a labeling substance to the blood collected in the collection step;
The biological observation method according to claim 1, further comprising a reduction step of returning the blood, to which the labeling substance has been added in the addition step, to the living body.   The biological observation method according to claim 3 or 4, wherein the labeling substance is a fluorescent dye.   5. The living body according to claim 3, wherein the labeling substance is a fluorescent substance and a labeling target recognition unit that binds to the fluorescent substance and a specific substance existing in a moving object or a region outside the moving object. Observation method.   The biological observation method according to claim 6, wherein the label recognition unit is an antibody.   The biological observation method according to claim 3 or 4, wherein the labeling substance is a fluorescent dye and a non-binding molecular compound to which the fluorescent dye is bound and does not bind to the substance in the living body.   The biological observation method according to claim 8, wherein the non-binding molecular compound is a polysaccharide.   The biological observation method according to claim 8, wherein the non-binding molecular compound is a polypeptide. An image acquisition unit that acquires a plurality of images of a moving object in a living body at different times in the same field of view;
An image synthesizing unit that synthesizes a projected image by superimposing a plurality of images acquired by the image acquiring unit;
A detection unit for detecting the moving object in the projection image synthesized by the image synthesis unit;
A measurement unit for measuring a distance between the moving objects detected by the detection unit;
A biological observation apparatus comprising: a calculation unit that calculates a speed of the moving object based on a distance measured by the measurement unit and an acquisition time of the image.

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