JP2009133697A - Luminescence measurement method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a luminescence measurement method capable of obtaining an accurate quantitative result of each luminescence color from each biological sample, and consequently enabling inspection of multiple items regarding the same biological sample. <P>SOLUTION: The luminescence measurement method comprises forming a biological sample, containing plural predetermined genes which are luminescence-labeled with a different luminescence color from each other; applying a predetermined stimulation to the formed biological sample from an outside of the biological sample; photographing a luminescence image of the biological sample, after the application of the predetermined stimulation by correlating, for each luminescence color, associating the luminescence color with the luminous intensity; and quantitatively measuring luminous intensity of each luminescence color based on the photographed image. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to luminescence imaging for observing a biological sample (for example, a sample containing cells), and more particularly to a method for performing a multi-item examination on the same biological sample.

  It is known that luciferase, which is a luminescent enzyme used for biological function analysis, has various luminescent colors depending on its type. At present, signals of different emission colors are detected from a large amount of ground cells using the luminometer in the same measurement time. However, it is difficult to obtain an accurate quantitative result for each emission color. Therefore, if Patent Document 1 is used to collect a large amount of cells in one lysis solution and simultaneously measure the relative light intensity of each luminescent enzyme in cells containing multiple luminescent enzymes, accurate quantitative results of each luminescent color can be obtained. Can be obtained.

JP 2007-218774 A

  However, in the prior art, since a large amount of cells are combined into one lysis solution, it is not possible to obtain an accurate quantitative result of each luminescent color from each cell. Therefore, it is necessary to perform a multi-item inspection on the same cell. There was a problem that could not.

  The present invention has been made in view of the above problems, and can obtain an accurate quantitative result of each emission color from each biological sample. As a result, the same biological sample is inspected in multiple items. An object of the present invention is to provide a luminescence measurement method that can be used.

  As a result of intensive studies by the present inventors, it has been found that the difference in emission intensity is large depending on the emission color of each cell. In addition, the difference exceeds the difference in transmittance for each filter when the spectral filter corresponding to each emission color is used, and is so large as to affect quantitative measurement and imaging performance. In view of the difference in emission intensity caused by the emission color, the present inventor completed the present invention by imaging or analyzing the relationship between both emission color and emission intensity for each emission color. It was. Specifically, for example, the above-described problem has been solved by adjusting the exposure time of the CCD camera to an appropriate range during imaging according to the type of luciferase and the dynamic range of the CCD camera.

  That is, in order to solve the above-described problems and achieve the object, the luminescence measurement method according to the present invention is a luminescence measurement method for measuring luminescence from a biological sample, and is luminescently labeled so that the luminescent colors are different from each other. A preparation step for preparing the biological sample including a plurality of predetermined genes, a stimulation step for applying a predetermined stimulus from outside the biological sample to the biological sample prepared in the preparation step, and the predetermined stimulation in the stimulation step. An imaging step of imaging a luminescent image of the biological sample after being given, in which the relationship between both the luminescent color and the luminescent intensity is associated with each luminescent color, and the luminescent image captured in the imaging step. And a measurement step of quantitatively measuring the emission intensity of each of the emission colors.

  The luminescence measurement method according to the present invention is the luminescence measurement method described above, wherein the imaging step depends on a dynamic range of a camera used for the imaging and a type of the luminescent protein used for the luminescence labeling. And adjusting the exposure time of the camera to capture the luminescent image of the biological sample.

  The luminescence measurement method according to the present invention is the luminescence measurement method described above, wherein the imaging step repeatedly captures the luminescence image of the biological sample, and the measurement step includes a plurality of images captured in the imaging step. Based on the light emission image, the time-dependent change of the light emission intensity of each of the light emission colors is quantitatively measured.

  According to the present invention, a biological sample including a plurality of predetermined genes that are luminescently labeled so that the luminescent colors are different from each other is prepared, a predetermined stimulus is applied to the prepared biological sample from outside the biological sample, and the predetermined stimulus is The luminescent image of a given biological sample is imaged by associating the relationship between the luminescent color and luminescent intensity for each luminescent color, and the luminescent intensity of each luminescent color is quantified based on the captured luminescent image. Measure automatically. Thereby, an accurate quantitative result of each luminescent color can be obtained from each biological sample, and as a result, there is an effect that a multi-item inspection can be performed on the same biological sample.

  According to the present invention, a luminescence image of a biological sample is captured by adjusting the exposure time of the camera according to the dynamic range of the camera used for the imaging and the type of photoprotein used for luminescent labeling. Thereby, an accurate quantitative result of each luminescent color can be obtained from each biological sample, and as a result, there is an effect that a multi-item inspection can be performed on the same biological sample.

  According to the present invention, a luminescence image of a biological sample is repeatedly taken, and a change with time of the luminescence intensity of each luminescent color is quantitatively measured based on the plurality of taken luminescence images. Thereby, there is an effect that it is possible to analyze the temporal change of the expression state of each predetermined gene by a predetermined stimulus.

  Embodiments of a light emission measuring method according to the present invention will be described below in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

  First, the configuration of the luminescence observation system 100 used in the luminescence measurement method according to the present invention (specifically, the imaging process and the analysis process) will be described with reference to FIGS. 1, 2, and 3. FIG. 1 is a diagram illustrating an example of the overall configuration of the light emission observation system 100.

  As shown in FIG. 1, the luminescence observation system 100 includes a container 103 (specifically, a petri dish, a slide glass, a microplate, a gel support, a fine particle carrier, etc.) containing a biological sample 102 and a stage on which the container 103 is arranged. 104, a luminescent image capturing unit 106, and an image analysis device 110. A light emission image capturing unit 106 for measuring weak light emission may be disposed below the stage 104. Thereby, disturbance light from above the sample due to opening and closing of the cover can be completely blocked, and the S / N ratio of the luminescent image can be increased. The light emission image capturing unit 106 may be a laser scanning optical system.

  The biological sample 102 is, for example, a living cell including a plurality of predetermined genes that are luminescently labeled with luminescence-related genes so that the luminescent colors are different from each other. The biological sample 102 is given a predetermined stimulus (for example, drug stimulation) from outside the biological sample immediately before imaging the biological sample.

The luminescence image capturing unit 106 is specifically an upright luminescence microscope and captures a luminescence image of the biological sample 102. As shown in the figure, the luminescent image capturing unit 106 includes an objective lens 106a, a dichroic mirror 106b, a CCD camera 106c, and an imaging lens 106f. Specifically, the objective lens 106a has a value of (numerical aperture / magnification) ² of 0.01 or more. The dichroic mirror 106b is used when light emitted from the biological sample 102 is separated for each color, and the amount of emitted light and the light intensity are measured for each color using two colors of light emission. The CCD camera 106c takes a light emission image and a bright field image of the biological sample 102 projected onto the chip surface of the CCD camera 106c via the objective lens 106a, the dichroic mirror 106b, and the imaging lens 106f. The CCD camera 106c is connected to the image analysis device 110 so as to be able to communicate with each other by wire or wirelessly. Here, when there are a plurality of biological samples 102 in the imaging range, the CCD camera 106c may capture the luminescent images and bright field images of the plurality of biological samples 102 included in the imaging range. The imaging lens 106f forms an image (specifically, an image including the biological sample 102) incident on the imaging lens 106f via the objective lens 106a and the dichroic mirror 106b. Note that FIG. 1 shows an example in which a light emission image corresponding to two light emissions separated by the dichroic mirror 106b is separately captured by the two CCD cameras 106c. The image capturing unit 106 may include an objective lens 106a, a single CCD camera 106c, and an imaging lens 106f.

  Here, when measuring the light emission amount and the light emission intensity for each color using the light emission of two colors, as shown in FIG. 2, the light emission image pickup unit 106 includes an objective lens 106a, a CCD camera 106c, and a split image unit 106d. And the imaging lens 106f. The CCD camera 106c may pick up a light emission image (split image) and a bright field image of the biological sample 102 projected onto the chip surface of the CCD camera 106c via the split image unit 106d and the imaging lens 106f. . The split image unit 106d separates the light emitted from the sample 102 by color, and is used when measuring the light emission amount and light emission intensity by color using two colors of light, similar to the dichroic mirror 106b.

  Further, when measuring the emission amount and emission intensity for each color using light emission of a plurality of colors (that is, when using multicolor light emission), the light emission image pickup unit 106 includes an objective lens 106a and an objective lens 106a as shown in FIG. The CCD camera 106c, the filter wheel 106e, and the imaging lens 106f may be used. Then, the CCD camera 106c may capture a light emission image and a bright field image of the biological sample 102 projected onto the chip surface of the CCD camera 106c via the filter wheel 106e and the imaging lens 106f. The filter wheel 106e is used when light emitted from the biological sample 102 is separated by color by exchanging filters, and the amount of light emitted and the intensity of light emitted are measured for each color using light of a plurality of colors.

  Returning to FIG. 1, the image analysis apparatus 110 is specifically a personal computer. As shown in FIG. 4, the image analysis device 110 is roughly divided into a control unit 112, a clock generation unit 114 that measures the system time, a storage unit 116, a communication interface unit 118, and an input / output interface unit. 120, an input device 122, and an output device 124. These units are connected via a bus.

  The storage unit 116 is a storage unit. Specifically, a memory device such as a RAM or a ROM, a fixed disk device such as a hard disk, a flexible disk, an optical disk, or the like can be used. And the memory | storage part 116 memorize | stores the data etc. which were obtained by the process of each part of the control part 112. FIG. The communication interface unit 118 mediates communication between the image analysis device 110 and the CCD camera 106c. That is, the communication interface unit 118 has a function of communicating data with other terminals via a wired or wireless communication line. The input / output interface unit 120 is connected to the input device 122 and the output device 124. Here, in addition to a monitor (including a home television), a speaker or a printer can be used as the output device 124 (hereinafter, the output device 124 may be described as a monitor). In addition to the keyboard, mouse, and microphone, the input device 122 can be a monitor that realizes a pointing device function in cooperation with the mouse.

  The control unit 112 has an internal memory for storing a control program such as an OS (Operating System), a program that defines various processing procedures, and necessary data, and executes various processes based on these programs. . The control unit 112 is roughly composed of a light emission image capturing instruction unit 112a, a light emission image acquisition unit 112b, an image analysis unit 112c, and an analysis result output unit 112d.

  The luminescent image capturing instruction unit 112a instructs the CCD camera 106c to capture a luminescent image and a bright field image via the communication interface unit 118. The luminescent image acquisition unit 112b acquires the luminescent image and bright field image captured by the CCD camera 106c via the communication interface unit 118. The control unit 112 controls the emission image capturing instruction unit 112a to repeatedly capture the emission image and the bright field image of the biological sample 102.

  Here, when the light emission image of the biological sample 102 is picked up by the CCD camera 106c, the relationship between both the light emission color and the light emission intensity is associated with each light emission color. In other words, the relationship between both the emission color and the emission intensity is associated with each emission color, and the emission image of the biological sample 102 is captured.

  Specifically, when the luminescent image of the biological sample 102 is captured by the CCD camera 106c, the CCD camera 106c is used in accordance with the dynamic range of the CCD camera 106c used for capturing the luminescent image and the type of luminescent protein used for luminescent labeling. Adjust the exposure time of the CCD camera. In other words, the exposure time of the CCD camera is adjusted according to the dynamic range of the CCD camera 106c used to capture the luminescent image and the type of the luminescent protein used for luminescent labeling, so that the luminescent image of the biological sample 102 is obtained. Take an image.

  The image analysis unit 112c quantitatively measures the emission intensity of each emission color based on the emission image acquired by the emission image acquisition unit 112b. The image analysis unit 112c quantitatively measures the temporal change in the emission intensity of each emission color based on the plurality of emission images acquired by the emission image acquisition unit 112b. The analysis result output unit 112d outputs the analysis result from the image analysis unit 112c to the output device 124. Specifically, the analysis result output unit 112d graphs the time-series data regarding the emission intensity of each emission color obtained by the image analysis unit 112c and displays the graph on the output device 124.

[Multicolor reporter assay (transcription response of AP-1 gene group)]
AP-1 (Activating Protein-1) is a transcription factor composed of Fos and Jun genes, and is known to control responses to various stimuli from outside the cell. When cells are stimulated, c-fos and c-jun are activated, and c-Fos and c-Jun proteins are synthesized. These proteins form a complex and function as an AP-1 transcription factor, bind to an AP-1 binding site in the promoter region of the target gene, and induce transcription of a downstream gene.

  In this example, signal transduction was performed by imaging the expression of luciferase under the control of c-fos and AP-1 promoters in multiple colors using a luminescence imaging system “LUMINOVIEW” (manufactured by Olympus Corporation). The changes over time in the response of the AP-1 gene group accompanying the above were followed.

The flow (procedure) of the experiment in this example will be described below.
[Procedure 1: Corresponding to the production process of the present invention] As a reporter for monitoring the activity of c-fos, the c-fos promoter region cloned from HeLa cells was inserted into MCS of pGL4.72 (Promega) and c- A fos promoter / RLuc expression vector (c-fos / hRL) was prepared.
[Procedure 2: Corresponding to the production process of the present invention] In order to observe the response of AP-1, the luciferase gene of TransLucent Reporter Vector (manufactured by Panomics) was transferred to SLG luciferase (manufactured by Toyobo Co., Ltd.) Substitution was carried out to produce an AP-1 enhancer / SLG expression vector (AP-1 / SLG).
[Procedure 3: Corresponding to the production process of the present invention] The two types of vectors prepared in Procedure 1 and Procedure 2 were transfected into HeLa cells seeded in an f35 mm-glass bottom dish and cultured overnight, and then the medium was added. It was replaced with serum-free medium and further cultured overnight. Subsequently, EnduRen (final concentration 60 μM) and luciferin (final concentration 500 μM) were added to the medium and allowed to stand for 1 hour.
[Procedure 4: Corresponds to the stimulation step of the present invention] FBS (final concentration 10%) was added to the medium to stimulate the cells.
[Procedure 5: Corresponding to the imaging step of the present invention] After setting the dish to LUMINOWVIEW, time-lapse photography of the luminescent image and the bright field image was performed at 15-minute intervals for 20 hours. As the spectral filter, a BA470-490 filter was used for c-fos expression analysis, and a BA530-550 filter was used for AP-1 expression analysis. As luminescence observation conditions, the magnification of the objective lens is 20 times, the exposure time is 10 minutes (for c-fos expression analysis) and 5 minutes (for AP-1 expression analysis), the binning is 1 × 1, the CCD camera is iXon (ANDOR ).
[Procedure 6: Corresponds to the measurement step of the present invention] Each c-fos / hRL emission image photographed in Procedure 5 and the bright field image corresponding thereto are superimposed, and a plurality of ROIs ( Region of Interest (region of interest) was specified (see FIG. 5). Further, each AP-1 / SLG emission image photographed in the procedure 5 and the corresponding bright field image were superimposed, and a plurality of ROIs were designated for the superimposed image (see FIG. 6). Then, the emission intensity of each designated ROI was measured based on each emission image, and the change over time of the emission intensity was displayed in a graph (see FIGS. 7 and 8).

  As a result of the above experiment, the response of each promoter after serum stimulation could be detected at the single cell level (see FIGS. 5 and 6). Moreover, when the maximum expression time is compared between FIG. 7 and FIG. 8 showing the time-dependent changes in the luminescence intensity of each gene expression, the response of AP-1 was delayed after the c-fos response started first. It was suggested to begin (see FIG. 7 and FIG. 8). Thus, by performing multicolor imaging using LUMINOVIEW, it was possible to capture the state of signal transmission in the same cell, and as a result, more accurate gene expression analysis was possible.

  As described above, the luminescence measurement method according to the present invention can be suitably used in various fields such as biotechnology, pharmaceuticals, and medicine.

1 is a diagram illustrating an example of an overall configuration of a light emission observation system 100. FIG. 2 is a diagram illustrating an example of a configuration of a light emission image capturing unit 106 of the light emission observation system 100. FIG. 2 is a diagram illustrating an example of a configuration of a light emission image capturing unit 106 of the light emission observation system 100. FIG. 2 is a block diagram illustrating an example of a configuration of an image analysis device 110 of the light emission observation system 100. FIG. It is a figure which shows the superimposition image of the bright field image imaged 7 hours after serum stimulation, and a c-fos / hRL light emission image. It is a figure which shows the superimposition image of the bright-field image imaged 7 hours after serum stimulation, and AP-1 / SLG light emission image. It is a figure which shows the graph showing the time-dependent change of the emitted light intensity of c-fos / hRL. It is a figure which shows the graph showing the time-dependent change of the emitted light intensity of AP-1 / SLG.

Explanation of symbols

100 Luminescence observation system
103 container (petri dish)
104 stages
106 Luminous image pickup unit
106a Objective lens (for light emission observation)
106b Dichroic mirror
106c CCD camera
106d Split image unit
106e Filter wheel
106f Imaging lens
110 Image analyzer
112 Control unit
112a Luminous image capturing instruction unit
112b Luminescent image acquisition unit
112c Image analysis unit
112d Analysis result output part
114 Clock generator
116 storage unit
118 Communication interface
120 Input / output interface
122 Input device
124 Output device

Claims (3)

A luminescence measuring method for measuring luminescence from a biological sample,
A production step of producing the biological sample containing a plurality of predetermined genes that are luminescently labeled so that the luminescent colors are different from each other;
A stimulation step of applying a predetermined stimulus from outside the biological sample to the biological sample prepared in the manufacturing step;
An imaging step of imaging a luminescent image of the biological sample after the predetermined stimulus is given in the stimulation step, by associating a relationship between both the luminescent color and the luminescent intensity for each of the luminescent colors;
A measurement step for quantitatively measuring the emission intensity of each of the emission colors based on the emission image captured in the imaging step;
A method for measuring luminescence, comprising: The imaging step adjusts the exposure time of the camera according to the dynamic range of the camera used for imaging the luminescent image and the type of photoprotein used for the luminescent labeling, and the luminescence of the biological sample. The luminescence measurement method according to claim 1, wherein an image is captured. The imaging step repeatedly captures the luminescent image of the biological sample,
3. The measurement step according to claim 1, wherein the measurement step quantitatively measures a temporal change in the emission intensity of each of the emission colors based on the plurality of emission images captured in the imaging step. Luminescence measurement method.

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