WO2023176174A1 - Analysis system and object analysis method - Google Patents

Abstract

The present invention addresses the problem of providing an analysis system capable of analyzing the state of an object in detail using a simple method. This analysis system for solving the aforementioned problem includes: a light source for emitting light of a given wavelength; an imaging section for obtaining the changes-over-time of an emission spectrum of a light emission control material that is capable of interacting with an object and that receives light from the light source and emits said light; and an analysis section for analyzing the state of the object from the changes- over-time of the emission spectrum. Regarding the light emission control material, the emission spectrum changes over time and/or the emission lifetime changes.

Description

Analysis system and object analysis method

The present invention relates to an analysis system and an object analysis method. In the field of fluorescence analysis and fluorescence imaging, which evaluates emission spectra, in addition to "wavelength", which has traditionally been widely used as an evaluation axis, "time" has been newly added as an evaluation axis, resulting in a quantitatively and qualitatively excellent multi-purpose method. This realizes dimensional data acquisition. This is intended to overcome the current saturation of data acquisition technology and provide analytical data that can maximize the benefits of advances in information technology such as AI. It is related to the various manufacturing and processing industries that are currently in operation, and related or incidental quality assurance, inspection, and analysis, and also includes the status of substances with respect to raw materials, manufacturing traceability, ID, etc. This invention aims to be applied to describing, recording, and evaluating information with high sensitivity.

When a substance is irradiated with light of a specific wavelength, light of a wavelength different from the irradiated wavelength may be emitted from the substance. Fluorescence is an example of light emitted from a substance. Fluorescence is light that is generated when electrons in a stable energy state (ground state) are temporarily brought into an excited state by irradiation with light, and then the excited electrons move to the ground state. The fluorescence usually occurs only for a short period of time, such as a few nanoseconds or a few picoseconds.

Here, the lifetime of the fluorescence emitted by various substances changes depending on the type of substance, the environment in which the substance is placed, and the transition process. Taking advantage of this property, fluorescence lifetime observation is performed as a method for evaluating the state of objects in physical property research of organic materials, solar cells, photocatalysts, biochemistry, etc. For example, an object or a fluorescent material that labels the object is irradiated with excitation light, and the lifetime of the fluorescence (so-called fluorescence lifetime) is measured. Then, based on the fluorescence lifetime, it is determined what state the object is in. Note that the fluorescence lifetime is the time when the fluorescence intensity at the fluorescence peak wavelength becomes 1/e with respect to its maximum value.

In recent years, in clinical trials of cancer cells, it has been discovered that canceration is promoted by proteins secreted by cells, and research to elucidate the mechanism of interactions between cells has become mainstream. The above-mentioned fluorescence lifetime measurement is performed as one method for observing protein interactions. In this method, a living tissue to be observed is irradiated with specific excitation light, and the lifetime of fluorescence unique to the living tissue is measured.

Additionally, Non-Patent Document 1 describes that the fluorescence lifetime of granules derived from a specific fluorescent protein (EGFP) and tubor protein is measured. This document states that the fluorescence lifetime of cells placed under stress decreases.

Furthermore, Patent Document 1 describes a method for measuring phosphorylation activity by binding an enzyme to a substrate labeled with a fluorescent substance in the presence of a phosphate donor and comparing the fluorescence intensity and fluorescence lifetime before and after binding the enzyme. is listed.

Patent No. 4615867

However, in the techniques described in Non-Patent Document 1 and Patent Document 1, light of all wavelengths emitted by a substance is collectively recognized as one light, and changes in the intensity are observed. Therefore, even if it is possible to recognize that the state of the object has changed from the standard state, it is difficult to analyze what is causing the state change. In other words, in the technique of Non-Patent Document 1, etc., the luminescence time and the like depend on the type of object, etc., and it is not intended to actively change the luminescence wavelength or luminescence time for observation. Furthermore, when an object emits autofluorescence, it may be difficult to distinguish between the autofluorescence and the fluorescence emitted by a fluorescent substance. Therefore, there has been a need to provide a method that allows more detailed analysis.

The current status and issues regarding these known technologies and data acquisition can be summarized as follows.

In the field of analysis and imaging using fluorescence, measurement techniques based on the manipulation of "wavelength" have been widely developed. For example, a technology that enables deep observation of living organisms by using "wavelengths" in the near-infrared region that have excellent biological permeability; There are techniques that simultaneously acquire information, and techniques that sweep the "wavelength" of the excitation light source and acquire information as three-dimensional data of observation wavelength and intensity (fluorescence fingerprint). In the present invention, the target of the emission spectrum is expressed as a spectrum including fluorescence, phosphorescence, and the like. Furthermore, "fluorescence lifetime" and "phosphorescence lifetime" are also expressed as luminescence lifetime.

There is also active development of fluorescent dyes that control the "wavelength" to facilitate the acquisition of fluorescence data. For example, there are "fluorescent labels" that emit fluorescence at different "wavelengths" in the near-ultraviolet-visible-near-infrared range, and "fluorescent probes" whose fluorescence "wavelength" and fluorescence intensity change depending on the presence of a specific substance.

On the other hand, with the development of computers and AI in recent years, the aspect of data required for analysis technology is changing. In other words, as it has become possible to store, communicate, and calculate large amounts of data, there is a need for more complex data acquisition with superior quantity and quality. This includes multidimensional data that exceeds the three dimensions that humans can grasp spatially.

The data acquisition technology by manipulating the above-mentioned "wavelength" is becoming saturated. In order to fully enjoy the benefits of advances in information technology such as computers and AI, it is desirable to acquire even more quantitatively and qualitatively superior data. For this purpose, it is essential to add another evaluation axis in addition to "wavelength" to make it multidimensional.

The present invention solves the above-mentioned problems by acquiring quantitatively and qualitatively superior multidimensional data using a simple method, and by utilizing informatization technology, the object can be advantageous in acquiring new knowledge. The purpose is to provide methods and systems for analyzing objects.

The means of the present invention will be explained.
In response to the aforementioned data acquisition technology that is becoming saturated, the present invention proposes multidimensionalization using the evaluation axis of "time."
Although there are already analysis and imaging techniques that focus on the luminescence lifetime of a dye as "time" (for example, Non-Patent Document 1), these techniques observe monotonically decaying fluorescence, and are based on "wavelength" x "time". It was not a technology to obtain multidimensional data. The present invention realizes quantitatively and qualitatively superior data acquisition by multidimensionalizing "wavelength" x "time."

Since the luminescence lifetime of general fluorescence is short on the order of nanoseconds, time resolution is difficult, and the effectiveness of using the time axis as an evaluation axis is limited. The use of long-lived luminescence, such as phosphorescence or thermally activated delayed fluorescence, facilitates time resolution and makes it possible to maximize the use of the time axis as an evaluation axis.

Even if we acquire multidimensional data of "wavelength" x "time" for fluorescence that decays monotonically, we can only obtain information about the constant "fluorescence lifetime", and the benefits of multidimensionalization are limited.
The present invention further proposes an ``analysis/imaging method using fluorescence whose emission spectrum fluctuates over time'' as a method for improving the ``quality'' of multidimensional data of ``wavelength'' x ``time.'' As a result, while conventional fluorescence analysis/imaging methods using a time axis could only obtain time changes in a simple scalar quantity called fluorescence intensity, the method of the present invention can obtain time changes in multidimensional data such as emission spectrum = color. It becomes possible to obtain.

In the present invention, a new "luminescent material" has been developed as a specific means for realizing the above analysis method. With this luminescent material, the above-mentioned "fluorescence whose emission spectrum fluctuates over time" can be realized. Furthermore, it is also possible to extend the fluorescence lifetime or to give this luminescent material the ability to show a sharp change due to external factors. Furthermore, the use of this luminescent dye has the advantage that data can be acquired with a simpler configuration (conventional measuring equipment) compared to ordinary fluorescent dyes that exhibit short-lived and monotonically decaying fluorescent dyes.

Note that the "wavelength" here refers to a specific wavelength range (for example, 400 nm to 700 nm), or may also refer to a specific wavelength (for example, 820 nm).

As mentioned above, the present invention realizes quantitatively and qualitatively superior multidimensional data acquisition. In order to achieve this, an analysis system that reflects one aspect of the present invention includes a light source that emits light of a predetermined wavelength, a light source that can interact with an object, and that emits light upon receiving light from the light source. The light emission control material has an imaging unit that acquires changes over time in the light emission state of the control material, and an analysis unit that analyzes the state of the object based on the change over time in the emission spectrum of the light emission state, and the light emission control material has a light emission lifespan. and/or the emission spectrum changes over time.

A method for analyzing a target according to one aspect of the present invention includes a method for analyzing a target, and a light emission control material that can interact with the target and emits light upon receiving light of a predetermined wavelength, in contact with the target. A step of irradiating the light emission control material with light, a step of obtaining a change over time in the light emission state of the light emission control material that has received the light, and a step of analyzing the state of the object from the change over time in the emission spectrum of the light emission state. The luminescence control material has a luminescence lifetime that changes and/or a luminescence spectrum that changes over time.

According to the analysis system and object analysis method according to an embodiment of the present invention, even in simple measurements, it is possible to obtain new quantitatively and qualitatively excellent multidimensional data such as "wavelength" x "time". It becomes possible to realize. With the development of information technology, by combining new multidimensional data that is superior in quantity and quality, we can now distinguish the type of object itself, which was previously impossible, and the structure of the object itself. This can be used in situations where changes or changes in the interaction between an object and a luminescence control material are observed.

FIG. 1 is a flowchart illustrating an example of a method for analyzing an object according to an embodiment of the present invention. FIG. 2A shows an emission spectrum, which is one of the indicators of the luminescent state, when light with a wavelength of 380 nm is irradiated on luminescent material 1 and luminescent material 2, and FIG. 2 is a graph showing changes over time in the light intensity of the emission peak wavelength when irradiating the light emitting material 2 and the light emitting material 2, respectively. FIG. 3 is a graph showing temporal fluctuations in the emission spectrum when the emission control material including the luminescent material 1 and the luminescent material 2 in FIG. 2 is irradiated with light having a wavelength of 380 nm. FIG. 4 is a diagram showing changes in the color of light emitted by the light emission control material shown in FIG. 3. FIG. 5 is a flowchart showing an example of a method for selecting a light emission control material. FIG. 6 is a graph schematically showing the relationship between luminescence intensity and time when two types of materials with different luminescence lifetimes are used. FIG. 7 is a schematic diagram showing an example of the configuration of an analysis system according to an embodiment of the present invention. FIG. 8 is a diagram in which the chromaticity of the data image acquired in Example 2 is measured and the chromaticity is expressed in a chromaticity diagram for each measurement time. FIG. 9 is a diagram in which the chromaticity of the data image acquired in Example 3 is measured and the chromaticity is expressed in a chromaticity diagram for each measurement time.

Hereinafter, the present invention will be described in detail based on embodiments. However, the present invention is not limited to these embodiments.

1. Analysis Method A method for analyzing an object according to an embodiment of the present invention will be described first, and then an analysis system that can be used in the analysis method will be described.

In this embodiment, as shown in the flowchart of FIG. 1, the light emission control material is irradiated with light while the target object and the light emission control material are in contact (S11, hereinafter also referred to as "light irradiation step"). Then, the change over time in the light emission state (so-called emission spectrum, etc.) emitted by the emission control material is acquired (S12, hereinafter also referred to as "data acquisition step"), and the state of the object is analyzed from the change over time in the emission spectrum. (S13, hereinafter also referred to as "analysis step"). Each step of the method for analyzing an object according to this embodiment will be described below.

(Light irradiation process)
In the light irradiation step S11, the target object and the emission control material are brought into contact with each other, and the object (especially the emission control material) is irradiated with light (this refers to irradiation with light having a so-called specific excitation spectrum). . The method of contact between the object and the emission control material is appropriately selected depending on the shape, condition, etc. of the object and the emission control material. For example, when both the target object and the emission control material are fluids such as liquids or particles, they may be mixed and brought into contact with each other. On the other hand, if either the target object or the emission control material is molded into a specific shape or fixed to various molded objects, the molded object containing one (for example, the emission control material) may The molded article may be applied with a liquid containing the object (object) or immersed in a liquid containing the other object.

Here, the "target object" in this specification may be either an organic substance or an inorganic substance, for example, it may be a natural substance such as a component derived from a living body, or it may be an artificially synthesized substance. good. Furthermore, it may be a substance with a known molecular structure or a substance with an unknown molecular structure. Further, the target object may be a single compound or a mixture of multiple compounds.

Further, only the target object may be brought into contact with the emission control material, but a composition containing the target object and other components (for example, a solvent, impurities, etc.) may be brought into contact with the emission control material.

On the other hand, the term "emission control material" as used herein refers to a material that emits light (emits an emission spectrum) upon receiving light of a predetermined wavelength (excitation spectrum), and whose emission spectrum changes upon interaction with an object. be. Further, the "emission control material" may be a material whose luminescence lifetime changes depending on changes in the surrounding environment, changes in the object itself, or differences in objects, and/or whose emission spectrum changes over time. The type of light emitted by the light emission control material upon receiving light of a predetermined wavelength is not particularly limited, and may be either fluorescence or phosphorescence.

The above phrase "the emission spectrum changes due to interaction with the target object" means that the emission control material changes due to changes in the target object itself or other changes (for example, changes in the structure, concentration, dispersion state, etc. of the target object). This refers to changes in the lifespan and emission spectrum of emitted light.

As mentioned above, the "emission lifetime" is the time during which the emission intensity at the emission peak wavelength becomes 1/e of its maximum value. When a substance whose luminescence lifetime changes is used as a luminescence control material, by measuring changes in light intensity over a certain period of time, it becomes possible to analyze the state of the object in detail. Note that two or more kinds of substances whose luminescence lifetimes change may be combined and used as a luminescence control material.

On the other hand, "the emission spectrum changes over time" means that the waveform of the emission spectrum changes depending on time when the emission spectrum is observed at regular intervals. Such a light emission control material can be obtained, for example, by combining two or more types of light emitting materials that have different maximum emission wavelengths and different light emission lifetimes. The number of luminescent materials contained in the luminescence control material is preferably 2 or more and 5 or less, more preferably 2 or more and 3 or less. However, if there is a material whose emission spectrum waveform changes over time, this may be used as the emission control material.

Changes in the emission spectrum of the emission control material of this embodiment over time will be explained using an example of a emission control material containing two types of luminescent materials. The luminescence control material contains luminescent material 1 (anthracene derivative) and luminescent material 2 (perylene bisimide derivative) at a ratio of 10:1 (mass ratio). FIG. 2A shows emission spectra when luminescent material 1 and luminescent material 2 are each irradiated with light having a wavelength of 380 nm. In addition, FIG. 2B shows the change over time in the light intensity of the light emission peak wavelength (wavelength 455 nm) when light with a wavelength of 380 nm is irradiated onto the light emitting material 1, and the light emission peak when the light emitting material 2 is irradiated with light with a wavelength of 380 nm. It shows the change over time in the light intensity of the wavelength (wavelength 600 nm). The luminescent lifetime of luminescent material 1 is 50 nanoseconds, and the luminescent lifetime of luminescent material 2 is 200 nanoseconds.

FIG. 3 shows changes in the emission spectrum when a light emission control material containing such luminescent material 1 and luminescent material 2 is irradiated with light at a wavelength of 380 nm. The time in FIG. 3 indicates the number of seconds that have elapsed since the moment when the irradiation of light with a wavelength of 380 nm ended as 0. As shown in FIG. 3, in the light emission control material, the intensity of each wavelength does not uniformly decrease over time, but the amount of decrease varies depending on the wavelength. Therefore, the waveform of the emission spectrum changes over time. For example, in the emission spectrum from 0 ns to 50 ns, light with a wavelength of about 450 nm is the strongest. On the other hand, in the emission spectrum after 100 ns, the wavelength of 600 nm is the strongest. In such a light emission control material, the observed color changes over time. Changes over time in the chromaticity of light emitted by the light emission control material are shown in the color coordinates of FIG. 4. The numbers in the color coordinates correspond to the times in FIG. In other words, by using such a light emission control material, it becomes possible to analyze the state of an object not only by changes in light intensity but also by unprecedented evaluation axes such as changes in color. It becomes possible to analyze the state in detail.

Here, in this embodiment, an example of a method for selecting a light emission control material whose light emission intensity distribution changes depending on the surrounding environment is shown in the flowchart of FIG. However, the method for selecting the emission control material is not limited to this method. First, a sample is prepared by bringing a desired object into contact with a material that is a candidate for an emission control material (hereinafter also referred to as "candidate material") (S101). The candidate material is preferably a combination of a plurality of light emitting materials. Then, the sample is irradiated with pulsed laser light (S102). After irradiation with the laser beam, the emission spectrum of the sample is photographed multiple times (timings a to n in FIG. 5) to obtain images a to n (S103). Then, the images a to n are analyzed to determine whether the output values could be separated on the time axis, that is, whether the color changed over time or the waveform of the emission spectrum changed (S104). If the output values cannot be separated on the time axis, the candidate material is changed (S106) and the same process is performed. In addition, after selecting a plurality of candidate materials using this method, it is confirmed whether the output value changes depending on the state change of the target object (S105), and if the output value can be separated on the time axis and the target material is A candidate material whose output changes depending on changes in the state of the object may be selected as the emission control material (S107).

The luminescent material used in the luminescence control material may be a known inorganic material that emits fluorescence, or may be an organic compound, an organometallic complex, or the like that emits fluorescence or phosphorescence. Among these, organic compounds and organometallics tend to interact with target objects (molecules, ions, etc.), and their luminescence lifetime, emission wavelength, and luminous efficiency tend to change depending on the environment in which the target object exists. Therefore, it is possible to analyze not only the target object but also the environment around the target object, so it is more preferable that the luminescent material is an organic compound or an organometallic complex. In addition, organic compounds and organometallic complexes in particular can have various functions (sensing properties, switching properties, absorption/emission colors, solubility, etc.) adjusted by molecular design, and are superior to inorganic compounds in terms of functional expandability. ing.

Among the above-mentioned organic compounds and organometallic complexes, it is preferable to combine at least one of a thermally activated delayed fluorescent dye and a phosphorescent dye because of their long luminescence lifetime. For example, if the luminescence lifetime of the luminescence control material is a few nanoseconds, a femtosecond laser may be required to oscillate a sufficiently short pulse of excitation light during the light irradiation described below, or a femtosecond laser may be required to oscillate a sufficiently short pulse of excitation light, or the data acquisition method described below. Processes may require fast gated image intensifier cameras with nanosecond time resolution. On the other hand, if the emission control material contains a thermally activated delayed fluorescent dye or a phosphorescent dye, it is possible to use a relatively low-spec optical system configuration, for example, it is possible to use an LED or a semiconductor laser as a light source. This may make it possible to use general-purpose high-speed cameras with time resolution from microseconds to milliseconds. Further, by using a thermally activated delayed fluorescent dye or a phosphorescent dye, even if the object emits autofluorescence, it becomes possible to easily distinguish it from this. Furthermore, it becomes easier to separate the light from the light source.

The heat-activated delayed fluorescent dye that can be used in this embodiment will be described below. Generally, a part of the dye in the singlet excited state generated by photoexcitation is converted to the more stable triplet excited state by internal intersystem crossing. In ordinary fluorescent dyes, the triplet excited state relaxes in a non-radiative process, but when the energy difference between the singlet excited state and triplet excited state is small enough to cause reverse intersystem crossing due to heat, the reverse It exhibits delayed fluorescence from the singlet excited state regenerated by intercrossing. In this specification, the fluorescence is referred to as thermally activated delayed fluorescence, and the dye that emits the thermally activated delayed fluorescence is referred to as a thermally activated delayed fluorescent dye. The heat-activated delayed fluorescent dye has a long luminescence lifetime compared to ordinary fluorescent dyes. Examples of heat-activated delayed fluorescent dyes include compounds represented by the following chemical formula.

Further, phosphorescent dyes that can be used in this embodiment will be explained below. Phosphorescence is the emission of light that occurs during the transition from the triplet excited state to the singlet ground state when a substance is excited. Compared to normal fluorescence, which originates from allowed transitions and occurs quickly, phosphorescence originates from spin-forbidden transitions, and thus has a long luminescence lifetime. In many dyes, the transition from the spin-forbidden triplet excited state to the singlet ground state is deactivated non-radiatively, and no phosphorescence is observed under room temperature conditions, but complexes of heavy elements are forbidden due to spin-orbit interactions. is relaxed and exhibits phosphorescence at room temperature. Materials exhibiting such phosphorescence are referred to herein as phosphorescent dyes. Specific examples of phosphorescent dyes include compounds represented by the following chemical formula.

Further, the method of irradiating the above-mentioned object and time evaluation with light while in contact with them is not particularly limited, as long as it is a method that can irradiate light of a desired wavelength for a certain period of time.

It is preferable that the light irradiation in this step be performed only for a short time. If you continue to irradiate light from the light source even after the light emission control material starts emitting light, the light emitted from the light emission control material and the light from the light source may mix, making it difficult to analyze the target object. . Specifically, the light irradiation time is preferably several nanoseconds to several tens of nanoseconds. By irradiating the light for this period of time, it is possible to sufficiently excite the emission control material. Further, within this range, it is unlikely to affect the observation of light emitted by the emission control material. Furthermore, in the case of several nanoseconds or more, a relatively inexpensive laser or the like can be used.

The wavelength of the light irradiated in this step is appropriately selected depending on the type of emission control material, sensor sensitivity, objective lens aberration, etc., but is usually preferably 200 nm to 1700 nm, more preferably 300 to 800 nm. When the wavelength of the irradiated light is within this range, there is no need to use a special light source, and the light emission control material and the object are less likely to be affected.

(Data acquisition process)
In the data acquisition step S12, data (hereinafter also simply referred to as "data") representing changes over time in the emission spectrum of the emission control material that received light in the above-described light irradiation step S11 is acquired. The data acquisition method is appropriately selected depending on the type of emission control material and the like. For example, images may be captured intermittently or continuously. Furthermore, the emission spectrum may be measured multiple times. Among these, a method of intermittently acquiring images is preferred.

When images are acquired intermittently, the number of times the images are acquired may be two or more times. In addition, the interval is not particularly limited; for example, in FIG. It may be selected as appropriate depending on the type of light, luminescence life, etc. Note that the data acquisition step of acquiring a series of data may be repeated. When the data acquisition step is repeated (for example, N times), the signal/noise ratio (SN ratio) improves according to the number of integrations N. The number of times the data acquisition is repeated is preferably selected as appropriate depending on the desired signal-to-noise ratio and analysis work time.

In addition, when repeating the data acquisition process to acquire a series of data, the intervals at which multiple images are acquired should be determined by the luminescence lifetime of the luminescence control material (if multiple luminescent materials are used, the luminescent material with the longest luminescence lifetime) When the luminescence lifetime of the material) is τ _2l , it is preferable to carry out the process at intervals proportional to τ _2l . By setting the image acquisition interval within this range, it is possible to more accurately understand the change over time in the light emitted by the light emission control material.

On the other hand, the upper limit of the time for performing one data acquisition step is preferably 1 second. In addition, when the luminescent lifetime of the luminescent control material (if multiple luminescent materials are used, the luminescent material with the shortest luminescent lifetime) is τ _1l , the time for performing one data acquisition process (data acquisition time ) is preferably τ _1l or more. By performing the data acquisition process for τ _1l or more, it becomes easier to understand the change in the luminescence control material over time, and it becomes easier to understand the state of the object more accurately. On the other hand, even if the above-mentioned heat-activated delayed fluorescent dye or phosphorescent dye is used as a light emission control material, it is usually difficult to obtain light emission longer than 1 second.

In particular, the timing of performing the data acquisition step is preferably set as follows, for example, when two types of luminescent materials with different luminescent lifetimes are used. FIG. 6 is a graph schematically showing the relationship between luminescence intensity and time when two types of materials with different luminescence lifetimes are used. As shown in FIG. 6, the luminescent lifetime of the material 1 with a short luminescent lifetime is τ _1l , the time when the luminescent intensity of the luminescent material 1 becomes 0 is τ _1e , and the luminescent lifetime of the luminescent material 2 with a long luminescent lifetime is τ _2l When the time when the luminescence intensity of the luminescent material 2 becomes 0 is τ _2e , the data acquisition start timing Ts is preferably τ _1l ≦Ts≦τ _1e . On the other hand, the end timing Te of data acquisition preferably satisfies τ _1e ≦Te≦τ _2e . Also, at this time, the ratio expressed by S2/S1 between the integral value S1 of the luminescent intensity of the luminescent material 1 from Ts to Te and the integral value S2 of the luminescent intensity of the luminescent material 2 from Ts to Te is large. Preferably, the data acquisition start timing Ts and the data acquisition end timing are set so that the numerical value becomes large.

(Analysis process)
In the analysis step S13, the state of the object is analyzed from the data acquired in the data acquisition step S12 (data regarding changes over time in the emission spectrum of the emission control material). In the analysis step S13, desired states such as the structure, concentration, dispersion state, bonding state of the target object and other substances, etc. of the target object are determined from the plurality of data or continuous data acquired in the data acquisition step S12 described above. , perform the analysis.

The data analysis method performed in the analysis step S13 is selected as appropriate depending on the type of data acquired in the data acquisition step S12. For example, when the data is a plurality of photographs or videos, the state of the object may be analyzed by identifying the emission spectrum of the light emitted by the luminescence control material from the photographs or videos at each time. Alternatively, for example, as shown in FIG. 4, the chromaticity of the light emitted by the light emission control material may be expressed in color coordinates, and the state of the object may be analyzed from the degree of change in chromaticity. In addition, for example, it is possible to create a histogram of fluorescence intensity on a pixel-by-pixel basis, or to identify and display the XY coordinate position of a pixel having a value above a certain threshold (for example, display a heat map, etc.), etc. Various analyzes for extracting the amount may be appropriately selected and performed.

In addition, in the above analysis, data when the target is in a standard state and data when the target is in a predetermined state are acquired in advance, and these data and the data acquired in the data acquisition process described above are used. The object may be analyzed by comparing the

Alternatively, analysis may be performed based on a learned model generated in advance by machine learning. When performing analysis based on a trained model, the state of the target object can be determined by applying the data regarding the change in the emission spectrum of the luminescence control material over time obtained in the data acquisition step S12 to the trained model. It is possible to judge (predict) from the accumulated data etc.

In machine learning, for example, a process similar to the data acquisition process S12 described above is performed multiple times. Then, multiple predictive models are constructed based on this. Then, by combining the results of the plurality of prediction models, a trained model that can predict information (for example, structure, etc.) regarding the state of the object is created.

If the state of the object is known in advance, the above prediction model can be constructed by performing machine learning using the features of multiple data as explanatory variables and the state of the object as the objective variable. . As explanatory variables, numerical values representing the characteristics of the data described above and numerical values calculated from them can be used. On the other hand, the objective variable can be selected as appropriate depending on the purpose of the analysis, and is not limited to the state of the object, but may be any other variable related to the object.

Machine learning may be supervised learning or unsupervised learning. Note that supervised learning refers to a learning method that learns the "relationship between input and output" from learning data with correct answer labels. Unsupervised learning refers to a learning method that learns the "structure of a data group" from training data without correct answer labels.

Additionally, machine learning may be reinforcement learning, deep learning, or deep reinforcement learning. Note that reinforcement learning is a learning method that learns the "optimal sequence of actions" through trial and error. Deep learning is a learning method that uses large amounts of data to learn features contained in the data in a step-by-step manner. Deep reinforcement learning refers to a learning method that combines reinforcement learning and deep learning.

General analysis methods (algorithms) can be applied to machine learning. Machine learning includes, for example, linear regression (multiple regression analysis, partial least squares (PLS) regression, LASSO regression, Ridge regression, principal component regression (PCR), etc.), random forests, decision trees, support vector machines (SVM), A prediction model constructed by an analysis method selected from support vector regression (SVR), neural network, discriminant analysis, etc. can be applied.

(effect)
As described above, in conventional methods for measuring fluorescence lifetime, light of all wavelengths emitted by a fluorescent substance is recognized as one light, and changes in the intensity are measured. Therefore, it was difficult to analyze the detailed state of the object. Furthermore, when the object emits autofluorescence, it may be difficult to identify the light originating from the fluorescent substance.

On the other hand, in this embodiment, the above-mentioned light emission control material is used, and the change over time of the emission spectrum of the light emission control material is obtained. In this method, since the emission spectrum of the emission control material changes over time, it is possible to evaluate the condition of the object not only by the lifespan of the light but also by unprecedented evaluation axes such as changes in color. It is possible to analyze the state of things in detail. Furthermore, with this method, even when the object emits autofluorescence, the light emitted by the emission control material can be identified.

In other words, according to the analysis method of this embodiment, it is possible to intentionally control the temporal fluctuation of the emission spectrum of light emitted by the target object and the system containing the emission control material using the emission control material. Acquire quantitatively and qualitatively excellent multidimensional data without using equipment or going through complicated processes, and use information technology such as computers and AI with the new multidimensional data mentioned above. By combining them, it becomes possible to analyze the target object in more detail.

2. Analysis System The method for analyzing the object described above can be performed by the following analysis system. However, the system that performs the above analysis method is not limited to this analysis system, and is not limited to the configuration shown below.

FIG. 7 shows a schematic diagram showing the configuration of the analysis system of this embodiment. The analysis system 200 of this embodiment includes a light source that can interact with a target object and emits light of a predetermined wavelength to a sample 20 that includes a light emission control material that emits light upon receiving light from the light source and the target object. 21, an imaging unit 22 that acquires changes over time in the emission spectrum of the emission control material, and an analysis unit 23 that analyzes the state of the object from the changes in the emission spectrum over time. The analysis system 200 also includes an optical unit 24 that guides the light emitted from the light source 21 so as to irradiate the sample 20 and guides the light emitted from the sample 20 to the imaging section 22 side; A movable stage 25 for placing the light source 21, an objective lens 26 for condensing the light emitted from the light source 21 onto the sample 20 or condensing the light emitted by the sample 20 (light emission control material), and the light emission timing of the light source 21. It further includes a control unit 27 that controls the imaging timing of the imaging unit 22 and the like. Each configuration will be explained below.

(sample)
The sample 20 used in the analysis system 200 of this embodiment may be in a state where the luminescence control material and the object interact. The light emission control material and the target object are the same as those in the above-described method for evaluating a target object. In this embodiment, as an example, the sample 20 is a plate on which a target object (aqueous solution) is impregnated with particulate luminescence control material, but the shape and state of the sample are not limited to this configuration. .

(light source)
The light source 21 is not particularly limited as long as it can irradiate the sample with light of a desired wavelength for a desired period of time. Examples of preferred light sources include picosecond diode lasers, wavelength tunable lasers, supercontinuum light sources, LED light sources, and the like. According to these light sources, the sample 20 can be irradiated with light of a predetermined wavelength only for a short time. In view of the desired signal/noise ratio (SN), it is preferable to select a light source that is sufficiently quenched before the emission control material emits light.

(Imaging unit)
The imaging unit 22 is not particularly limited as long as it is capable of acquiring changes over time in the emission spectrum of the emission control material, and is appropriately selected according to the type of data to be acquired. The imaging unit 22 may be a known CCD camera, CMOS camera, or the like that captures images of the sample 20 multiple times intermittently or continuously.

(Analysis Department)
The analysis section 23 may be any means that can analyze the data acquired by the above-mentioned imaging section. For example, the target object may be analyzed by reading separately acquired reference data and comparing the reference data with data acquired by the imaging unit 22. The analysis unit 23 also reads out the learned model from an external storage device (not shown) or internal storage means (not shown), and combines the learned model with the data acquired by the imaging unit 22. You may perform a comparison operation.

Such an analysis unit includes storage means such as a hard disk drive (HDD), solid state drive (SSD), and read-only memory (ROM) that store programs and data, and a central processing unit that executes programs and performs calculation processing. A general computer (general-purpose computer) equipped with a device (CPU) can be used. Further, the computer may further include input means such as a keyboard and mouse, and output means such as a monitor and a printer.

(Other configurations)
As described above, the analysis system 200 of this embodiment includes the optical unit 24. The optical unit 24 includes an excitation light filter 222 for cutting unnecessary wavelength light emitted from the light source 21, and a dichroic filter that guides the light from the light source 21 to the sample 20 side while transmitting the light emitted by the emission control material. The mirror 223 or a fluorescence filter 224 that cuts unnecessary wavelengths of light from the light transmitted through the dichroic mirror 223 may be provided. Although not shown, the optical unit 24 may further include a light source for alignment, an optical path switching element, and the like. Further, it may include a beam expander for expanding the laser spot and a diffuser plate for reducing speckles. Furthermore, in order to perform observations for purposes other than this one within the same device, it may be separately equipped with general fluorescence, polarization, or differential interference related elements, light sources suited to these uses, and the like.

The analysis system 200 also includes a movable stage 25 for holding the sample 20. The movable stage 25 is a member for focusing the imaging section 22 on the sample 20, and is configured to be freely movable.

Further, the analysis system 200 includes an objective lens 26 for condensing the light emitted from the light source 21 onto the sample 20 or condensing the light emitted by the sample 20 (light emission control material). The objective lens 26 is similar to a known objective lens, and its magnification is appropriately selected.

The analysis system 200 further includes a control unit 27 that adjusts the timing at which the light source 21 emits light and controls the timing at which the imaging unit 22 starts and ends imaging. The control unit also includes storage means such as a hard disk drive (HDD), solid state drive (SSD), and read-only memory (ROM) that store programs and data, and a central processing unit (CPU) that performs program execution and calculation processing. ) can be used.

(effect)
In the analysis system of this embodiment, the above-mentioned light emission control material is used, and the imaging unit acquires the change over time in the light emission spectrum of the light emission control material. With this system, it is possible to analyze a target object in detail based on changes in the emission spectrum of the emission control material, changes in chromaticity of light emitted by the emission control material, and the like. Further, the analysis system does not require complicated configurations or operations, and is extremely useful in various fields.

(others)
"Multidimensionalization of acquired data" is not limited to the above, and possible uses and specific examples regarding multidimensionalization of fluorescence analysis and fluorescence imaging data are listed below.

Fluorescence intensity plotted on a two-dimensional plane of "excitation wavelength" x "emission wavelength" is called a "fluorescence fingerprint," and technology that uses it as a "fingerprint" to represent the unique properties of a substance is becoming common. . On the other hand, material-specific data obtained as four-dimensional data by adding the time axis to this three-dimensional data is defined as a "phosphorescent fingerprint." In order to expand the time axis range, it is desirable to use a luminescent dye with a long luminescence lifetime compared to fluorescence, so the name uses the typical "phosphorescence", but this luminescence mechanism is not necessarily limited to phosphorescence. The main idea is that it is not a physical object, but four-dimensional data that includes a time axis. This is the same as the currently widely recognized "fluorescent fingerprint" in which the emission mechanism is not limited to fluorescence in the narrow sense.

While the three-dimensional data of a fluorescent fingerprint can be depicted in a three-dimensional space that can be grasped by humans, the four-dimensional data of a phosphorescent fingerprint cannot be depicted directly in a three-dimensional space. However, this difference is not a problem in AI that calculates multidimensional data.

Additionally, if humans need to understand the meaning of data, it is useful to slice it along a certain axis and present it as multiple three-dimensional data. The axis for slicing is selected according to the purpose. For example, slicing along the time axis allows you to understand the temporal changes in "excitation wavelength x emission wavelength x emission intensity," and slicing along the emission wavelength allows you to understand the temporal changes in "excitation wavelength x emission time x The dependence of the emission intensity on the emission wavelength can be understood.

The wavelength range that can be used for efficient analysis and imaging is limited due to the absorption of visible light by hemoglobin, water scattering, and autofluorescence of biomolecules. By using phosphorescent fingerprints, it is possible to maximize the amount of information in a limited wavelength range using a new axis, the time axis, making it an analysis and imaging method with a high degree of superiority in the biological field.

It is also possible to apply multicolor staining, which has traditionally been performed using the wavelength axis in analysis and imaging in the biofield, to using the time axis. While there was a limit to the number of colors in a limited wavelength range, by using a luminescence control material whose emission color changes over time, it is possible to have a virtually unlimited number of colors.

Phosphorescent fingerprints are superior in identifying whether two samples come from the same source or not. With phosphorescent fingerprints, the amount of information per sample can be greatly increased by adding a time axis. This allows identification, which is normally possible by combining multiple measurements, to be made possible with a single measurement. For example, difference analysis is important in forensic investigation of crimes, but the use of phosphorescent fingerprints reduces the number of measurements required for identification, leading to a reduction in the time required for evidence investigation. In addition, phosphorescent fingerprint measuring devices can be lighter and more portable than analytical devices such as mass spectrometers that are often used for transfer identification analysis, and can be used as a simple analysis method in the field.

We have already discussed the effectiveness of using long-lived luminescent species as a means of utilizing time as a new evaluation axis, and specific luminescent species include ``phosphorescence'' and ``thermally activated delayed fluorescence.'' It will be done. It is known that these luminescent species behave differently from normal fluorescence due to differences in the luminescence principle, and by utilizing this, it is also possible to add a further evaluation axis (≈dimension). For example, phosphorescence is a phenomenon of light emission from a triplet excited state, and it is known that this triplet excited state is deactivated by the presence of dissolved oxygen. Furthermore, it is known that the luminous efficiency and luminescent lifetime of thermally activated delayed fluorescence change depending on the temperature. These phenomena can be used passively as sensors to know the environment in which the luminescent material exists, but by using them more actively, they can be utilized as a new evaluation axis (≒ dimension). That is, by performing measurements while performing operations such as blowing in oxygen or heating, it becomes possible to acquire multidimensional data with new dimensions such as "oxygen concentration" and "temperature."

[Example 1]
As luminescence control materials, luminescent material 1 (anthracene derivative) and luminescent material 2 (perylene bisimide derivative) were dissolved in toluene at a ratio of 10:1 (mass ratio). Silica gel was added to the solution and the solvent (toluene) was distilled off to obtain a particulate luminescence control material in which luminescent materials 1 and 2 were adsorbed. Furthermore, soft drink 1 was prepared as a target object, and a dispersion liquid in which the time evaluation material was dispersed in the target object was placed on a plate, which was designated as sample 20.

Then, the analysis system 200 shown in FIG. 7 was prepared, the plate was placed on the movable stage 25, and the objective lens 26 was focused. Then, the sample 20 was irradiated with excitation light from the light source 21 . After the excitation light irradiation from the light source 21 was completed, six data images were acquired by the imaging unit 22 every 50 ns. The chromaticity of the data image acquired by the imaging unit 22 was measured and represented in a chromaticity diagram for each measurement time. The results are similar to the graph shown in FIG.

[Example 2]
A chromaticity diagram was obtained in the same manner as in Example 1, except that the type of soft drink was changed and soft drink 2 was used. The results are shown in FIG.

[Example 3]
A chromaticity diagram was obtained in the same manner as in Example 1, except that the type of soft drink was changed and soft drink 2 was used. The results are shown in FIG.

This application claims priority based on Japanese Patent Application No. 2022-041452 filed on March 16, 2022. All contents described in the application specification and drawings are incorporated herein by reference.

According to the analysis system and analysis method of the present invention, it is possible to easily analyze a target object without going through complicated steps. The analysis method and analysis system are useful in various fields. For example, in the industrial field, it can be used to control the functionality and quality of materials. It can also be used in the development of materials, etc. On the other hand, in the medical field, it can also be applied to acquiring information regarding living organisms.

21 Light source 22 Imaging unit 23 Analysis unit 24 Optical unit 25 Movable stage 26 Objective lens 27 Control unit 222 Excitation light filter 223 Dichroic mirror 224 Fluorescence filter

Claims (12)

1. a light source that emits light of a predetermined wavelength;
   an imaging unit that is capable of interacting with a target object and acquires changes over time in a light emission state of a light emission control material that emits light upon receiving light from the light source;
   an analysis unit that analyzes the state of the object based on changes over time in the emission spectrum of the light emission state;
   The luminescence control material is an analysis system in which the luminescence lifetime changes and/or the luminescence spectrum changes over time.
2. The luminescence control material includes two or more types of luminescent materials,
   The analysis system according to claim 1.
3. The emission control material includes at least one type of heat-activated delayed fluorescent dye or phosphorescent dye.
   The analysis system according to claim 1 or 2.
4. When the luminescent lifetime of the material with the longest luminescent lifetime among the two or more types of luminescent materials is τ _1l , the acquisition time of the temporal change in the emission spectrum of the imaging unit is τ _1l or more;
   The analysis system according to claim 2.
5. The imaging unit continuously or intermittently acquires changes over time in the emission spectrum of the emission control material.
   The analysis system according to any one of claims 1 to 4.
6. The acquisition time of the temporal change in the emission spectrum of the imaging unit is within 1 second;
   The analysis system according to any one of claims 1 to 5.
7. irradiating the light emission control material with light while in contact with a target object and a light emission control material that can interact with the target object and emits light upon receiving light of a predetermined wavelength;
   obtaining a change over time in a light emission state of the light emission control material that has received the light;
   Analyzing the state of the object based on the temporal change in the emission spectrum of the light emission state;
   including;
   A method for analyzing a target object, wherein the luminescence control material has a luminescence lifetime that changes and/or a luminescence spectrum that changes over time.
8. The luminescence control material includes two or more types of luminescent materials,
   The method for analyzing an object according to claim 7.
9. The emission control material includes at least one type of heat-activated delayed fluorescent dye or phosphorescent dye.
   The method for analyzing an object according to claim 7 or 8.
10. When the luminescent lifetime of the material with the longest luminescent lifetime among the two or more types of luminescent materials is τ _1l , the acquisition time of the temporal change in the step of acquiring the temporal change in the emission spectrum is τ _1l or more is,
    The method for analyzing an object according to claim 8.
11. The step of obtaining the change in the emission spectrum over time is a step of continuously or intermittently obtaining the change in the emission spectrum of the emission control material over time.
    The method for analyzing an object according to any one of claims 7 to 10.
12. In the step of acquiring the temporal change of the emission spectrum, the acquisition time of the temporal change is within 1 second.
    The method for analyzing an object according to any one of claims 7 to 11.

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