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Luminescence measurement method and luminescence measurement system

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Luminescence measurement method and luminescence measurement system


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.


Browse recent Olympus Corporation patents - Tokyo, JP
Inventors: Ryutaro AKIYOSHI, Hirobumi SUZUKI
USPTO Applicaton #: #20120270247 - Class: 435 8 (USPTO) - 10/25/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Luciferase

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The Patent Description & Claims data below is from USPTO Patent Application 20120270247, Luminescence measurement method and luminescence measurement system.

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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).

BRIEF

SUMMARY

OF THE INVENTION Problems to be Solved by the Invention

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.

Means for Solving the Problems

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).

Effects to be Obtained from the Invention

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.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

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;



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stats Patent Info
Application #
US 20120270247 A1
Publish Date
10/25/2012
Document #
File Date
10/31/2014
USPTO Class
Other USPTO Classes
International Class
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