JP6049305B2 - Method for acquiring an image of a test sample derived from a biological sample - Google Patents

Description

  The present invention relates to a method for acquiring an image of a test sample derived from a biological sample.

  It has been reported that the Wnt / β-catenin pathway is involved in cell carcinogenesis. Wnt is a secreted glycoprotein that is conserved in a wide range of biological species and has been confirmed to be involved in development, morphogenesis, and the like. β-catenin is a protein expressed in cells, and when transferred into the nucleus, it binds to a transcription factor and promotes expression of various genes. In the Wnt / β-catenin pathway, the intracellular concentration of β-catenin is regulated in response to Wnt stimulation.

  The Wnt / β-catenin pathway is regarded as a promising target in the search for new anticancer agents. In addition, the Wnt / β-catenin pathway has been reported to be associated with various diseases other than cancer. New and effective means of studying the Wnt / β-catenin pathway are important in the progress of these studies.

  As methods for obtaining information from a biological sample, there are a method for detecting luminescence using a luminometer and a method for detecting a gene expression pattern using an immunohistological staining method.

  In luminescence detection using a luminometer, a method for measuring changes in promoter activity during the development process of embryos, larvae, pupae and adults of biological samples such as Drosophila is known. However, data obtained by spectroscopic measurement such as a luminometer is only numerical data of promoter activity, and image data cannot be obtained. For this reason, it is not possible to measure gene expression patterns in embryos, larvae, pupae and adults.

  In addition, immunohistological staining methods (such as in situ hybridization, promoter assay using β-galactosidase and X-gal) have been used as a method for detecting gene expression patterns for a long time, but sample fixation was performed during staining. Therefore, it is impossible to continuously observe biological samples such as living Drosophila.

  Patent Document 1 discloses gene expression distributed in the vicinity of the outer surface of a different sample by acquiring information about the gene expression from a different angle using light emission by luciferase for a biological sample such as a thick embryo having poor light transmission. A method of analyzing is disclosed.

JP 2008-089521 A

Cell (1992) Vol.26: 1073-1087

  An object of the present invention is to provide useful information about a test sample derived from a biological sample.

  A method for acquiring an image of a test sample derived from a biological sample using the optical imaging means according to the present invention comprises a biological sample having a nucleic acid comprising a promoter binding region and a region comprising a luminescent enzyme gene located downstream thereof. Detecting a luminescence derived from the luminescent enzyme emitted from a test sample from which the test sample is derived, obtaining a first image showing the intensity and distribution of the activity of the promoter in the test sample, and obtaining the first image Simultaneously or continuously with the acquisition, the test sample is irradiated with irradiation light, and the light emitted from the test sample is detected according to the irradiation light, and the internal structure of the test sample and / or Obtaining a second image showing the appearance.

  According to the present invention, useful information about a test sample derived from a biological sample is provided.

FIG. 1 is a diagram schematically showing a signal transduction pathway involving Wnt and β-catenin. FIG. 2 is a diagram illustrating an example of the first image and the second image. FIG. 3 is a diagram schematically showing how images are acquired from each of two different faces of an embryo. FIG. 4 is a diagram showing an example of crossing for producing Drosophila that can be used in the present invention. FIG. 5 is a diagram illustrating an example of a first image and a second image acquired over time. FIG. 6 is a diagram showing the effects of administering ionomycin. FIG. 7 is a diagram showing the effects of administering 6-bromoindirubin-3-oxime. FIG. 8 is a diagram showing the effects of administration of rapamycin.

  1st Embodiment of this invention is related with the method of acquiring the image of the test sample derived from a biological sample using an optical imaging means.

  The optical imaging means is means for acquiring an image by optically detecting an observation target on a predetermined imaging plane, for example. The optical imaging means preferably has an imaging optical system for specifically acquiring an optical signal generated on the imaging surface. Further, the optical imaging means may include means for selectively detecting light having a specific wavelength among light irradiated from the sample. Further, the optical imaging means may include means for selectively irradiating the sample with light having a specific wavelength. An example of these means is a filter that can mainly transmit light having a specific wavelength. The optical imaging means may include an image sensor for converting the detected image into electronic data. The optical imaging means is connected to a computer, and the electronic data of the image may be displayed or recorded by the computer. An example of the optical imaging means is an optical microscope. A specific example of the optical imaging means is a light emission imaging system LV200 (Olympus Corporation).

  A biological sample means any living organism except humans. As the biological sample, for example, an organism whose internal structure, appearance shape and / or size, etc. change in development and whose genetic activity also changes in the course of the change can be used. The biological sample may be an organism that undergoes transformation, in particular an insect. The biological sample may be a living organism. A specific example of a biological sample is Drosophila.

  Drosophila means flies belonging to the order Drosophilidae. The fruit fly that can be used in the present invention is, for example, Drosophila melanogaster. The test sample derived from Drosophila is, for example, an individual of Drosophila. An individual may be in any state during the growth from a fertilized egg to an adult. The test sample is, for example, a fertilized egg, embryo, larva, pupa or adult. The test sample can be used alive.

  The method according to the present invention detects luminescence derived from a luminescent enzyme emitted from a test sample derived from a biological sample having a nucleic acid comprising a promoter binding region and a luminescent enzyme gene located downstream thereof. Obtaining a first image showing the intensity and distribution of the activity of the promoter in the test sample.

  This nucleic acid may be a nucleic acid contained in the genome of the biological sample, or may be a nucleic acid contained in a vector contained in the cells of the biological sample.

  A promoter binding region is a region of a nucleic acid having a specific base sequence. In general, a specific gene exists downstream of the promoter binding region, and when a promoter protein is bound to the promoter binding region, expression of this gene is promoted. The promoter binding region may be, for example, an endogenous one inherent in a biological sample. For example, a promoter binding region is a promoter binding region for a protein that is conserved in various biological species. Alternatively, the promoter binding region corresponds to a promoter having responsiveness to a specific drug. The promoter binding region is a promoter binding region for a protein related to specific signal transduction. The promoter binding region can be selected according to the purpose of research. For example, when studying the development of a biological sample, a promoter binding region for a protein whose activity varies as the development progresses can be used. Furthermore, the promoter binding region may be an exogenous one that is not inherently contained in the biological sample.

  An example of a promoter binding region is a promoter binding region for the Armadillo gene. Armadillo possessed by Drosophila has high homology with β-catenin possessed by mammals.

  FIG. 1 schematically shows a signal transduction pathway involving Wnt and β-catenin.

  FIG. 1 (a) shows a state where there is no stimulation by Wnt on the cells. In the absence of Wnt2, the receptor / cell membrane protein 11 for Wnt2 present on the cell membrane 100 cannot bind to Wnt2 and is not activated. In this case, casein kinase 1α (CK1α) 12, Adenomatous Polyposis Coli (APC) 13, Axin14, glycogen synthase kinase 3β (GSK-3β) 15 and the like bind to β-catenin 1 in the cell. To do. By the action of these proteins bound to β-catenin 1, β-catenin 1 is ubiquitinated and a plurality of ubiquitin 16 are bound. Due to this ubiquitin 16 signal, β-catenin 1 is transported to the proteasome and degraded. FIG. 1 (a) shows metabolized β-catenin 1 'degraded.

  On the other hand, FIG.1 (b) has shown the state which the cell received the stimulation by Wnt2. Wnt2 binds to and activates the receptor / cell membrane protein 11. The activated receptor / cell membrane protein 11 then activates the disheveled protein (Dvl) 17, and the activated Dvl17 binds to Axin14 and the like and suppresses the binding between Axin14 and the like and β-catenin 1 To do. Thereby, (beta) -catenin 1 escapes decomposition | disassembly by a proteasome, As a result, the intracellular density | concentration of (beta) -catenin 1 rises. Next, β-catenin 1 moves into the nucleus 101 and binds to T cell factor / Lymphocyte enhancing factor (Tcf / Lef) 18 which is a transcription factor to form a complex. It binds to the regulatory region and promotes gene expression. Details of the Wnt signal pathway are described in, for example, Biochemistry Vol. 80 No. 12, pp. Reference is made to 1079-1093, 2008.

  Genes whose expression is regulated by the Wnt / β-catenin pathway include genes involved in cell proliferation, differentiation, body axis formation, and organ formation. Therefore, β-catenin has an important function in these phenomena.

  A luminescent enzyme means an enzyme that catalyzes a chemical reaction in which luminescence occurs. An example of a luminescent enzyme is luciferase. A substance that becomes a luminescent substrate for luciferase is called luciferin. Luminescence occurs when luciferin undergoes a chemical change by the catalytic action of luciferase in the presence of ATP. As the luciferase, one derived from fireflies, one derived from bacteria, one derived from a click beetle (click beetle), or the like can be used. A specific example of luciferase is Emerald Luc contained in a pELuc vector commercially available from Toyobo Co., Ltd. The luminescent enzyme expressed in the cells of the test sample promotes the luminescent reaction of the luminescent substrate separately supplied to the cells, and as a result, generates luminescence.

  The positional relationship between the luminescent enzyme gene and the promoter binding region is such that expression of the luminescent enzyme gene is promoted when the promoter is bound to the promoter binding region. The promoter binds to the promoter binding region, for example, due to the original life activity of the test sample or due to external stimuli on the test sample. In response to the binding of the promoter to the promoter binding region, expression of the gene for the luminescent enzyme located downstream thereof is promoted.

  The first image is acquired by detecting luminescence generated by a chemical reaction catalyzed by a luminescent enzyme using an optical imaging means. At this time, the luminescent substrate can be provided to the test sample by any method. When the test sample is an embryo of an insect that undergoes transformation such as Drosophila, for example, the embryo can be immersed in a solution containing a luminescent substrate, and the luminescent substrate can be infiltrated into the embryo. Alternatively, the parent adult worm can be fed with a luminescent substrate to provide the luminescent substrate within the embryo born from the parent. When the test sample is a larva or an adult, the luminescent substrate can be provided in the body of the test sample, for example, by giving a food mixed with the luminescent substrate. When the test sample is a pupa, a feed containing a luminescent substrate can be given at the larval stage to provide the luminescent substrate in the body of the transformed pupae. By using the microinjection method, it is also possible to provide a luminescent substrate directly in the body of an individual. When an embryo is used as a test sample and immersed in a solution containing a luminescent substrate to provide the luminescent substrate, the concentration of the luminescent substrate is preferably 5 mM or less, more preferably 3 mM or less. If the concentration of the luminescent substrate is too high, abnormalities may occur in the growth of the test sample. The luminescent enzyme and the luminescent substrate can be arbitrarily selected from solutions of various origins or compositions depending on the purpose.

  FIG. 2 shows an example of the first image and the second image. FIG. 2A shows an example of the second image, which will be described later. 2B and 2C are examples of the first image. In this example, a Drosophila embryo is used as a test sample, a promoter binding region for the Armadillo gene is used as a promoter binding region, and an ELuc gene is used as a gene for a luminescent enzyme. In embryos, pupae, and adults of biological samples with relatively high light transmission like Drosophila, information inside the living body can be imaged as a luminescent image by appropriately combining the luminescent enzyme, luminescent substrate, and exposure time at the time of image acquisition. I found that I can get it. Regarding the relationship between the luminescent substrate and the biological tissue of the test sample, JP-A-2007-248050 by the applicant may be referred to. FIGS. 2B and 2C are first images obtained after 1 hour 20 minutes and 10 hours from the start, respectively. 2B shows that a part of the test sample emits light, and FIG. 2C shows that the entire test sample emits light.

  The intensity of the activity of the promoter in the test sample can be identified from the amount of luminescence in the first image, for example. Moreover, the distribution of the activity of the promoter in the test sample can be specified from the position of luminescence in the first image, for example.

  The method according to the present invention irradiates the test sample with irradiation light, detects light emitted from the test sample according to the irradiation light, and shows the internal structure and / or appearance of the test sample Obtaining a second image. In particular, in the present invention, it is preferable to acquire an image using illumination light or illuminate excitation light to acquire a fluorescence image. Moreover, by acquiring both the first image and the second image for the same imaging plane inside the living body of the test sample, and collating the internal structures derived from the first image and the second image from the respective images, It is possible to know the shape and / or promoter activity in the living body which cannot be grasped from the surface or the vicinity of the epidermis. Furthermore, by analyzing the shape and / or promoter activity in the living body using the first image and the second image in time series, it is possible to accurately grasp the change actually occurring in the living body.

  Acquisition of the first image and acquisition of the second image can be performed almost simultaneously. That is, the acquisition of the first image and the acquisition of the second image can be performed simultaneously, or the acquisition of the first image and the acquisition of the second image can be performed continuously in a short time. When performing continuously, for example, the acquisition of the second image can be started immediately after the acquisition of the first image is completed, or the images can be acquired in the reverse order.

  For example, the irradiation light may be a mixture of light of various wavelengths. For example, the irradiation light may be light emitted from a general light source such as a tungsten lamp or a halogen lamp. In this case, a bright field image is acquired as the second image. That is, for the acquisition of the second image, light generated by transmitting the irradiation light through the test sample and / or light generated by reflecting the irradiation light to the test sample is detected. The illumination light is, for example, visible light, and is irradiated to the test sample from a light source provided in the optical imaging means, for example.

  FIG. 2 shows an example of the first image and the second image. FIG. 2A shows an example when the second image is acquired as a bright field image. In this example, a Drosophila embryo is used as a test sample, a promoter binding region for the Armadillo gene is used as a promoter binding region, and an ELuc gene is used as a gene for a luminescent enzyme. As a result of collating the first image and the second image on the same imaging plane, it can be seen that an internal structure that can be confirmed has not yet been formed.

  When detecting the light generated when the irradiation light passes through the test sample, a process for increasing the transparency of the test sample may be performed. For example, an image can be acquired by an optical imaging means in a state where a test sample is placed on a glass plate and silicon oil, glycine, mineral oil or the like is dropped on the test sample.

  Or illumination light may mainly contain the light of a specific wavelength. For example, the illumination light may be light that can excite a specific fluorescent protein. In this case, the test sample that expresses the specific fluorescent protein can be used, and the fluorescent protein can be excited by the illumination light to generate fluorescence. In this case, a fluorescence image can be acquired as the second image by detecting this fluorescence.

  The expression of the fluorescent protein can be performed by introducing a fluorescent protein gene into the nucleic acid of the test sample. Known fluorescent proteins can be used. An example of a fluorescent protein is GFP, for example.

  The fluorescent protein can be expressed alone in the cells of the test sample. Alternatively, the fluorescent protein can be expressed in the cells of the test sample in a fused state with a protein different from the fluorescent protein. That is, fluorescent proteins can be used as fluorescent tags for different proteins. Here, different proteins can be selected according to the purpose. For example, a protein that is expressed only in a specific cell at a specific timing can be selected.

  As the second image, the bright field image as described above and the fluorescent image as described above can be obtained together.

  When acquiring a bright field image, the internal structure of the test sample can be specified based on the light transmitted through the test sample. On the other hand, when acquiring a fluorescent image, the fluorescence emitted from the fluorescent protein is expressed by expressing a fluorescent protein fused to a protein that is known to be specifically expressed in a specific tissue or organ in the test sample. The tissue or organ can be specified based on the above.

  In addition, when a bright field image is acquired, the appearance of the test sample can be specified based on light generated by reflection on the test sample. On the other hand, when acquiring a fluorescence image, a fluorescent protein is expressed on the outer surface of the test sample, and the appearance can be specified based on the fluorescence. In order to express the fluorescent protein on the outer surface of the test sample, the fluorescent protein may be expressed alone in the test sample or fused to a protein known to be specifically expressed on at least the outer surface of the test sample. The fluorescent protein may be expressed in a state.

  In the second embodiment of the present invention, the nucleic acid comprises at least one of a second promoter-binding region different from the above-mentioned promoter-binding region and a gene of a second luminescent enzyme located downstream thereof and different from the above-mentioned luminescent enzyme. It further includes one area. And 2nd Embodiment detects the light emission derived from the 2nd luminescent enzyme radiated | emitted from a test sample, and acquires the 3rd image which shows the intensity | strength and distribution of the activity of the 2nd promoter in a test sample. In addition.

  The second promoter binding region can be selected in the same manner as the promoter binding region for acquiring the first image. However, these promoter binding regions are located upstream of different genes.

  The second luminescent enzyme can be selected in the same manner as the luminescent enzyme for acquiring the first image. However, the wavelength of fluorescence generated due to the luminescent enzyme for acquiring the first image is different from the wavelength of fluorescence generated due to the second luminescent enzyme. In particular, these wavelengths are preferably different to the extent that they can be distinguished by optical imaging means.

  In the third embodiment of the present invention, acquisition of the first image and the second image is repeated a plurality of times.

  The acquisition timing can be set arbitrarily. For example, it can be acquired at regular intervals. That is, in this case, the first image and the second image are acquired almost simultaneously, the first image and the second image are acquired again almost at the same time after a predetermined time, and then such acquisition is performed as necessary. Can be repeated. Alternatively, image acquisition can be performed selectively at a particular stage of the test sample. That is, for example, the first image and the second image are acquired almost simultaneously at the start stage of the present invention, and the first image and the second image are acquired again almost simultaneously at the stage where the change occurs in the test sample. Acquisition can be performed whenever any change occurs.

  In the fourth embodiment of the present invention, the first image and the second image are acquired from each of a plurality of different surfaces of the test sample.

  An example of this embodiment is shown in FIG. FIG. 3 shows how images are acquired from two different faces of a Drosophila embryo, respectively. FIG. 3A shows a state in which a Drosophila embryo 200 is observed with the objective lens 201 from the side. The observation surface 202 a to be observed is perpendicular to the optical axis of the objective lens 201. FIG. 3B shows a state in which the embryo 200 is observed with the objective lens 201 from the upper surface. The observation surface 202 b to be observed is perpendicular to the optical axis of the objective lens 201.

  In this embodiment, for example, the first image and the second image can be acquired from one surface of the test sample, and the first image and the second image can be acquired from another surface almost simultaneously. Or after acquiring from one surface, you may acquire from another surface after time. Images for a plurality of surfaces may be acquired from the same test sample, or test samples may be prepared according to the number of surfaces to be acquired, and images for different surfaces may be acquired.

  The fifth embodiment of the present invention further includes providing a stimulus to the test sample before and / or after acquiring the first image and the second image.

  The time between the stimulus to the test sample and the acquisition of the image is arbitrarily set. In addition, images can be acquired before and after stimulation.

  The stimulus can be at least one selected from the group consisting of administration of a drug, irradiation of light, electrical stimulation, exposure to gas, and heating.

  When a drug is administered as a stimulus, for example, a drug whose influence on the test sample is known can be used. When a region for a protein involved in the Wnt / β-catenin pathway such as β-catenin (Armadillo) is used as the promoter binding region, an inhibitor of the protein involved in such a pathway can be used. For example, ionomycin that inhibits complex formation between β-catenin and Tcf, 6-bromoindirubin-3-oxime (BIO) that is a GSK-3β inhibitor, and the like can be used. Alternatively, a drug having an unknown or uncertain influence on the test sample can be used. For example, a substance that is a drug candidate such as an anticancer drug can be administered.

  When light is irradiated as a stimulus, the light may be visible light or light other than visible light. The light may be continuously irradiated for a certain period of time, may be irradiated once for a short time, or may be irradiated in a pulse form.

  When electrical stimulation is performed, for example, an electrode can be inserted into the test sample for stimulation. When an adult insect such as Drosophila is used as a test sample, an electrode can be inserted into or around the adult nervous system in order to confirm the response to nerve cells.

  When the test sample is exposed to gas as a stimulus, for example, the test sample can be exposed to the gas by placing the test sample in a container filled with gas. As the gas, a gas having an anesthetic effect such as diethyl ether can be used. Alternatively, a gas for which the influence on the test sample is to be studied can be selected as the gas. For example, ethanol gas can be used for the study of alcoholism.

  When heating a test sample as a stimulus, the test sample may be heated directly or heated to that temperature by moving the test sample to a hot environment.

  In the case where the first image and the second image are acquired at least once before and after applying the stimulus, this embodiment is acquired after applying the stimulus with at least one first image acquired before applying the stimulus. Comparing at least one first image obtained and / or comparing at least one second image obtained before applying the stimulus and at least one second image obtained after applying the stimulus Can further be included.

  For example, when images are acquired a plurality of times after giving a stimulus, it is possible to see the change in the state of the test sample by arranging all the acquired images along the progress of time. In addition, images can be acquired at regular intervals, and stimulation can be given multiple times at specific timing.

  The sixth embodiment of the present invention further includes extracting corresponding portions of the first image and the second image.

  Extraction can be performed by surrounding the portion with a square or by surrounding it with freehand. A plurality of portions can be extracted from one image. This part can also be extracted as, for example, a so-called region of interest (ROI). For the portion extracted from the first image, the light emission amount can be digitized. Extraction and quantification of the amount of luminescence can be performed using a computer.

  When extracting the portion, the internal structure and appearance information included in the second image can be used. For example, a portion corresponding to a specific tissue or organ in the test sample can be extracted.

  When acquiring a plurality of images, it is not necessary to extract a part from all the images, and a part according to need can be extracted from only a specific image. For example, based on information obtained from the second image, only the image in which a specific internal structure is confirmed can be acquired as a part.

  The seventh embodiment of the present invention further includes obtaining an image obtained by superimposing the first image and the second image.

  For example, the first image and the second image acquired almost simultaneously can be superimposed. Moreover, the 1st image and 2nd image which image | photographed the same surface of the test sample can be piled up. Superposition can be performed using a computer.

  This embodiment may further include displaying or recording the superimposed image. Display and recording can be performed using a computer.

  According to the first embodiment, useful information on a test sample derived from a biological sample can be obtained. Since the first image provides information on the intensity and distribution of the promoter activity and the second image provides information on the internal structure and / or appearance of the test sample, the promoter activity and internal structure are obtained from these two types of information. And the relationship with the appearance.

  Moreover, fertilized eggs, embryos, larvae, pupae or adults of Drosophila can be used as test samples, and information on various stages of Drosophila growth can be obtained.

  When a bright field image is acquired as the second image, information on the internal structure and appearance of the test sample can be easily acquired.

  On the other hand, when a fluorescent protein is expressed in a test sample and a fluorescent image is acquired as the second image, it becomes possible to label a specific internal structure or appearance with fluorescence, and the structure or appearance is specified more clearly. It becomes possible. When moths and adults are used as test samples, it is difficult to capture the internal structure from a bright-field image, but it is easy to identify the internal structure even in such test samples by acquiring a fluorescence image. Become.

  Furthermore, when a fluorescent protein is expressed in a state of being fused with a specific protein in a test sample, only cells that specifically express the specific protein or only sites in cells where the specific protein is localized It can be fluorescently labeled. Thereby, the position and shape of the specific structure in the test sample can be known from the second image.

  As in the second embodiment, by using a test sample having a nucleic acid further containing a second promoter binding region and a gene of the second luminescent enzyme, obtaining a third image based on the second luminescent enzyme, Information about the activities of a plurality of promoters can be obtained from one first image. From such a first image, it is possible to know the difference in intensity and distribution of each promoter activity.

  As in the third embodiment, by acquiring the first image and the second image repeatedly a plurality of times, it is possible to know changes in information such as information on promoter activity and the internal structure and appearance of the test sample. In particular, it is possible to know changes in promoter activity, changes in internal structure, etc. accompanying embryogenesis and growth of biological samples.

  When acquiring a 1st image and a 2nd image from each of several different surfaces of a test sample like 4th Embodiment, more information is acquired compared with the case where an image is acquired from a single surface. be able to. That is, the position where the promoter activity is increased and the internal structure can be grasped three-dimensionally. Thereby, such a position and structure can be specified more accurately.

  As in the fifth embodiment, by applying a stimulus to the test sample, the first image and the second image can be acquired for the test sample that has received such a stimulus.

  Furthermore, when a drug is administered, light is irradiated, electrical stimulation, gas exposure, or heating is performed as a stimulus, the response of the test sample to the stimulus can be examined. In particular, when a drug known to give a specific effect as a stimulus is administered, the state of the test sample in the state of receiving the effect can be examined. Further, drug screening can be performed by examining what effect the administered drug has. When electricity is used as a stimulus, for example, the influence of a test sample on nerve cells can be examined.

  Furthermore, by acquiring images before and after stimulation, changes in the test sample due to the stimulation can be examined. When images are acquired a plurality of times after stimulation, the state of the test sample according to the elapsed time from when the stimulation is received can be examined.

  When extracting corresponding portions of the first image and the second image as in the sixth embodiment, it is possible to acquire information by paying attention to such portions. In addition, the amount of promoter activity of only such a portion can be quantified.

  When acquiring an image obtained by superimposing the first image and the second image as in the seventh embodiment, information on light emission derived from the first image, information on the internal structure and appearance derived from the second image, and Can be directly matched in the superimposed image.

  Furthermore, by displaying or recording such superimposed images, it is possible to improve the usability of the acquired images.

[Example 1: Preparation and observation of test sample]
In this example, Drosophila embryos were prepared as test samples, and the first and second images were acquired over time. The promoter binding region for the armadillo gene was used as the promoter binding region, and the luciferase gene derived from Crick Beetle was used as the luminescent enzyme gene.

  The armadillo promoter region and click beetle luciferase gene Emerald Luc (Toyobo Co., Ltd.) were introduced into the KP124 vector to prepare an arm :: ELuc vector.

  This arm :: ELuc vector was introduced into a fertilized egg of Drosophila melanogaster of 86Fb strain by a microinjection method. The composition of the injection solution was 200 ng / ul arm :: ELuc vector and 0.5% Brilliant Blue FCF. Fertilized eggs were generated and adults were obtained.

  About this adult, crossing shown by FIG. 4 was performed and the luminescence Drosophila strain | arm :: ELuc strain | stump | stock was produced.

  Luminescence imaging was performed on this arm :: ELuc embryo. Embryos were placed in a 3 mM luciferin solution. Two embryos were prepared, one imaged from the side of the embryo and the other imaged from the top surface of the embryo. A luminescence imaging system LV200 (Olympus Corporation) was used, and Andor, EM-gain 255, binning 1 × 1, and 60 × objective were set. The exposure time for acquiring the bright field image was 100 ms, and the exposure time for acquiring the luminescent image was 4.5 minutes. Imaging was continued for 22 hours at 5-minute intervals.

  FIG. 5 shows representative four-stage images of the acquired first and second images. FIG. 5A is an image acquired at the time of 1 hour 55 minutes from the start, the second image seen from the side (bright field) in the upper left, and the first image seen from the side in the lower left (light emission). ), The second image (bright field) seen from the upper surface is shown in the upper right, and the second image (light emission) seen from the side is shown in the lower right. FIGS. 5B to 5D are images acquired at the time points of 6 hours 45 minutes, 9 hours, and 10 hours 5 minutes, respectively, and the arrangement of the four images at each time point is as shown in FIG. The same. The first image was acquired as a pseudo color image colored according to the emission intensity, but FIG. 5 represents it in monochrome.

  From the second image (bright field), the appearance and internal structure of the embryo can be seen. In particular, it can be seen that the structure is formed inside the embryo over time. Moreover, the structure can be grasped three-dimensionally from the images of the side surface and the top surface.

  From the first image (fluorescence), it can be seen when and where the promoter activity increases. From the state of promoter activity, the state of expression of armadillo protein (β-catenin homolog) can be estimated. As shown in FIGS. 5 (a) to 5 (b), the armadillo protein is hardly expressed at the initial stage, whereas, as shown in FIG. It can be seen that the expression increases at the position of, and as shown in FIG. 5 (d), at the stage where 10 hours have passed, the position of expression spreads and the amount thereof also increases. In addition, in FIG. 5 (d), it can be seen that one place of the embryo is strongly expressed when viewed from the side, but when viewed from the top, the portions showing the strong expression are present on the left and right sides of the embryo, respectively. I understand that

[Example 2: Observation of response to drug (ionomycin) administration]
A drug (ionomycin) was administered to the embryo produced as in Example 1 above, and the appearance of the embryo after administration was observed.

  The structural formula of ionomycin is shown in FIG. Ionomycin inhibits the binding of β-catenin and TCF. As a result of inhibition of this binding, binding of the β-catenin / TCF complex to the nucleic acid 102 does not occur in the nucleus 101 as shown in FIG. As a result, it is considered that the transcriptional activity of genes whose expression is induced by the Wnt / β-catenin pathway is reduced.

  The embryo prepared in Example 1 was immersed in a solution containing 10 μM ionomycin. As a control, another embryo was immersed in a solution without ionomycin. These embryos were observed over time under the same conditions as in Example 1 above. However, in this example, an image was acquired only from the side.

  FIGS. 6A and 6B show images acquired in this way. FIG. 6 (a) is an image obtained from a control embryo not administered with ionomycin. The left is an image acquired at 7 hours and 15 minutes from the start, and the right is an image acquired at 16 hours and 20 minutes from the start. In each, the top is the second image (bright field) and the bottom is the first image (light emission). FIG.6 (b) is the image acquired from the embryo which administered ionomycin, and arrangement | positioning of each image is the same as that of Fig.6 (a).

  From comparison between FIGS. 6 (a) and (b), it can be seen that the control embryo is slightly expressed on the head side (left side of the embryo) at the time point of 7 hours and 15 minutes, but ionomycin was administered. It can be seen that such expression is suppressed to such an extent that it cannot be observed in the embryo. In addition, at 16 hours and 20 minutes, the control embryos are generally expressed, especially in the abdomen, whereas in the administered embryos, the expression of the head and front chest cannot be observed. It is suppressed to a certain extent, and further, the expression level of the abdomen is decreased as compared with the control, and it can be seen that there is a difference in the distribution.

  As a result of continuing observation, embryos administered with ionomycin hatched in the same manner as control embryos.

[Example 3: Observation of response to drug (BIO) administration]
A drug (6-bromoindirubin-3-oxime) was administered to the embryo prepared as in Example 1 above, and the state of the embryo after administration was observed.

  The structural formula of 6-bromoindirubin-3-oxime (BIO) is shown in FIG. 7 (c). BIO inhibits the function of GSK-3β. As shown in FIG. 1A, GSK-3β15 binds to β-catenin 1 and promotes the degradation of β-catenin 1 in the absence of stimulation by Wnt2. To do. When the function of GSK-3β15 is inhibited by BIO, it is expected that the degradation of β-catenin 1 is suppressed.

  The embryo prepared in Example 1 was immersed in a solution containing 2 μM BIO. As a control, another embryo was immersed in a solution without BIO. These embryos were observed over time under the same conditions as in Example 1 above. However, in this example, an image was acquired only from the side.

  FIGS. 7A and 7B show images acquired in this way. FIG. 7A is an image obtained from a control embryo not administered with BIO. The left is an image acquired at 1 hour 20 minutes from the start, and the right is an image acquired at 10 hours from the start. In each, the top is the second image (bright field) and the bottom is the first image (light emission). FIG. 7B is an image acquired from an embryo administered with BIO, and the arrangement of each image is the same as that in FIG.

  7 (a) and 7 (b), it can be seen that very strong expression occurs in the head from the early stage of development of 1 hour 20 minutes in the embryo administered with BIO. Furthermore, as time progresses, it can be seen that in the embryo administered with BIO, the expression is increased throughout the embryo.

  In addition, even if 30 hours passed, development did not progress in embryos administered with BIO, and hatching did not occur.

[Example 4: Observation of response to drug (rapamycin) administration]
A drug (rapamycin) was administered to the embryo produced as in Example 1 above, and the appearance of the embryo after administration was observed.

  The structural formula of rapamycin is shown in FIG. Rapamycin specifically inhibits Mammalian target of rapamycin (mTOR). It is used as an immunosuppressant, and is known to have a cancer treatment effect and to have a life extension effect (yeast, nematode, drosophila, mouse).

  The embryo produced in Example 1 was immersed in a solution containing 1 μM rapamycin. As a control, another embryo was immersed in a solution without rapamycin. These embryos were observed over time under the same conditions as in Example 1 above. However, in this example, an image was acquired only from the side.

  FIGS. 8A and 8B show images acquired in this way. FIG. 8A is an image obtained from a control embryo not administered with rapamycin. The left is an image acquired at 1 hour 20 minutes from the start, and the right is an image acquired at 17 hours 5 minutes from the start. In each, the top is the second image (bright field) and the bottom is the first image (light emission). FIG. 8B is an image obtained from an embryo administered with rapamycin, and the arrangement of each image is the same as in FIG.

  8 (a) and (b), at the stage of 1 hour and 20 minutes, the embryo administered with rapamycin develops in the same manner as the control, and the expression pattern is almost the same, but the expression is higher than that of the control. I understand that. Furthermore, at 17 hours and 20 minutes, it was found that the control embryo had invaginated (bright field image), but the administered embryo did not have normal development, and the expression pattern was also different from the control. I understand that.

  Even after 50 hours, the embryos administered with rapamycin did not hatch.

  1 ... β-catenin, 1 ′ ... degraded β-catenin, 2 ... Wnt, 11 ... receptor / cell membrane protein, 12 ... CK1α, 13 ... APC, 14 ... Axin, 15 ... GSK-3β, 16 ... ubiquitin, 17 ... Dvl, 18 ... Tcf / Lef, 100 ... cell membrane, 101 ... nucleus, 102 ... nucleic acid, 200 ... embryo, 201 ... objective lens, 202a, 202b ... observation surface.

Claims (14)

Detecting luminescence derived from the luminescent enzyme emitted from a test sample derived from a biological sample having a nucleic acid containing a promoter binding region and a luminescent enzyme gene located downstream thereof, Acquiring a first image showing the intensity and distribution of the activity of the promoter on the imaging plane, and irradiating the test sample with irradiation light simultaneously with the acquisition of the first image or continuously with the acquisition And optical imaging means including detecting light emitted from the test sample in response to the irradiation light and obtaining a second image showing an internal structure and appearance of an imaging surface inside the test sample a method for obtaining a test image of a sample from a biological sample using,
The method in which the first image and the second image are acquired on the same imaging plane inside the test sample .   The method according to claim 1, wherein the test sample is a Drosophila fertilized egg, embryo, larva, moth or adult.   3. The second image according to claim 1, wherein the second image is obtained by detecting light generated when the irradiation light is transmitted through the test sample and / or light generated when the irradiation light is reflected by the test sample. Method.   The nucleic acid further includes a gene for a fluorescent protein, the irradiation light is light capable of exciting the fluorescent protein, and the second image shows fluorescence emitted from the fluorescent protein expressed in the test sample. The method according to claim 1 or 2, which is obtained by detection.   The method according to claim 4, wherein the fluorescent protein is present in the test sample in a fused state with a protein different from the fluorescent protein. The nucleic acid further includes at least one region consisting of a second promoter binding region different from the promoter binding region and a gene of a second luminescent enzyme located downstream thereof and different from the luminescent enzyme,
The method further comprises detecting luminescence derived from the second luminescent enzyme emitted from the test sample to obtain a third image showing the intensity and distribution of the activity of the second promoter in the test sample. 6. The method according to any one of 1 to 5. The method according to any one of claims 1 to 6, wherein the acquisition of the first image and the second image is repeatedly performed a plurality of times over time .   The method according to any one of claims 1 to 7, wherein the first image and the second image are acquired from each of a plurality of different surfaces of the test sample.   The method according to any one of claims 1 to 8, further comprising applying a stimulus to the test sample before and / or after acquiring the first image and the second image.   The method according to claim 9, wherein the stimulation is at least one selected from the group consisting of administration of a drug, irradiation with light, electrical stimulation, exposure to a gas, and heating. The first image and the second image are acquired at least once before and after applying the stimulus,
Comparing at least one of the first images acquired before applying the stimulus with at least one of the first images acquired after applying the stimulus and / or acquired before applying the stimulus 11. The method of claim 9 or 10, further comprising comparing at least one of the second images and at least one of the second images obtained after applying the stimulus.   The method according to claim 1, further comprising extracting corresponding portions of the first image and the second image.   The method according to any one of claims 1 to 12, further comprising obtaining an image obtained by superimposing the first image and the second image.   The method of claim 13, further comprising displaying or recording the superimposed image.

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