Luminescence measurement method and luminescence measurement system   

Abstract: Disclosed is a luminescence measuring method which can produce a luminous intensity depending on the amount of a substance to be measured even when the substance occurs in a biological sample in an amount equal to or more than a given amount, and which can achieve quantitative measurement. The method is characterized by includes preparing a biological sample containing a luminescence-associated protein which is can react with a substance occurring in the biological sample in amount equal to or more than a given amount and which has a Km value equal to or higher than a predetermined value so that the luminous intensity can be quantified depending on the amount of the substance, measuring the luminescence intensity emitted from the biological sample, and outputting a result of the measurement on a regions and/or part of the biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent application Ser. No. 12/725,878, filed on Mar. 17, 2010, which is a Continuation Application of PCT Application No. PCT/JP2008/072456 filed on Dec. 10, 2008, which was published under PCT Article 21(2) in Japanese, the entire contents of each of which are incorporated herein by reference.

This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2007-319000, filed Dec. 10, 2007, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a luminescence measurement method and a luminescence measurement system for observing biological samples (for example, samples including cells). In particular, this invention relates to a method and a luminescence measurement system for performing the quantitative measurement of substances that may exist excessively in a biological sample.

2. Description of the Related Art

Conventionally, luciferase which is a luminescence enzyme or GFP which is a fluorescence enzyme has been employed in a biological function analysis. In particular, an assay utilizing the luminescence from a luciferin-luciferase reaction, etc. is widely employed as an experimental technique since the assay is advantageous, as compared with the method of employing fluorescence, in many respects such as (1) excellent S/N ratio; (2) excellent quantitative performance; (3) non-cytotoxicity in the employment of exciting light; etc.

For example, the luciferase assay is employed for quantitatively measure the quantity of ATP in a biological sample by measuring the intensity of luminescence which is steadily generated by luciferase or employed for observing the level of manifestation of a specified gene through the determination of luminescence intensity that can be performed by introducing luciferase gene, together with a reporter sequence, into cells.

On this occasion, as one example of the modification of the luciferase assay, there is employed a genetic engineering method of modifying the luciferase, i.e. luminescence enzyme itself, thereby providing the luciferase with heat resistance or high luminescence properties (see Bruce R. Branchini et al. Biochemistry, 2003, 42, pp. 10429-10436).

SUMMARY

However, the conventional measuring method using a luminescence enzyme is accompanied with a problem that if a substance to be used as a substrate is existed more than a prescribed level in a biological sample, it becomes difficult to detect differences or fluctuation of luminescence intensity that will be caused in proportion to the quantity of the substrate, thereby making it difficult to quantitatively measure an object to be measured.

Especially, when it is desired to quantitatively measure ATP in an assay system utilizing a luciferin-luciferase reaction, the quantity of ATP is required to vary according to reaction rate-limiting. However, as the quantity of ATP becomes close to a state of saturation relative to luciferase, it becomes difficult to obtain an ATP-dependent luminescence intensity.

Further, when the substance to be used as a substrate is existed excessively relative to a luminescence enzyme, a difference in luminescence intensity relative to the luminescence intensity to be brought about by the manifestation of gene is caused to generate extremely, thereby bringing about a problem that it becomes difficult to concurrently detect the quantity of the substance (for example, within the same exposure time) by making use of the same device which is designed to detect a very weak beam.

The present invention has been accomplished in view of the aforementioned circumstances and, therefore, objects of the present invention are to provide a luminescence measurement method and a luminescence measurement system, which are capable of obtaining luminescence intensity in proportion to the quantity of an object substance even in a case where the object substance is existed more than a prescribed level in an biological sample, thereby making it possible to quantitatively measure the quantity of the object substance. Further objects of the present invention are to provide a luminescence measurement method and a luminescence measurement system, which are capable of overcoming the aforementioned problem of the generation of extreme difference in luminescence intensity, thereby making it possible to concurrently detect the quantity of an object substance existing more than a prescribed level in an biological sample by making use of the same device which is designed to detect a very weak beam.

As a result of extensive studies performed by the present inventor, it has been found out that it is possible to more accurately measure the quantity of an object substance by selectively employing a luminescence-associated material which is low in affinity to the object substance provided that the object substance such as ATP is existed more than a prescribed level in a biological sample (for example, in cells). Especially, it has been found possible to obtain an object-depending luminescence intensity by suitably selecting a luminescence-associated material which is high in a Km value so as to prevent the concentration of the substance from approaching to the vicinity of Vmax in the Michaelis-Menten equation on the occasion of quantitatively measuring an object substance such as ATP. Further, with regard to the sequence of gene, it has been found out that Genji firefly (scientific name: Luciola cruciata; the name of luciferase thereof is referred to as Genji in this specification) among several kinds of firefly belonging to Luciola which are known to exist in the territory of Japan exhibits a difference in Km value as described in the experiments conducted as an embodiment of the present invention. The employment of luciferase as a luminescence marker in conformity with the intended purpose by taking advantage of this difference in Km value is one of the important subject matters of the present invention.

Namely, to solve the problems mentioned above and achieve the objectives, the luminescence measuring method for measuring luminescence emitted from a biological sample according to the present invention is characterized by comprising the step of preparing a biological sample containing a luminescence-associated protein which is capable of reacting with a substance existing more than a prescribed quantity in the biological sample, the protein having a Km value which is higher than a prescribed value which enables to quantitatively measure a luminescence intensity in dependence with the substance, the step of measuring the luminescence intensity emitted from the biological sample prepared in above-described preparing step and the step of outputting a measured result obtained from each of regions and/or sites of the biological sample, that is, a measured result in regard to the luminescence intensity obtained in above-described measuring step.

Further, the luminescence measuring method according to the present invention is characterized by the substance being ATP, the luminescence-associated protein being luciferase, and the Km value being not less than 364.

Further, the luminescence measuring method according to the present invention is characterized by the luciferase being Yaeyama Hime firefly-originated luciferase to be created based on the DNA sequence of Sequence No. 1.

Further, the luminescence measuring method according to the present invention is characterized by the step of measurement including a step of picking up a luminescence image based on the biological luminescence of the biological sample including a plurality of cells.

Further, the luminescence measuring method according to the present invention is characterized by the step of measurement including a step of measuring the luminescence intensity of each of the cells.

Further, the luminescence measuring method according to the present invention is characterized by the step of preparation comprising a step of preparing the biological sample by making use of a plurality of luminescence-associated proteins differing in the Km value from each other.

Further, the luminescence measuring method according to the present invention is characterized by the step of measurement being performed depending on the Km value.

Further, the luminescence measuring method according to the present invention is characterized by the step of output being performed depending on the Km value.

Moreover, the present invention is a luminescence measurement system for executing the luminescence measuring methods mentioned above, the system is characterized by comprising a picking up section for obtaining a luminescent image from a biological sample, an image analysis section for executing image processing for analyzing the luminescent image obtained from the picking up section, an output device for outputting a result of the analysis of image obtained from the image analysis section, and a dynamic range adjusting section for executing the picking up section and the image analysis section in conformity with the Km value of luminescent protein used in the biological sample.

Further, in the luminescence measurement system according to the present invention, it is characteristic that the dynamic range adjusting section is provided with a plurality of control modes.

Moreover, in the luminescence measurement system according to the present invention, it is characteristic that the system further comprises an input device for designating a desired region and/or a desired site in the biological sample, and a memory section for storing information input from the input device, wherein the dynamic range adjusting section is designed to output an output content in which an image and an analyzed image are formulated in conformity with the information stored in the memory section (in correspondence with the dynamic range, the picking up section and the image analysis section execute the processing based on information stored in a memory section, and an output apparatus outputs the results of imaging corresponding to the information to be output).

According to the method of the present invention, a biological sample containing a luminescence-associated protein is prepared. In this case, the protein which is capable of reacting with a substance existing more than a prescribed level in the biological sample and which has a higher Km value than a predetermined level is selected, thereby making it possible to quantitatively measure luminescence intensity in proportion to the quantity of the substance. Then, the luminescence intensity to be generated from the biological sample thus prepared is measured, thus making it possible to output measured results of each of region and/or site of the biological sample. By doing so, it is possible to perform quantitative measurement in proportion to the quantity of the substance even in a case where the substance to be measured is existed more than a prescribed level in the biological sample. Further, since it is possible to adjust the luminescence intensity so as to prevent the generation of an extreme difference in luminescence intensity, it is possible to realize the merit that the examination of many items can be concurrently performing by making use of the same very weak beam detecting apparatus. Furthermore, it is also possible to realize the merit that a plurality of regions of a biological sample or a plurality of sites in the same cell can be concurrently measured and hence it is now possible to perform the analysis of each of regions (or each of sites) which are related to a luminescence picture image that has been obtained.

According to the present invention, since the substance to be measured may be ATP and the luminescence-associated protein may be luciferase and the Km value is not less than 364 μM, it is possible to realize the merit that ATP can be rate-determined, thus making it possible to obtain a quantitative luminescence intensity depending on the existence of ATP.

According to the present invention, since the luciferase originated from Yaeyama-hime firefly that can be created based on the DNA sequence of Sequence No. 1 is employed, it is possible to realize the merit that a large ATP-dependent difference in luminescence intensity and hence a glow type luminescence pattern. Especially, as the concentration of ATP within cells is decreased by a chemical treatment from 1.35 mM to 0.65 mM, the reaction velocity is expected to decrease from about 80% of Vmax to about 60% according to Michaelis-Menten equation when luciferase (Yaeyama) originated from Yaeyama-hime firefly is employed, thereby generating a difference of 20% in the reaction velocity thus further facilitating the detection of Yaeyama as compared with the case where GL3 is employed (a difference of about 5% in reaction velocity).

According to the present invention, in the step of measuring the luminescence intensity, a luminescence picture image of biological sample containing a plurality of cells is pictured based on bioluminescence. By doing so, it is possible to obtain the merit that the regions of a plurality of cells and/or a plurality of sites within the same cell can be concurrently measured.

According to the present invention, in the step of measuring the luminescence intensity, it is performed for each one of cells. By doing so, it is possible to obtain the merit that it is possible to designate the region and/or site to be measured for each cell and to quantitatively measure a plurality of regions and sites at the same time.

According to the present invention, in the step of preparing a biological sample, the biological sample is prepared by making use of a plurality of luminescence-associated proteins differing in Km value from each other. By doing so, it is possible to obtain the merit that it is possible to perform quantitative measurement concurrently even when there is a large difference in the quantity of object substance to be measured.

According to the present invention, in the step of measuring the luminescence intensity, the measurement is performed in correspondence with the Km value. By doing so, it is possible to obtain the merit that it is possible to perform quantitative measurement by changing the intervals of image pick-up or exposure time in correspondence with the Km value of the luminescence-associated proteins. For example, the kinetic analysis as to how the dynamics of a bioactive substance which is wide in dynamic range has been changed and also the analysis of the expression/fluctuation of a specific gene as to how the transcription of the specific gene related to the dynamics of the bioactive substance has been controlled can be performed quickly or at real-time on the same cell (or cell group).

According to the present invention, in the step of outputting the results of analysis, the out is executed in correspondence with the Km value. By doing so, it is possible to obtain the merit that the results of analysis can be output after they have been subjected to conversion processing based on various parameters (coloration, contrast, dimension, display speed of moving images, etc.) in conformity with the dynamic range based on the Km value.

According to the present invention, a luminescent picture image to be derived from a biological sample is obtained in correspondence with the Km value of luminescent protein used in the biological sample and the image processing for analyzing the luminescent picture image is performed in correspondence with the Km value of luminescent protein used in the biological sample before outputting the results of the image analysis. By doing so, it is possible to obtain the merit that a plural kinds of measurement differing in dynamic range from each other in correspondence with Km value can be carried out to the same or different objects to be analyzed.

According to the present invention, the adjustment of dynamic range having a plurality of control modes is performed. By doing so, not only a measuring item having a wide dynamic range such as ATP but also a measuring item having a relatively narrow dynamic range such as the expression of a specific gene, for example, can be carried out to the same object to be analyzed, thereby making it possible to track concurrently or at real-time each of regions and/or sites on the same picture image.

According to the present invention, a desired region and/or site in a biological sample is designated through an input apparatus, and information that has been input by the input apparatus is stored in a memory section, after which, based on the information stored in the memory section, the image pick-up section and the image analysis section are actuated by means of the dynamic range adjustment section, thereby enabling the results of imaging corresponding to the information to be output by means of an output apparatus. By doing so, it is possible to obtain the merit that the dynamic range can be adjusted in conformity with the Km value of luminescence-associated proteins so as to carry out the image pick-up processing, analytical processing and output processing in correspondence with the dynamic range, thereby enabling a plural kinds of measurement differing in dynamic range from each other in correspondence with Km value to be carried out to the same or different objects to be analyzed.

FIG. 1 is a diagram illustrating one example of the overall construction of a luminescence observation system 100;

FIG. 2 is a diagram illustrating one example of the construction of a luminescent image pick-up unit 106 of the observation system 100;

FIG. 3 is a diagram illustrating another example of the construction of a luminescent image pick-up unit 106 of the observation system 100;

FIG. 4 is a block diagram illustrating one example of the construction of an image analyzer 110 of the observation system 100;

FIG. 5 is a table showing the Km value of D-luciferase and the Km value of various kinds of luciferase to ATP;

FIG. 6 is a graph showing the ultraviolet/visible light absorption spectrum of D-luciferase;

FIG. 7 is a graph showing the ultraviolet/visible light absorption spectrum of ATP;

FIG. 8 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of D-luciferase of CBG;

FIG. 9 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of D-luciferase of CBG;

FIG. 10 is a graph showing Hanes-Woolf plots obtained relative to the concentration of D-luciferase of CBG;

FIG. 11 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of D-luciferase of CBR;

FIG. 12 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of D-luciferase of CBR;

FIG. 13 is a graph showing Hanes-Woolf plots obtained relative to the concentration of D-luciferase of CBR;

FIG. 14 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of D-luciferase of ELuc;

FIG. 15 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of D-luciferase of ELuc;

FIG. 16 is a graph showing Hanes-Woolf plots obtained relative to the concentration of D-luciferase of ELuc;

FIG. 17 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of D-luciferase of Genji;

FIG. 18 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of D-luciferase of Genji;

FIG. 19 is a graph showing Hanes-Woolf plots obtained relative to the concentration of D-luciferase of Genji;

FIG. 20 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of D-luciferase of GL3;

FIG. 21 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of D-luciferase of GL3;

FIG. 22 is a graph showing Hanes-Woolf plots obtained relative to the concentration of D-luciferase of GL3;

FIG. 23 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of D-luciferase of Yaeyama;

FIG. 24 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of D-luciferase of Yaeyama;

FIG. 25 is a graph showing Hanes-Woolf plots obtained relative to the concentration of D-luciferase of Yaeyama;

FIG. 26 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of ATP of CBG;

FIG. 27 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of ATP of CBG;

FIG. 28 is a graph showing Hanes-Woolf plots obtained relative to the concentration of ATP of CBG;

FIG. 29 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of ATP of CBR;

FIG. 30 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of ATP of CBR;

FIG. 31 is a graph showing Hanes-Woolf plots obtained relative to the concentration of ATP of CBR;

FIG. 32 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of ATP of ELuc;

FIG. 33 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of ATP of ELuc;

FIG. 34 is a graph showing Hanes-Woolf plots obtained relative to the concentration of ATP of ELuc;

FIG. 35 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of ATP of Genji;

FIG. 36 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of ATP of Genji;

FIG. 37 is a graph showing Hanes-Woolf plots obtained relative to the concentration of ATP of Genji;

FIG. 38 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of ATP of GL3;

FIG. 39 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of ATP of GL3;

FIG. 40 is a graph showing Hanes-Woolf plots obtained relative to the concentration of ATP of GL3;

FIG. 41 is a graph showing the fluctuation of luminescence intensity due to an increase in concentration of ATP of Yaeyama;

FIG. 42 is a graph showing Lineweaver-Burk plots obtained relative to the concentration of ATP of Yaeyama;

FIG. 43 is a graph showing Hanes-Woolf plots obtained relative to the concentration of ATP of Yaeyama;

FIG. 44 is a table illustrating the summary of the results wherein the Km values are calculated from the Lineweaver-Burk plots and the Hanes-Woolf plots created on the basis of the photon count values obtained by means of a luminometer;

FIG. 45 is a graph showing the fluctuation of luminescence of ELuc obtained on the basis of the quantity of ATP in cells measured using a luminometer (Chronos);

FIG. 46 is a graph showing the fluctuation of luminescence of GL3 obtained on the basis of the quantity of ATP in cells measured using a luminometer (Chronos);

FIG. 47 is a photograph showing a picture of luminescence image taken immediately after the stimulation using chemicals in an ELuc-expressing HeLa cell;

FIG. 48 is a graph showing the fluctuation of luminescence intensity after the STS stimulation in each of cells (ELuc expressing HeLa cells: 1-7) that has been analyzed from the images (1-7) each rectangularly encircled in FIG. 47;

FIG. 49 is a photograph showing one example of the luminescent image photographed prior to the stimulation (before the Apoptosis induction by way of the stimulation of cell), which was performed according to the process and conditions of experiment performed in Example 4;

FIG. 50 is a photograph showing an image obtained as three measurement regions (ROI: region of interest) were designated in the luminescent image shown in FIG. 49; and

FIG. 51 is a graph showing the brightness of luminescence in three measurement regions and a table thereof.

100 Luminescence observation system 103 Vessel (Petri dish) 104 Stage 106 Luminescence image pick-up unit 106a Objective lens (for observing luminescence) 106b Dichroic mirror 106c CCD camera 106d Split image unit 106e Filter wheel 106f Imaging lens 108 Dynamic range adjusting section 110 Image analyzer 112 Control section 112a Luminescent image pick-up instruction section 112b Luminescent image acquisition section 112c Image analysis section 112d Analysis result output section 114 Clock-generating section 116 Memory section 118 Communication interface section 120 Input/Output interface section 122 Input apparatus 124 Output apparatus

DETAILED DESCRIPTION

Next, various embodiments of the luminescence measurement method and the luminescence measurement system according to the present invention will be explained in detail with reference to drawings. Incidentally, these embodiments are not intended to limit the scope of the present invention.

Especially, in the following embodiments, there may be explained cases where the present invention is applied to luminescent imaging. However, the present invention is not limited to such a luminescent imaging, but can be applied likewise to the measuring method using a luminometer, for instance.

First of all, the construction of a luminescence observation system (luminescence measuring system) 100 to be employed in the luminescence measurement method (specifically, a measuring step and an output step) according to the present invention will be explained with reference to FIG. 1, FIG. 2 and FIG. 3. FIG. 1 shows a diagram illustrating one example of the overall construction of luminescence observation system 100.

As shown in FIG. 1, the luminescence observation system 100 is constituted by a vessel 103 (specifically, it may be a Petri dish, a slide glass, a microplate, a gel-supporting member, a fine particle carrier, etc.) housing a biological sample 102, a stage 104 for mounting the vessel 103, a luminescence image pick-up unit 106, and an image analyzer 110. Herein, the luminescence observation system 100 may be constructed such that the luminescence image pick-up unit 106 for measuring a weak luminescence is disposed on the underside of the stage 104 so as to completely intercept the disturbing light from the direction above the sample on the occasion of opening or closing the cover, thereby making it possible to increase the S/N ratio of luminescent image. The luminescence image pick-up unit 106 may be formed of a laser scanning type optical system.

The biological sample 102 is formed of a living cell containing luminescence-associated protein that can be obtained by introducing a luminescence-associated gene into the protein. This biological sample 102 contains more than a prescribed quantity of a substance which is capable of reacting with the luminescence-associated protein. As for the luminescence-associated protein, it is selected from those exhibiting more than a prescribed level of Km value so as to make it possible to quantitatively determine the luminescence intensity in correspondence with the quantity of the substance. As for the object to be analyzed in this case, it may be a biological tissue including cells, or various kinds of internal organs or organ including such a biological tissue. Alternatively, the object to be analyzed may be an embryo or a bion having such a biological tissue, internal organ or organ. The stage 104 for sustaining the object to be analyzed may be designed in such a manner that specific cell(s) (one or more) to be analyzed would not be moved out of the visual field (preferably, the optical axis) for observing the luminescence of the object during a desired time period of analysis (for example, an object-fixing tool or a tracking mechanism for the stage).

The luminescence image pick-up unit 106 is, specifically, formed of an upright type luminescence microscope which is capable of picking up the luminescent image of the biological sample 102. As shown in FIG. 1, the luminescence image pick-up unit 106 is constituted by an objective lens 106a, a dichroic minor 106b, a CCD camera 106c and an imaging lens 106f. The objective lens 106a is, specifically, constructed to have a value of (the number of apertures/magnification)2 which is confined to 0.01 or more. The dichroic mirror 106b is employed for separating, color by color, the luminescence emitted from the biological sample 102, thereby measuring, color by color, the quantity of luminescence and the luminescence intensity by making use of the luminescence of two colors. The CCD camera 106c is used for taking the luminescent image and the brightfield image of the biological sample 102 that have been projected, through the objective lens 106a, the dichroic minor 106b and the imaging lens 106f, on the chip surface of the CCD camera 106c. Further, the CCD camera 106c is connected with an image analyzer 110 to thereby enable it to communicate, through a wire or wireless circuit, with the image analyzer 110. In this case, if a plurality of biological samples 102 are existed within the range of picking up, the CCD camera 106c may be designed so as to perform the image pick-up of luminescence images and brightfield images of the plurality of biological samples 102. The imaging lens 106f is employed for picking up the image (specifically, an image including the biological sample 102) that has been entered, through the objective lens 106a and the dichroic minor 106b, into the imaging lens 106f. Incidentally, in FIG. 1, there is illustrated one example wherein luminescent images each corresponding to a couple of beams separated by the dichroic minor 106b are individually taken up by a couple of CCD cameras 106c. Therefore, in a case where only one beam is employed, the luminescence image pick-up unit 106 may be constituted by the objective lens 106a, a single CCD camera 106c and the imaging lens 106f.

When it is desired to measure the quantity of luminescence and the intensity of luminescence color by color by making use of two color beams, the luminescence image pick-up unit 106 may be constituted by the objective lens 106a, the CCD camera 106c, the split image unit 106d and the imaging lens 106f as shown in FIG. 2. Further, the CCD camera 106c may be used for taking the luminescent image (split image) and the brightfield image of the biological sample 102 that have been projected, through the split image unit 106d and the imaging lens 106f, on the chip surface of the CCD camera 106c. The split image unit 106d is used for separating beam emitted from the sample 102 color by color and for measuring the quantity of luminescence and the intensity of luminescence color by color by making use of two color beams.

Further, when it is desired to measure the quantity of luminescence and the intensity of luminescence color by color by making use of a plurality of color beams (namely, when a multi-color beam is employed), the luminescence image pick-up unit 106 may be constituted by the objective lens 106a, the CCD camera 106c, a filter wheel 106e and the imaging lens 106f as shown in FIG. 3. In this case, the CCD camera 106c may be used for taking the luminescent image and the brightfield image of the biological sample 102 that have been projected, through the filter wheel 106e and the imaging lens 106f, on the chip surface of the CCD camera 106c. The filter wheel 106e is used for separating beam emitted from the sample 102 color by color by way of filter exchange and for measuring the quantity of luminescence and the intensity of luminescence color by color by making use of a plurality of color beams.

Now turn back to FIG. 1, the image analyzer 110 is, specifically, formed of a personal computer. This image analyzer 110 is roughly constituted as shown in FIG. 4 by a control section 112, a clock-generating section 114 for measuring the time of the system, a memory section 116, a communication interface section 118, an input/output interface section 120, an input apparatus 122 and an output apparatus 124, wherein all of these sections are connected with each other through a bus. The details of these constructions shown in FIGS. 1 to 4 can be understood by referring to International Patent Publication WO2006/106882 (the title thereof: A method of measuring a quantity of luminescence at a prescribed site, An apparatus of measuring a quantity of luminescence at a prescribed site, A method of measuring a quantity of manifestation, and A measuring apparatus). Since this International Patent Publication discloses a method of analyzing two kinds of medical information on the same cell by making use of both of the fluorescent image and luminescent image thereof, the method can be also applied, as another embodiment of the present invention, to the method of analysis wherein a plural kinds of fluorescent marker substance differing in dynamic range (fluorescence-associated protein such as GFP, CFP, YFP, RFP, etc., for example) are employed. Further, in the case of BRET (bioluminescence resonance energy transfer), since it is an optical phenomenon wherein bioluminescence is combined with fluorescence, it is possible to obtain the advantage that a system for exciting fluorescence can be dispensed with. Furthermore, it is also possible to utilize, as fluorescence-associated protein, Oberlin, etc. other than luciferase.

The memory section 116 is formed of storage means, so that it may be, as a specific example, a memory device such as RAM, ROM, etc., a stationary disk device such as hard disk, a flexible disk, an optical disk, etc. This memory section 116 is designed to store data obtained by the processing of each of the sections of the control section 112. The communication interface section 118 acts to mediate the communication between the image analyzer 110 and the CCD camera 106a. Namely, the communication interface section 118 is provided with a function to communicate with other terminals so as to receive or send data through a wire or wireless communicating circuit. The input/output interface section 120 is connected with an input apparatus 122 and with an output apparatus 124. As for the output apparatus 124 in this case, it is possible to employ not only a monitor (including a home television) but also a speaker or a printer (incidentally, in the following description, the output apparatus 124 may be referred to as a monitor). Further, as for the input apparatus 122, it is possible to employ a key board, a mouse, a microphone as well as a monitor which is capable of functioning as a pointing device in cooperation with a mouse. In this case, based on a luminescent image displayed in a monitor employed as the output apparatus 124, an interested region including one or more of specific cells (or a cell group) to be analyzed within a desired time period of analysis or an interested site in a cell as well as measuring item(s) are designated through the input apparatus 122 by a user, thereby enabling the positional information (adress) of the region (or site) designated in the observing visual field to be stored in the memory section 116. Due to the information thus stored in this manner, it is now possible to perform image analysis which makes it possible to check up a plurality of regions (or sites) or temporally check up the specific cells (or a cell group) on the basis of time series.

Further, the image analyzer 110 is constructed in such a manner that when the kind (or the Km value itself) of luminescence-associated protein used as an object to be placed on the stage 104 is input through the input apparatus 122 by a user, the dynamic range of each of measuring item related to one of more of luminescence-associated protein to be used is specifically selected by a dynamic range adjusting section 108 from memory information that has been stored in advance such as a look-up table, thereby enabling a control mode corresponding to the collated dynamic range to be instructed to the control section 112. In this case, once the kind of luminescence-associated protein is specified, the kind of ground substance which causes the luminescence-associated protein to radiate can be univocally determined, so that the Km value to the ground substance may be also stored in advance in the look-up table. The control section 112 is designed such that each of processes (an imaging process, an image-obtaining process, a picture image processing for analysis and a process of outputting analyzed results) according to the instructed control mode can be executed at each of the sections (a luminescent image pick-up instruction section 112a, a luminescent image acquisition section 112b, an image analysis section 112c and an analysis result output section 112d) while coordinating with the address of each of the designated regions (or sites) that have been stored in the memory section 118. Furthermore, the information related to the luminescent image and/or the analyzed results thus obtained is displayed on the picture plane of the output apparatus 124 after the information has been converted, through the dynamic range adjusting section 108, to an output format corresponding to the dynamic range. Incidentally, when it is desired to combine the information with a measuring item wherein luminescence-associated protein is employed, it is preferable to input the kind (or Km value itself) of the luminescence-associated protein. In this case however, since it is conceivable that, due to the modification of the luminescence-associated protein or fluorescence-associated protein, the Km value thereof may be varied from the Km value before the modification thereof, it is preferable to input the Km value of the protein to be actually used.

As for the instruction of picking up corresponding to the dynamic range and to be executed by the luminescent image pick-up instruction section 112a, it includes picking up intervals (for example, a video mode of not more than 5 seconds, a video mode consisting of intermediate intervals ranging from 6 seconds to 10 minutes, a time lapse mode consisting of long picking up intervals ranging from 11 minutes to 120 minutes, or a combination of these modes). As for the instruction of acquisition corresponding to the dynamic range and to be executed by the luminescent image acquisition section 112b, it includes for example the exposure time (a short time exposure mode of not more than one second, an intermediate exposure time exposure mode ranging from 2 seconds to 10 minutes and a long time exposure mode ranging from 6 minutes to 120 minutes) of an image pick-up device (for example, a CCD camera, a CMOS camera, etc.). At the image analysis section 112c, the analysis of each of the regions (or sites) related to the obtained luminescent image is executed based on such a computing algorithm that makes it possible to analyze each kind of measuring items in correspondence with the dynamic range. At the analysis result output section 112d also, the output of the output format (an image format, a numerical format, a graphic format, etc.) corresponding to each kinds of measuring items is executed. Finally, at the dynamic range adjusting section 108, the result of each kind of analyzed results that has been transmitted from the analysis result output section 112d is subjected to a conversion processing wherein the same or different output contents (image, numeral, graph, etc.) are converted based on a parameter (selected from the group consisting of color, color tone, gradation, brightness, dimension and video display speed) corresponding to the dynamic range before the result is displayed at the output apparatus 124. According to this system, a plural kinds of objects to be measured and varying in dynamic range or in Km value with respect to a substance to be measured and corresponding to measuring item can be applied to the same or different object to be analyzed. For example, a measuring item having a wide dynamic range such as ATP and a measuring item having a relatively narrow dynamic range such as a specific kind of gene expression may be applied to the same object to be analyzed, thereby realizing the advantage that each of the regions and/or site on the same picture image can be tracked concurrently and at real time. Although it is made possible to identify cells one by one as a luminescent image by superimposing the luminescent image with a bright visual field image which has been also obtained in this example, the luminescent image may not be superimposed with the bright visual field image, provided that the image pick-up device or luminescent reagent (luciferase, luciferin or other kinds of additives) is high in sensitivity. Further, as described hereinafter, depending on a purpose, even if various kinds of luminescent protein such as a glow type or flash type luminescent protein are prepared to thereby enable the same biological sample to be simultaneously labeled, it is possible to carry out the picking up and the analysis by means of the aforementioned system. Therefore, it is possible to realize a combination of assays or a multi-assay.

The control section 112 is provided with a control program such as OS (Operating System), a program regulating various kinds of procedures and an internal memory for storing data required, thereby making it possible to execute various kinds of processes based on these programs. This control section 112 is roughly constituted by the luminescent image pick-up instruction section 112a, the luminescent image acquisition section 112b, the image analysis section 112c and the analysis result output section 112d.

The luminescent image pick-up instruction section 112a is designed to instruct, through the communication interface section 118, the CCD camera 106c to execute the picking up of luminescent image and bright visual field image. The luminescent image acquisition section 112b is designed to receive, through the communication interface section 118, the luminescent image and the bright visual field image that have been taken by means of the CCD camera 106c. The control section 112 is designed to control the luminescent image pick-up instruction section 112a so as to execute repeated picking up of the luminescent image and the bright visual field image of biological sample 102.

In this case, on the occasion of performing the picking up of the luminescent image of biological sample 102 by means of the CCD camera 106c, a luminescence-associated protein having an appropriate Km value so as to prevent the generation of an extreme difference in luminescence intensity among the luminescence-associated proteins (for example, in a case where one of them is luciferase for quantitatively measure ATP and the other is luciferase for analyzing the gene expression) is selected (for example, luciferase having a higher Km value (Km>364 μM) as compared with the luciferase for analyzing the gene expression is selected as the luciferase for quantitatively measure ATP), thereby making it possible to concurrently perform the picking up in the same exposure time.

The image analysis section 112c is designed to quantitatively measure the luminescence intensity of each of luminescent colors on the basis of the luminescent image that has been obtained at the luminescent image acquisition section 112b. Further, the image analysis section 112c is designed to quantitatively measure fluctuation with time of the luminescence intensity of each of luminescent colors on the basis of a plurality of luminescent images that have been obtained at the luminescent image acquisition section 112b. The analysis result output section 112d is designed to feed the result of analysis obtained at the image analysis section 112c to the output apparatus 124. In this case, the analysis result output section 112d may be designed such that the time series data related to the luminescence intensity of each of luminescent colors that have been obtained at the image analysis section 112c are turned into a graph, which is then displayed at the output apparatus 124.

The above description illustrates one example of the construction of the luminescence observing system (luminescence measuring system) to be employed in the luminescence measuring method of the present invention. Incidentally, the output apparatus 124 may be designed such that a plurality of luminescent images corresponding to at least a portion of the time series numerical data can be fed in the form of video or parallel display to a monitor. As described above, according to the present invention, not only the kinetic analysis as to how the dynamics of a bioactive substance which is wide in dynamic range has been changed but also the analysis of the expression/fluctuation of a specific gene as to how the transcription of the specific gene related to the dynamics of the bioactive substance has been controlled can be performed quickly or at real-time on the same cell (or cell group). Therefore, it is possible to provide information accurately and quickly for use in the medical research or for clinical use (for example, response tests of drugs for the purpose of treatment, diagnosis and preventive medicine). Incidentally, in the case where a fluorescence image-taking unit is co-used in the analysis system for executing the method of the present invention, the fluorescence image-taking unit and the luminescent image pick-up unit may be placed on the same stage in such a manner that they are respectively disposed on a different optical axis or these units may be respectively constituted by a different apparatus (for example, a fluorescence microscope and a luminescence microscope) which is disposed on a different stage. Alternatively, these units may be designed to perform the picking up and the analysis while allowing a plurality of analyzing objects to successively move on the same or different stage. As for the analysis system, it can be applied also to a different kind of picking up system (various kinds of fiber scope (for example, an endoscope) and an image analysis type spectrometer (for example, a luminometer)) other than the aforementioned microscope-based system as long as the analysis system is equipped at least with the image analyzer as shown in FIG. 4. Further, in the case where the object is formed of a biological sample which has been isolated from a living body and incubated or artificially processed (cells, living tissue, internal organs (or organs), etc.), the analysis system should preferably be constructed in combination with a suitable culture apparatus so as to maintain the biological activity of the object during a prescribed period of analysis. However, when the object is an individual, the picking up can be intermittently performed while appropriately supplying or feeding oxygen and nutrition to the individual in place of the culture apparatus, thereby making it possible to execute the analysis in the same manner as described above.

(Enzymological Properties of Various Kinds of Luciferase and Application of Luciferase to Luminescence Measurement)

In this example 1, with a view to find out appropriate luciferase having a suitable Km value for the application of the present invention, the enzymological properties (Km value relative to D-luciferin and ATP) of luciferase available in the market (CBG, CBR, Eluc, Genji, GL3) were determined.

(Experiment Method 1: Calculation of Km Value of Various Kind of Luciferase Relative to D-luciferin)

D-luciferin was added to a 0.1M ATP solution (Tris-HCl (pH=8.0)) to obtain various kinds of solutions differing in ultimate concentration of D-luciferin from each other, i.e. 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM, 640 μM, respectively, thus preparing 8 kinds of solutions. Then, a 100 μg/ml luciferase solution was prepared by making use of 0.1M Tris-HCl (pH=8.0).

Then, D-luciferin solutions having the aforementioned concentrations were respectively aliquoted to a vessel having 96 wells, thus creating wells each containing 50 μl of D-luciferin solution. Then, the luciferase solution was connected with a standard pump of luminometer, after which a program was prepared so as to initiate the measurement concurrent with the addition of 50 μl of the luciferase solution to each of the wells.

Subsequently, the program was started to measure the photon-count value at each D-luciferin concentration. Based on the results obtained, Lineweaver-Burk plot and Hanes-Woolf plot were prepared to determine the Km value of each of luciferase relative to D-luciferin. In this case, the Lineweaver-Burk plot can be represented by the following formula (1) and the Hanes-Woolf plot can be represented by the following formula (2).

Formula   ( 1 )  1 v = Km v ma   x × 1 [ S ] + 1 v m   ax Formula  

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