JP2010032338A - Concentration detecting method of luminous substance in biosample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method which enables the detection of the concentration of a fluorescent reagent or the like in a biosample of an experimental small animal or the like by an imaging technique using an optical microscope or the like. <P>SOLUTION: The method for detecting the concentration of the luminous substance in the biosample contains the process for injecting the luminous substance and a first luminous solid particle into the biosample, the process for arranging a second luminous solid particle on the surface of the biosample, the process for measuring the intensities of the lights emitted from the first luminous solid particle and luminous substance in the biosample and the second luminous solid particle arranged on the surface of the biosample using an imaging device, the process for calculating the intensity of light in a case that it is supposed that the luminous substance is present in the outside of the biosample on the basis of the intensities of the lights, and the process for determining the concentration of the luminous substance distributed in the biosample on the basis of the calculated intensity of light. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention uses a luminescent substance such as a fluorescent reagent to emit light inside a biological sample such as an individual of an experimental small animal (eg, mouse, rat, guinea pig, rabbit, etc.), embryo, isolated organ, or cultured tissue. More particularly, the present invention relates to a method of detecting the concentration of a luminescent substance such as a fluorescent reagent in or inside a biological sample as described above.

  In the field of medical, pharmaceutical or biological research, due to recent advances in imaging technology using optical microscopes (including stereomicroscopes), comparison of small experimental animals, embryos, isolated organs, cultured tissues, etc. Experiments for observing the inside or deep part of biological samples having a large target volume using light such as a fluorescent reagent have been carried out in various modes. As an example of such an experiment, for example, a fluorescent reagent or the like is administered to a blood vessel of a biological sample, and the structure of the vascular system is visualized by light of the fluorescent reagent or the like. Experiments have been conducted such as observing the state of leakage from (see, for example, Non-Patent Documents 1 and 2 and Patent Document 1). Experiments such as visualization of the vascular system and observation of leakage of a fluorescent reagent from the blood vessel are used, for example, for investigating the structure and strength of the vascular system in the vicinity of tumor cells in the living body or the production of new therapeutic agents. It is implemented as a model experiment for observation of dynamics in the body, and is useful for evaluating the efficiency of drug delivery to tumor cells.

As described above, when observing the inside or the deep part of a thick sample such as an experimental small animal individual, an embryo, an isolated organ, or a cultured tissue using an optical microscope, the light reaches the photodetector from the site to be observed (observation site) The optical path until the light passes through the tissue or culture medium in the sample, so the intensity of the light reaching the photodetector from the observation site is the absorption of part of the light by the sample in the optical path. It will be attenuated by. In other words, the intensity of light that actually reaches the photodetector from the observation site varies depending on the distance or depth of the observation site from the sample surface, and there is a risk that the structure of the observation site cannot be observed accurately. As understood by those skilled in the art, in general, in the observation of a certain structure using light, the structure is basically grasped by the brightness and contrast of the detected light. However, a clear or accurate image cannot be obtained, and it is difficult to grasp the structure.) Therefore, for example, in Patent Document 2, images of fluorescent beads arranged at various distances (sample depths) from a sample surface in a culture solution sample are taken in advance, and the brightness of those images and the sample are taken. It has been proposed to obtain the relationship with the depth of the observation site, and to correct the variation in the intensity of the detection light due to the depth from the sample surface to the observation site based on the relationship obtained there (in the same document) Used for shading correction in the optical axis direction in a three-dimensional imaging system using a fluorescent confocal microscope.) Patent Document 3 proposes imaging an observation site having a depth from the sample surface by a tomography technique.
JP 2006-227000 JP 2005-275199 A US Pat. No. 6,615,063 Dreher, Matthew R and 5 others Journal of the National Cancer Institute 98, No.5 335-344 March 1, 2006 Nitta, Takehiro and 7 others The Journal of Cell Biology 161, 3, 653-660 May 12, 2003 VisEn Internet homepage http://www.visenmedical.com/technologies/near_infra_red/index.html

  By the way, in an experiment in which a fluorescent reagent is given to a biological sample such as an experimental small animal individual, an embryo, an isolated organ, or a cultured tissue, and light from the fluorescent reagent is observed, often in a biological sample. There are cases where one wants to know the absolute concentration or concentration distribution of a fluorescent reagent. For example, in a model experiment in which leakage from a blood vessel is observed using a fluorescent reagent as exemplified above, the concentration of the fluorescent reagent in the blood vessel or the concentration or concentration distribution of the fluorescent reagent leaked around the blood vessel is calculated. For example, it will be possible to quantitatively determine the degree of leakage from the blood vessel.

  However, in the observation by imaging inside or deep of a biological sample having a thickness as described above using light such as a fluorescent reagent, it is not possible to quantitatively detect the absolute concentration of the fluorescent reagent distributed inside the sample. It has become difficult. As already mentioned, the tissue of a biological sample generally has a property of absorbing a part of the light transmitted therethrough, and when detecting light from an observation site by fluorescence observation, it is applied to the observation site. The excitation light to be emitted and the fluorescence emitted from the observation site are attenuated while passing through the sample. Accordingly, it is impossible to quantitatively detect the amount or concentration of the fluorescent reagent actually present in the observation site from the fluorescence intensity measured there. Of course, as proposed in Patent Document 2, by correcting the intensity of the detection light from the relationship between the depth of the observation site in the sample obtained using the fluorescent beads and the detection light intensity, In principle, it is possible to eliminate unevenness in brightness or contrast in luminance. However, the light absorption characteristics of the sample depend on the wavelength of the light, and the length of the sample in the optical path from the observation site to the photodetector (the distance between the photodetector and the observation site is the same). The absolute concentration of the fluorescent reagent or the like cannot be detected even by the method of Patent Document 2 because the degree of light absorption by the sample varies depending on the size of the sample. Therefore, in the past, when the absolute concentration of a fluorescent reagent or the like distributed inside or deeply in a biological sample is quantitatively detected, a technique such as taking a section from the observation site and measuring the fluorescence intensity is taken. However, in such a case, it is necessary to slaughter experimental animals, or it becomes impossible to continue experiments with the same sample after fluorescence measurement. Further, according to the observation method using the tomography technique as in Patent Document 3, it is possible to detect the concentration of a fluorescent reagent or the like in the deep part of the biological sample, but the configuration of the tomography apparatus or equipment is complicated. Yes, and very expensive.

  Thus, one object of the present invention is to provide a concentration of a fluorescent reagent or the like applied to a thick biological sample such as an experimental small animal individual, an embryo, an isolated organ, or a cultured tissue, particularly in the interior or deep part of the biological sample. It is an object of the present invention to provide a method capable of detecting the concentration of a fluorescent reagent or the like at an observation site using an imaging technique using an optical microscope or the like.

  Another object of the present invention is a method for detecting the concentration of a fluorescent reagent or the like at an observation site located in the deep or deep part of the biological sample as described above, and inside the biological sample of any size. That can detect the concentration of a fluorescent reagent, etc. at an observation site or an observation site at an arbitrary depth from the sample surface and / or the concentration of a fluorescent reagent, etc. having various excitation light wavelength characteristics or emission wavelength characteristics It is to provide a method capable of detecting.

  Furthermore, another object of the present invention is a method for detecting the concentration of a fluorescent reagent or the like in an observation site located in or deep in a biological sample having a thickness as described above. To provide a method capable of detecting the concentration of a fluorescent reagent or the like at an observation site invasively or non-invasively.

  According to the present invention, in imaging using a relatively simple and inexpensive optical microscope (including a stereomicroscope), a luminescent substance such as a fluorescent reagent capable of diffusing in or inside a biological sample having a relatively large thickness A novel method is provided that allows detection of the concentration of.

  According to the present invention, a method for detecting the concentration of a luminescent substance capable of diffusing inside a biological sample includes a step of injecting a solution containing the luminescent substance and first luminescent solid particles into the biological sample; It is possible to obtain a microscopic image of a biological sample by combining a process of arranging a second luminescent solid particle having the same emission wavelength characteristic as that of the solid particle on the surface of the biological sample, and an optical microscope and a light detection device. Using the imaging device, the light emitted from the first luminescent solid particles present inside the biological sample, the light emitted from the luminescent material distributed inside the biological sample, and the second luminescence arranged on the surface of the biological sample The process of measuring the light intensity of each light emitted by the solid particles, the light intensity of the first luminescent solid particles, the light intensity of the second luminescent solid particles, and the light intensity of the luminescent substance distributed inside the biological sample And based on Based on the process of calculating the light intensity when it is assumed that the luminescent material was present outside the biological sample and the light intensity when the luminescent material is assumed to be present outside the biological sample, And a step of determining the concentration of the light-emitting substance to be distributed.

  In the above configuration, the “biological sample” is a biological sample that is usually used in this field (excluding the human body) such as a small experimental animal individual, its embryo, its excised organ or cultured tissue. It's okay. In addition, “a luminescent substance that can diffuse inside a biological sample” means a fluorescent reagent, a phosphorescent reagent, or a chemiluminescent reagent usually used in this field for optical observation or imaging of a biological sample. Or a substance that emits light by excitation light or other means. The first and second “luminescent solid particles” are typically solid microparticles often used in the field known as fluorescent or luminescent microspheres or fluorescent or luminescent beads, A solid particle labeled with a substance or a solid particle carrying a luminescent substance, and the luminescent substance does not substantially diffuse or leak out of the solid particle even after being injected or administered into a biological sample. Any type of particle that maintains particle morphology. In the embodiment of the method of the present invention, typically, the luminescent material is a fluorescent material, and the first and second luminescent solid particles have substantially the same excitation light wavelength characteristics and An optical microscope that is a solid particle that emits fluorescence having emission wavelength characteristics and that forms an image of a biological sample is a so-called fluorescence microscope.

  The above-described method of the present invention is a method of determining the concentration of a luminescent substance such as a fluorescent reagent that is administered or injected into a biological sample and distributed in or deeply from the biological sample based on the light intensity emitted from the luminescent substance. As will be appreciated by those skilled in the art, the light intensity of a luminescent material is a function of the concentration of the luminescent material. Therefore, if the relationship between the light intensity and the concentration of the luminescent substance is known, the concentration of the luminescent substance can be determined from the light intensity of the luminescent substance. However, as already mentioned, the intensity of light from a luminescent material present in or deep within a biological sample is absorbed or attenuated by the biological sample present in the optical path by the time the light reaches the photodetector. The degree of absorption or attenuation by the biological sample varies depending on the wavelength of the light and the size of the biological sample (if the luminescent material emits light by being irradiated with the excitation light, the excitation light reaches the luminescent material. Since it is absorbed by the sample, the intensity of the excitation light received by the luminescent material also varies depending on the wavelength of the light and the size of the biological sample, thereby varying the luminescence intensity of the luminescent material. Therefore, in order to determine the concentration of a luminescent substance in or inside a biological sample from the light intensity emitted by the luminescent substance, first, absorption or attenuation of light by the biological sample actually present in the optical path of light from the luminescent substance. It is necessary to consider the influence of the above or eliminate the influence.

  Therefore, in the above-described method of the present invention, when the light intensity of the luminescent substance inside the biological sample is measured in order to consider or eliminate the influence of light absorption or attenuation by the biological sample existing in the optical path. At the same time, the (first and second) luminescent solid particles such as so-called fluorescent microspheres or fluorescent beads are substantially the same as the inside of the biological sample (particularly, the region (observation site) where the luminescent substance whose concentration is to be detected is distributed). It is preferable that they are in the same area.) And the outside or surface of the biological sample (area not affected by light absorption or attenuation by the biological sample), and the light intensity emitted by the luminescent solid particles is measured respectively. Is done. The relationship between the light intensity values of the luminescent solid particles thus measured is as follows: light intensity when affected by light absorption or light attenuation by the biological sample and light intensity when not affected by light absorption or light attenuation by the biological sample. Represents the relationship. Therefore, by using the relationship between the light intensity values of the (first and second) luminescent solid particles and the light intensity from the luminescent substance actually present inside the biological sample, the luminescent substance is outside the biological sample. The light intensity of the luminescent material inside the biological sample when the effect of light absorption or light attenuation by the biological sample is excluded (the luminescent material inside the biological sample) The light intensity when the light from the light source is not affected by light absorption or light attenuation by the biological sample can be calculated, and the absolute concentration of the luminescent substance can be calculated from the calculated light intensity. It is possible to calculate.

  It should be noted that the light emission whose concentration should be determined when calculating the light intensity of the light-emitting substance inside the biological sample when the influence of light absorption or light attenuation by the biological sample is excluded. The light intensity of the luminescent solid particles placed on the biological sample in which the substance is present is used. According to this, since the influence of light absorption or light attenuation in the biological sample actually subjected to observation is eliminated, the degree of light absorption or light attenuation for each biological sample subjected to observation. It is not a problem that the two are different, and the calculation result of the light intensity when there is no influence of light absorption or light attenuation can be obtained with high accuracy.

  In the above-described method of the present invention, when determining the concentration of the luminescent substance existing inside the biological sample from the light intensity when the influence of light absorption or light attenuation is excluded, the concentration of the luminescent substance is: Typically, the light intensity of light emitted by a solution of a known concentration of luminescent material measured using an imaging device that measures the light intensity of the luminescent material inside the biological sample and the first and second luminescent solid particles, and The light-emitting substance may be determined based on the calculated light intensity when it is assumed that the luminescent substance exists outside the biological sample. In this regard, when the concentration of the luminescent material is relatively low, generally, it may be considered that the concentration of the luminescent material and the light intensity thereof are proportional to each other. As the light intensity of the emitted light, it is only necessary to measure the light intensity at an appropriate concentration. However, when it is desired to determine the concentration more accurately, or when the concentration of the luminescent substance is detected by the method of the present invention in a concentration range that is high enough to break the linearity between the concentration of the luminescent substance and the light intensity. A calibration curve that gives a concentration value of the luminescent material corresponding to the light intensity of the luminescent material of any size is the light intensity of the light of a plurality of solutions with different concentrations of the luminescent material measured using the imaging device The concentration of the luminescent substance present inside the biological sample corresponding to the light intensity value calculated as the light intensity when the effect of light absorption or light attenuation is excluded using the calibration curve is prepared based on the value. It may be determined.

  In the method of the present invention described above, the light intensity when the luminescent substance is assumed to exist outside the biological sample (the light intensity when the influence of light absorption or attenuation of the biological sample is excluded) Regarding the calculation, the calculated light intensity is the ratio of the light intensity of the first luminescent solid particles and the light intensity of the second luminescent solid particles, the light intensity from the luminescent material distributed inside the biological sample, It may be determined based on the extinction coefficient of the biological sample at the emission wavelength of the first and second luminescent solid particles and the extinction coefficient of the biological sample at the emission wavelength of the luminescent substance. As already mentioned, the degree of light absorption or light attenuation of a biological sample varies with the wavelength of light passing through the biological sample. The wavelength dependency of the degree of light absorption or light attenuation of the biological sample appears in the extinction coefficient of the biological sample. Therefore, when the emission wavelengths of the luminescent substance and the luminescent solid particles whose concentrations are to be examined are different from each other, the light intensity of the luminescent substance in the biological sample and the light intensity of the luminescent solid particles are further reduced as described above. And using the extinction coefficient of the biological sample at the emission wavelength of the second luminescent solid particle and the extinction coefficient of the biological sample at the emission wavelength of the luminescent material, the wavelength dependence of the light absorption or attenuation of the biological sample is determined. The light intensity when it is assumed that the luminescent substance exists outside the biological sample may be calculated in the considered mode. As described in the section of the embodiment, a better detection result can be obtained when the luminescent substance and the first and second luminescent solid particles are selected to have different emission wavelengths.

Further, when performing the above-described method of the present invention, when the luminescent material and the first and second luminescent solid particles emit light by irradiating excitation light, the luminescent material and the first and second luminescent materials are used. In addition to the different emission wavelengths of the luminescent solid particles, the luminescent material and the first and second luminescent solid particles may be excited with excitation light having different wavelengths. Then, the intensity of the excitation light received by the luminescent substance in the biological sample and the first luminescent solid particles will also differ from each other. Therefore, when the luminescent substance and the first and second luminescent solid particles are excited with excitation light having different wavelengths, the intensity of the excitation light that reaches the luminescent substance and the first luminescent solid particles is reduced. In consideration of the difference, the light intensity assuming that the luminescent material was present outside the biological sample is the light intensity of the luminescent material in the biological sample, the light intensity of the first and second luminescent solid particles, The excitation light wavelengths of the first and second luminescent solid particles together with the extinction coefficient of the biological sample at the emission wavelength of the first and second luminescent solid particles and the extinction coefficient of the biological sample at the emission wavelength of the luminescent material. It is also possible to calculate using the extinction coefficient of the biological sample in the light source and the extinction coefficient of the biological sample at the excitation light wavelength of the luminescent substance. In this case, the light intensity X when the luminescent material is assumed to exist outside the biological sample is
X = C · (A / B) ^{(εx ′ + εm ′) / (εx + εm)} (a)
May be given by Here, A is the light intensity of the first luminescent solid particles, B is the light intensity of the second luminescent solid particles, and C is the luminescent substance distributed inside the biological sample. Εx and εm are the extinction coefficients of the biological sample at the excitation wavelength and emission wavelength of the first and second luminescent solid particles, and εx ′ and εm ′ are the excitation wavelength and emission of the luminescent substance. The extinction coefficient of the biological sample at the wavelength. Each of the extinction coefficients of the biological sample at each excitation light and emission wavelength may have been examined in advance for the biological sample used, and the value given in any known material may be used. It may be used. [Derivation of Formula (a) is described in the column of the embodiment. ]

  The above-described method of the present invention is typically advantageous in a model experiment for observing the behavior of a luminescent substance applied to a biological sample such as an individual laboratory animal having blood vessels, an embryo, an isolated organ, and a cultured tissue. Can be used. Therefore, in one embodiment of the method of the present invention, in the step of injecting the solution containing the luminescent substance and the first luminescent solid particles into the inside of the biological sample, First luminescent solid particles are injected into the blood vessel. Then, while the first luminescent solid particles are circulated in the vascular system while maintaining the state of the particles as they are, the luminescent material is circulated while diffusing in the vascular system, and the molecular size of the luminescent material or Depending on the characteristics (properties such as blood vessel wall permeability), it will be distributed outside the blood vessel. If the steps of the above-described method of the present invention are executed, the concentration of the luminescent substance in the biological sample is detected. In particular, the above-described method of the present invention may be used for experiments in which the concentration of a luminescent substance leaked from a part of a blood vessel after being injected into the blood vessel is detected.

  In the above-described method of the present invention, the luminescent substance inside the biological sample can be relatively easily obtained in vivo or in situ without externally extracting or exposing the tissue or region whose concentration is to be detected. The concentration can be detected. Therefore, according to the present invention, it is possible to perform various types of experiments or measurements that could not be performed in the past, and various types that could not be obtained in the field of medical, pharmaceutical, or biological research. It is expected that information can be obtained. For example, in the experiment for visualizing the vasculature of small experimental animals as described above and observing leakage of fluorescent substances from blood vessels, according to the method of the present invention, the concentration of the fluorescent substance leaked can be determined. From the volume of the spatial distribution area of the leaking reagent, the absolute amount of the leaking reagent can be detected.

  Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

  The present invention will now be described in detail with reference to a few preferred embodiments with reference to the accompanying drawings. In the figure, the same reference numerals indicate the same parts.

  The method for detecting the concentration of a luminescent substance in a biological sample according to the present invention is a method of injecting or administering a luminescent substance into a biological sample such as an individual of an experimental small animal such as a mouse, rat, guinea pig, or rabbit, an embryo, an isolated organ, or a cultured tissue. However, it may be carried out as part of various observations or measurements made by visualizing the interior or depth of a biological sample using light of a luminescent material. In particular, in this embodiment, a fluorescent reagent is leaked from a blood vessel in an experiment in which a fluorescent reagent is administered as a luminescent substance into a blood vessel of a laboratory animal (mouse) and the vascular system and surrounding tissues are imaged. Although a method for detecting the concentration of the present invention is described, it should be understood by those skilled in the art that the method of the present invention can be applied in other experiments.

Outline of Experiment In the method for detecting the concentration of a luminescent substance in a biological sample in this embodiment, basically, a fluorescent reagent is first administered into a blood vessel of a laboratory animal, The fluorescence image is taken using a fluorescence imaging apparatus (which may be normally used in this field) having a stereomicroscope and a CCD camera. As is well known in the art, fluorescent reagents administered into the blood vessels of laboratory animals are expected to circulate within the vascular system and diffuse throughout the system. Therefore, in the fluorescence image of the experimental animal to which the fluorescent reagent has been administered in this way, the vasculature part appears to shine due to the light emitted from the fluorescent reagent (visualization of the vascular system). In addition, if there is a leaking part in a part of the blood vessel, the fluorescent reagent diffuses from the leaking part to the periphery of the blood vessel, so that the degree of such blood leaking can be observed in the fluorescence image. Become.

  However, as already mentioned, the light emitted from the fluorescent reagent reaches the objective lens of the microscopic optical system (such as a stereomicroscope) until the excitation light for exciting the fluorescent reagent reaches the blood vessel or the fluorescent reagent around it. By the time, each of the excitation light and the fluorescence is absorbed by the biological sample (experimental animal), the amount of the fluorescence in the blood vessel or its surroundings (observation site) can be measured only in the blood vessel or its surroundings. It is not possible to know whether a fluorescent reagent is present, that is, the concentration of the fluorescent reagent in or around such blood vessels. Therefore, in the present embodiment, as described below, fluorescent beads (first luminescent solid particles) are administered into a blood vessel of a laboratory animal together with a fluorescent reagent for visualizing a vascular system. (Similar to the fluorescent reagent, the fluorescent beads are also circulated in the vascular system.) Further, the same fluorescent beads (second luminescent solid particles) are arranged on the surface of the laboratory animal, and the blood vessels of the laboratory animal are arranged. Take fluorescent images of inner and surface fluorescent beads. Then, using the fluorescence intensity of the fluorescent beads on these images, the fluorescence reagent in the blood vessel or its surroundings (excitation light and fluorescence) is not affected by light absorption by the experimental animal. The fluorescence intensity is estimated (calculated), and the concentration of the fluorescent reagent is determined from the estimated fluorescence intensity.

Procedure of Experiment FIG. 1A shows a procedure of a method for detecting the concentration of a fluorescent reagent administered into a blood vessel of an experimental animal using a fluorescence imaging apparatus in the form of a flowchart. Is a schematic representation of the situation.

(I) Preparation of sample Referring to the figure, first, a fluorescent reagent for visualizing leakage from a vascular system or blood vessel was dissolved and fluorescent beads were dispersed in a laboratory animal 10 that may be arbitrarily selected. The solution is injected by intravenous injection or the like (step 100, reference numeral 12 in FIG. 2A). Intravenous injection may be performed in the usual manner. The fluorescent reagent may be any reagent usually used for visualization of the vascular system (particularly, one having an emission wavelength in the near infrared region). In addition, fluorescent beads having a size that can be distributed in the blood vessels of laboratory animals may be selected. The concentration of the fluorescent reagent and the amount of fluorescent beads in the solution injected into the experimental animal may be appropriately adjusted for each experiment. The emission wavelength band of the fluorescent beads is preferably different from the wavelength band of the fluorescent reagent. This is because the fluorescence intensity of the fluorescent beads and the fluorescence intensity of the fluorescent reagent are independent in the estimation calculation of the fluorescence intensity of the fluorescent reagent when not affected by light absorption by the sample described below. This is because the accuracy of the estimated fluorescence intensity is improved. Furthermore, it should be understood that the intravenously injected solution may optionally contain other substances or drugs depending on the purpose of various experiments.

  2A, the same fluorescent beads as those injected into the blood vessel are placed on the surface of the experimental animal (step 110). Such an arrangement may be made by dropping or applying a solution in which an appropriate amount of fluorescent beads are dispersed using a dropper 14 or the like onto the body surface of a laboratory animal. It should be understood that either step 100 or step 110 may be performed first.

(Ii) Imaging of fluorescence image Thus, imaging of the fluorescence image of the sample 10 (experimental animal) prepared as described above is executed using the fluorescence imaging apparatus 20 (step 120). The fluorescence imaging apparatus 20 is a known arbitrary type of imaging apparatus (for example, Olympus IV100, OV110, etc.) configured to take an image obtained by a microscopic optical system with a CCD camera (photodetection apparatus) 26. Good. In the imaging apparatus 20, the microscopic optical system may be a normal type fluorescent stereomicroscope, in which the excitation light or illumination light Iex from the light source 24 is reflected by the dichroic mirror 28, The light is condensed on the observation site of the sample 10 by the objective lens system 22. Then, the fluorescence Iem emitted from the fluorescent reagent (or fluorescent bead) at the observation site is collected by the objective lens system 22, passes through the dichroic mirror 28, and passes through the light receiving surface (not shown) of the CCD camera 26. And a fluorescent image of the sample is formed. It should be noted that filters 24a and 26a that selectively transmit a specific wavelength are disposed in the light source 24 in the optical path of the excitation light and in the optical path of the detection light from the sample to the CCD camera, respectively. The operation of the light source 24 and the CCD camera 26 is controlled by the controller 30, and the image acquired by the CCD camera 26 is processed by the controller in a normal manner and recorded in an image recording device (not shown). The image may be displayed on the monitor 30a. Further, the controller may be configured to acquire a luminance value (in this case, fluorescence intensity) in the acquired image and use the luminance value for arithmetic processing such as arbitrary image processing. .

  When photographing the fluorescent image of the sample 10, the focal point of the objective lens system 22 is focused on the surface of the sample 10, an image (a) of the fluorescent beads on the surface of the sample 10 is photographed, and the focal point of the objective lens system 22 is focused on the sample. An image (b) of the fluorescent beads inside the sample 10 and an image (c) of the vascular system are taken in accordance with the inside of the sample 10 (see the lower right diagram in FIG. 2B). In photographing these fluorescent images, when the excitation light wavelength and emission wavelength of the fluorescent reagent are different from the excitation light wavelength and emission wavelength of the fluorescent beads, the sample is irradiated with a suitable excitation light, and Photographing is executed by switching the filters 24a and 26a and the dichroic mirror 28 placed in the light path of the light source 24 or the CCD camera 26 so that a microscope image can be photographed in a suitable emission wavelength band. Therefore, in that case, an image of the fluorescent beads on the surface of the sample 10 (a), an image of the fluorescent beads inside the sample 10 (b), and an image of the vascular system inside the sample 10 (c) are taken separately. And obtained and stored as a fluorescence image (see FIG. 3A). On the other hand, when the excitation light wavelength and emission wavelength of the fluorescent reagent are the same as the excitation light wavelength and emission wavelength of the fluorescent bead, the image (b) of the fluorescent beads inside the sample 10 and the vascular system inside the sample 10 The image (c) may be imprinted on the same image. As will be understood from the principle of estimation calculation of the fluorescence intensity of the fluorescent reagent when not affected by light absorption by the sample, which will be described later, the image (b) of the fluorescent beads inside the sample 10 and the vascular system In photographing the image (c), when fluorescent beads are present in the region of the vascular system to be observed, the fluorescence intensity of the fluorescent reagent when not affected by light absorption is accurately estimated. The Therefore, when photographing the image (b) of the fluorescent beads and the image (c) of the vascular system, first, the fluorescent beads circulating in the blood vessel are searched for under the microscope, the images are photographed, and the fluorescence found thereafter. Preferably, an image of the vasculature around the bead is taken.

(Iii) Estimation of fluorescence intensity of fluorescent reagent when not affected by light absorption by sample As described above, fluorescent image (a) of fluorescent beads on the surface of sample 10 and fluorescent image of fluorescent beads inside sample 10 ( b) When the fluorescence image (c) of the vascular system inside the sample 10 is acquired, the fluorescence intensity and concentration of the fluorescent beads and the concentration of the fluorescent beads on the surface of the sample 10 and the inside of the sample 10 are to be detected. Is measured (step 130). The fluorescence intensity is measured as shown in FIG. 3A. The fluorescent beads (bead A) on the surface of the sample 10, the fluorescent beads (bead B) inside the sample 10, the vascular system inside the sample 10, or the surroundings thereof. This is done by referring to the luminance value in each image of the fluorescent reagent (at the site where the concentration is to be detected) (in each image, the part that is not actually shown is indicated by a broken line) Yes.) In addition, the site | part or area | region where a density | concentration should be detected may select arbitrary site | parts or area | regions in the fluorescence image (c) in which the vascular system is reflected.

When it is assumed that the fluorescent reagent at the site where the concentration is to be detected is outside the sample 10 from the fluorescence intensity values of the beads A, beads B and the fluorescent reagent at the site where the concentration is to be detected. Fluorescence intensity, ie, fluorescence intensity when the influence of light absorption by a biological sample is removed from the fluorescence intensity emitted by the fluorescent reagent at the site where the concentration should be detected (assuming that the fluorescent reagent was not affected by light absorption) Of fluorescence intensity) is estimated (step 140). Specifically, the fluorescence intensity X when it is assumed that the fluorescent reagent at the site where the concentration is to be detected is outside the sample 10 is:
X = C · (A / B) ^{(εx ′ + εm ′) / (εx + εm)} (1)
Given by. Here, A is the fluorescence intensity of the bead A, B is the fluorescence intensity of the bead B, and C is the fluorescence intensity of the fluorescent reagent distributed in and around the blood vessel of the biological sample. Further, εx and εm are the extinction coefficients of the sample at the excitation light wavelength and detection light wavelength (bead emission wavelength) at the time of imaging the beads A and B, and εx ′ and εm ′ are at the time of imaging the fluorescent reagent. Is the extinction coefficient of the sample at the excitation light wavelength and the detection light wavelength (the emission wavelength of the fluorescent reagent).

  The above formula (1) indicates that the absorption of light by the sample when the excitation light irradiated to the fluorescent reagent distributed in or around the blood vessel inside the bead B and the sample 10 passes through the sample, and the bead B and the fluorescent reagent. In consideration of light absorption by the sample as it passes through the sample until it reaches the objective lens system 22. FIG. 3B shows a schematic cross-sectional view of a sample for explaining the principle of calculating the formula (1).

With reference to the figure, as already described, in the sample of this embodiment, the fluorescent beads A are arranged on the surface of the sample, and the fluorescent beads B are present in the blood vessels inside the sample. A fluorescent reagent is present in and around the blood vessel. Therefore, first, assuming that the excitation light Iexo is irradiated to the beads A and B, the fluorescence intensity A of the beads A is considered to be proportional to the received excitation light intensity.
A = κIexo (2)
And the fluorescence intensity Bo emitted from the bead B inside the sample is
Bo = κIex (3)
Given by. Here, κ is a proportional coefficient, and Iex is the intensity of the excitation light attenuated by the light absorption of the sample before reaching the bead B. Regarding the above excitation light intensity Iexo and Iex, from Lambert-Beer law,
log (Iexo / Iex) = εx · ξ (4)
The relationship is established. Note that ξ is an amount determined by the thickness of the sample and the density of the tissue. Further, since the fluorescence from the bead B inside the sample travels in the same optical path as the excitation light and enters the objective lens system 22, the fluorescence intensity Bo emitted from the bead B and the fluorescence output to the outside of the sample. From the Lambert-Beer law between the strength B
log (Bo / B) = εm · ξ (5)
The relationship is established. Thus, from the relationship of the formulas (2) to (5), between the fluorescence intensity A of the beads A and the fluorescence intensity B of the beads B measured via the objective lens system 22,
log (A / B) = (εx + εm) · ξ (6)
The relationship is established.

Next, considering the fluorescence intensity of the fluorescence reagent distributed in or around the blood vessel inside the sample, the fluorescence intensity C from the fluorescence reagent inside the sample measured through the objective lens system 22 and the fluorescence reagent inside the sample Is assumed to be outside (surface) of the biological sample, that is, when the sample is not affected by light absorption by the sample, the relationship between the fluorescence intensity A of the beads A and the fluorescence of the beads B is as follows. It is the same as that between the strength B. Therefore, between the fluorescence intensity X and the fluorescence intensity C,
log (X / C) = (εx ′ + εm ′) · ξ (7)
The relationship is established. Therefore, the equation (1) is calculated from the equations (6) and (7) by eliminating the constant ξ. In such formula (1),
(A / B) ^{(εx ′ + εm ′) / (εx + εm)} (1a)
However, it can be said that this is a correction coefficient for eliminating the influence of light absorption by the sample. That is, in the present embodiment, a correction coefficient for eliminating the influence of light absorption by the sample is given for each sample to be observed.

  In the above formula (1), the extinction coefficient of the sample at the excitation light / detection light wavelengths of the beads A and B and the fluorescent reagent may be obtained in advance by an arbitrary experiment, but is illustrated in FIG. As described above, a value obtained from the wavelength characteristic data of the extinction coefficient of a tissue sample such as a known experimental animal may be used. In addition, as understood from FIG. 4, the beads A and B and the fluorescent reagent have excitation light / detection light wavelengths in the near infrared region (650 nm or more) in order to suppress light absorption by the excitation light and the fluorescent sample as much as possible. It is desirable to be selected so that

In addition, when the excitation light and detection light wavelengths of the beads A and B and the fluorescent reagent are the same, the expression (1) is
X = C (A / B) (8)
Thus, the fluorescence intensity X can be calculated without using the extinction coefficient value. However, in that case, as understood from (b) or (c) of FIG. 3 (A), the fluorescence of the beads B and the fluorescent reagent overlap in the same image, and the respective fluorescence intensities should be accurately measured. Can be difficult. Therefore, it is preferable that at least the wavelength of the detection light is different between the beads A and B and the fluorescent reagent.

  Moreover, when estimating the value of the fluorescence intensity X using Formula (1), it is preferable that the image of (b) or (c) of FIG. 3 (A) is an image of the same area | region. As understood from the above description, when the expression (1) is derived, it is assumed that the amounts ξ in the expressions (6) and (7) are equal. Since the amount ξ is an amount determined by the thickness of the sample and the density of the tissue as described above, if the position of the fluorescent beads B and the position of the distribution region of the fluorescent reagent whose concentration is detected are greatly shifted, The shift of ξ increases, and the accuracy of the calculated fluorescence intensity X value decreases.

(Iv) Determination of concentration of fluorescent reagent inside sample As described above, when the fluorescence intensity X is estimated when it is assumed that the fluorescent reagent inside the sample is outside (surface) of the biological sample, the estimated fluorescence Based on the value of the intensity X, the concentration of the fluorescent reagent is determined (step 150). Determination of the concentration of the fluorescent reagent from the fluorescence intensity X is performed under the same conditions (lens magnification, excitation light wavelength, excitation light intensity, A fluorescent image of a fluorescent reagent solution having a known concentration in the detection light wavelength band, etc.), and the fluorescence intensity value of the fluorescent reagent solution obtained from the fluorescent image is compared with the previously estimated fluorescent intensity X May be done. Typically, since the fluorescence intensity and the fluorescence reagent concentration are considered to be proportional, if the fluorescence intensity of a reagent solution with a known concentration Yo is Xo, the fluorescence reagent concentration Y in the sample is
Y = Yo · X / Xo (9)
Given in.

  However, when it is desired to determine the concentration of the fluorescent reagent more accurately, a calibration curve for determining the concentration of the fluorescent reagent from the fluorescence intensity of the fluorescent reagent measured by the fluorescent imaging device 20 (see FIG. 5C). )) May be prepared, and the concentration of the fluorescent reagent in the sample 10 may be determined using the calibration curve. FIGS. 1B, 5A, and 5B are steps of a process of preparing a calibration curve for determining the concentration of the fluorescent reagent from the fluorescence intensity of the fluorescent reagent measured in the fluorescence imaging apparatus 20. And the situation. Referring to these drawings, first, as illustrated in FIG. 5A, a preparation is prepared in which a plurality of fluorescent reagent solutions having different concentrations are placed (step 200). Next, as schematically shown in FIG. 5B, fluorescence images of the fluorescent reagent solution on the preparation are sequentially photographed using the fluorescence imaging apparatus 20 (step 210). Thereafter, the fluorescence intensity is measured from the luminance value of the fluorescence image of each fluorescent reagent solution (step 220), then each fluorescence intensity is plotted against the concentration, and the plot points are interpolated by an arbitrary method. A calibration curve of 5 (C) is prepared (step 230). Thus, when the fluorescence intensity X is determined when it is assumed that the fluorescent reagent inside the sample is outside the biological sample, the corresponding concentration value Y can be read on the prepared calibration curve.

  As described above, according to the embodiment of the present invention described above, fluorescence administered into a blood vessel of a biological sample such as a small experimental animal noninvasively or minimally invasively by using a fluorescence imaging technique. The reagent concentration can be detected in vivo. Further, according to the method of the present invention, fluorescent beads are given to a sample to be observed, and the fluorescence intensity of the fluorescent reagent inside the sample is corrected using the fluorescence intensity (formula (1)). See), and the fluorescence intensity and concentration of the fluorescent reagent in the sample from which the influence of light absorption by the sample is removed are determined. That is, it is to be understood that the effect of light absorption by the sample is corrected for each sample, and therefore it does not matter that the degree of light absorption differs for each sample.

  Although the above description has been made in relation to the embodiment of the present invention, many modifications and changes can be easily made by those skilled in the art, and the present invention is limited to the embodiment exemplified above. It will be apparent that the invention is not limited and applies to various methods without departing from the inventive concept.

  For example, when detecting the concentration of a luminescent substance in a biological sample from an image obtained by imaging, it should be understood that the light used for imaging is not limited to fluorescence but may be phosphorescence or chemiluminescence. Yes (in the case of imaging that does not require excitation light, the influence of absorption of excitation light need not be considered). In addition, the method of the present invention may be applied not only to imaging of blood vessels or their surroundings, but also to imaging of a site or region where a luminescent substance and luminescent solid particles can be administered into a biological sample. It should be understood as belonging to the scope of the present invention.

FIG. 1 (A) shows a procedure of a method for detecting the concentration of a fluorescent reagent in a biological sample according to the present invention in the form of a flowchart, and FIG. 1 (B) is used in (A). The procedure for preparing a calibration curve of concentration versus fluorescence intensity is shown in the form of a flowchart. FIG. 2 (A) schematically shows the preparation of a small experimental animal (mouse) used as a sample in the method according to the present invention, and FIG. 2 (B) shows a fluorescence image of the sample. The schematic structure and operation of the fluorescence imaging apparatus 20 for imaging | photography are typically represented. In the figure, after an image of the surface of the sample 10 is taken, the focal position of the objective lens system 22 is changed downward and the inside of the sample 10 is taken. However, the order of such photographing may be arbitrary. FIG. 3A shows a fluorescent image (a) of the fluorescent beads A arranged on the surface of the sample 10 assumed to be acquired by the fluorescence imaging apparatus in the method according to the present invention, into the blood vessel of the sample 10. Fluorescent image (b) of the fluorescent bead B that is administered and distributed in the blood vessel, and a fluorescent image (c) of the fluorescent reagent that is administered into the blood vessel of the sample 10 and leaks around the blood vessel. is there. In these figures, the portion indicated by a broken line is a portion that cannot be seen in the corresponding fluorescent image. In the image (c), the hatched area indicates the area where the fluorescent reagent is distributed (in the example of the figure, the state in which the fluorescent reagent leaks from a part of the blood vessel is schematically shown). Is drawn in.) FIG. 3B is a schematic cross-sectional view of the sample for explaining the principle of estimating the fluorescence intensity when it is assumed that the fluorescent reagent is outside (surface) of the sample. FIG. 4 is a wavelength characteristic spectrum of the extinction coefficient of a general biological sample (Non-patent Document 3). The spectral value of the extinction coefficient is used for the calculation of the fluorescence intensity when it is assumed that the fluorescent reagent is outside (surface) of the sample. FIG. 5 (A) schematically shows a preparation on which a fluorescent reagent solution used for preparing a calibration curve of concentration versus fluorescence intensity is placed (the numbers in the figure indicate the concentration of the fluorescent reagent). (MM).). FIG. 5B schematically shows a state in which fluorescent images of the fluorescent reagent solution on the slide are sequentially photographed using the fluorescence imaging apparatus 20. In the figure, components other than the objective lens system 22 are omitted. FIG. 5C shows an example of a calibration curve of concentration versus fluorescence intensity.

Explanation of symbols

10 ... Sample (small experimental animal (mouse))
DESCRIPTION OF SYMBOLS 12 ... Syringe 14 for intravenous injection ... Dropper 16 ... Fluorescent bead solution 20 dripped on the sample surface ... Fluorescence imaging device 22 ... Objective lens system 24 ... Light source 24a ... Filter 26 for excitation light ... CCD camera (light detection device)
26a ... Filter for detection light 30 ... Controller 30a ... Monitor

Claims (8)

A method for detecting the concentration of a luminescent substance that can diffuse within a biological sample,
Injecting a solution containing the luminescent substance and first luminescent solid particles into the biological sample;
Placing a second luminescent solid particle having the same emission wavelength characteristic as the first luminescent solid particle on the surface of the biological sample;
Using an imaging device that combines an optical microscope and a light detection device to obtain a microscopic image of the biological sample, the light emitted from the first luminescent solid particles present inside the biological sample and the biological sample Measuring the light intensity of each of the light emitted from the luminescent material distributed inside the light and the light emitted from the second luminescent solid particles disposed on the surface of the biological sample;
Based on the light intensity of the first luminescent solid particles, the light intensity of the second luminescent solid particles, and the light intensity of the luminescent substance distributed inside the biological sample, the luminescent substance is the biological sample. The process of calculating the light intensity assuming that it existed outside the
And determining a concentration of the luminescent material distributed inside the biological sample based on the calculated light intensity when it is assumed that the luminescent material is present outside the biological sample. And how to.   2. The method according to claim 1, wherein in the step of determining the concentration of the luminescent material distributed inside the biological sample, the solution of the luminescent material having a known concentration measured using the imaging device. The concentration of the luminescent substance existing inside the biological sample is determined based on the light intensity of the emitted light and the calculated light intensity when the luminescent substance is assumed to exist outside the biological sample. A method characterized by being made.   3. The method according to claim 1, wherein the luminescent substance is a fluorescent substance, and the first and second luminescent solid particles emit fluorescence having substantially the same excitation light wavelength characteristic and emission wavelength characteristic. A method characterized in that the optical microscope is a fluorescent microscope.   4. The method according to claim 1, wherein the light intensity when it is assumed that the luminescent material is present outside the biological sample is equal to the light intensity of the first luminescent solid particles and the second light intensity. The ratio of the light-emitting solid particles to the light intensity, the light intensity from the light-emitting substance distributed inside the biological sample, and the absorption of the biological sample at the emission wavelength of the first and second light-emitting solid particles A method characterized in that it is determined based on a coefficient and an extinction coefficient of the biological sample at an emission wavelength of the luminescent material. 5. The method according to claim 1, wherein the luminescent substance and the first and second luminescent solid particles emit light when irradiated with excitation light, and the luminescent substance is exposed to the outside of the biological sample. The light intensity X when it is assumed that the light is present is distributed in the ratio of the light intensity A of the first luminescent solid particles and the light intensity B of the second luminescent solid particles and the inside of the biological sample. The light intensity C of the luminescent material, the extinction coefficients εx and εm of the biological sample at the excitation wavelength and emission wavelength of the first and second luminescent solid particles, and the excitation wavelength and emission wavelength of the luminescent material. Using the extinction coefficients εx ′ and εm ′ of the biological sample, X = C · (A / B) ^{(εx ′ + εm ′) / (εx + εm)}
A method characterized by being given by:   6. The method according to any one of claims 1 to 5, wherein the biological sample is a sample selected from the group consisting of a small laboratory animal having blood vessels, an embryo, an isolated organ, and a cultured tissue. In the step of injecting the solution containing the luminescent material and the first luminescent solid particles, the solution containing the luminescent material and the first luminescent solid particles are injected into a blood vessel. Method.   The method according to claim 6, wherein after the injection into the blood vessel, the light intensity of the light emitted from the luminescent material leaked from a part of the blood vessel is measured, and the concentration of the luminescent material leaked from the blood vessel is determined. A method characterized by being made.   2. The method according to claim 1, wherein in the step of determining the concentration of the luminescent material distributed inside the biological sample, the solution of the luminescent material having a known concentration measured using the imaging device. Using a calibration curve that gives a concentration value of the luminescent material corresponding to the light intensity of an arbitrary size of the luminescent material prepared based on the light intensity value of the emitted light, the above-mentioned corresponding to the calculated light intensity A method, characterized in that the concentration of said luminescent material present inside a biological sample is determined.

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