JP2009150665A - Single-molecule fluorescence/phosphorescence analysis method and device therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect light emission from a detection object substance, in the presence of a foreign substance of emitting self-fluorescence, in a technology for measuring and analyzing fluorescence or phosphorescence by using an optical system of a confocal optical microscope. <P>SOLUTION: This method is performed by arranging a flow passage for flowing a sample including a focal area of an objective lens and a means for irradiating to the sample with the exciting light on the upstream side of the focal area, and preparing the sample including a substance to be tested of adding a light emitting substance having the light emitting service life longer than the self-fluorescence. In measurement of the light emission, the sample is made to flow in the flow passage, and reaches the focal area of the objective lens after the self-fluorescence of the foreign substance disappears after a predetermined time passes after irradiating the exciting light, and the light emission from the substance to be tested is detected there. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to proteins, peptides, nucleic acids, lipids, amino acids and other physiologically active substances or biomolecules (hereinafter referred to as “biomolecules”) or other molecules emitting fluorescence or phosphorescence labeled with other substances or The present invention relates to a fluorescence / phosphorescence analysis method for measuring the emission intensity of a substance and analyzing the measured light intensity to detect characteristics, reactions or interactions of biomolecules. The present invention relates to a fluorescence / phosphorescence analysis method that employs a system and analyzes light intensity by measuring light emission from one molecule (or several molecules) of fluorescence or phosphorescence.

  With the recent development of optical measurement technology, nowadays fluorescence such as Fluorescence Correlation Spectroscopy (FCS) and Fluorescence-Intensity Distribution Analysis (FIDA) which measure and analyze fluorescence from one fluorescent molecule. Analytical methods are available. In these fluorescence analysis methods (single molecule fluorescence analysis technology) that measure fluorescence at the single molecule level, the optical system of a laser confocal microscope and photon counting (one-photon detection) are possible. The fluorescence intensity at the fluorescence single molecule level is measured using an apparatus, and the fluctuation of the intensity is analyzed by various methods to determine the speed of movement of the fluorescent molecule or the fluorescently labeled molecule or the size of the molecule (FCS ), Number density of molecules (or particles) or fluorescence intensity per molecule (in the case of FIDA). Therefore, based on such information, various phenomena such as a change in the structure or size of the molecule, a binding / dissociation reaction or dispersion / aggregation of the molecule can be detected. In the field of biological science, medicine or pharmacy, this single-molecule fluorescence analysis technology is applied to the detection and observation of the state and movement of biomolecules, etc., and the phenomena and reactions of various biomolecules, such as the binding reaction between proteins and nucleic acids. In addition, attempts have been made to elucidate the antigen-antibody reaction at the cellular level or the molecular level (for example, Patent Document 1-2 and Non-Patent Document 1-2). In addition, single-molecule fluorescence analysis technology can measure in a very short amount of sample and in a short time compared to conventional biochemical methods, so in various fields such as medicine and pharmacology, It is also expected to be applied in clinical diagnosis of illness and schooling of bioactive substances.

With the single-molecule fluorescence analysis technique as described above, it is possible to measure weak fluorescence at the fluorescent single molecule level (weak light at the fluorescence intensity level emitted from single or several fluorescent molecules). One of the reasons is that the optical system of the confocal optical microscope is adopted for measuring light. As is understood by those skilled in the art, in the optical system for fluorescence measurement, even if the sensitivity of the photodetector is sufficiently high, blind fluorescence and reflected / scattered light of excitation light generated by various factors are light. When the light enters the detector, the weak light at the fluorescence single molecule level is buried in the light, so it is extremely difficult to measure the weak light at the fluorescence single molecule level with a normal incident light optical system (Patent Literature). 4). However, according to the optical system of the confocal microscope, the light received by the photodetector can be substantially only the light from within the focal region of the objective lens. In addition, it is possible to accurately measure the weak fluorescence at the fluorescence single molecule level in the focal region of the objective lens without being affected by the reflected / scattered light of the excitation light.
JP 2003-275000 A JP 2005-337805 A Japanese Patent No. 3517241 Patent No. 3676212 3 outside casks, PNAS 96, 24, 13756-13761, November 23, 1999 Three people outside Kobayashi, Analytical Biochemistry, 2004, 332, 58-66 3 people outside Goush, Analytical Chemistry, 2000, 72, 3260-3265 3 people outside Kohler, Journal of Cell Science, 2000, 113, 3921-3930

  By using the optical system of the confocal microscope as described above, it is possible to block the light from other than the fluorescence observation region of the apparatus (the focal region of the objective lens) so that they do not enter the photodetector. Become. However, when light other than the light to be detected is emitted from the inside of the fluorescence observation region of the apparatus, that is, there is a contaminant that generates autofluorescence in the sample including the measurement target. When the background light is generated from the inside of the fluorescence observation region of the apparatus, the background light cannot be removed even with the optical system of the confocal microscope. In particular, biological samples or biological samples (serum, plasma, etc.) that are used for observation of interactions such as biomolecules or clinical diagnosis of diseases generally have various configurations that are not related to the reaction to be detected. In this case, the background light increases due to autofluorescence from a number of molecules other than the target molecule in the sample, which reduces the accuracy or reliability of the measurement results. Will end up.

  The effects of other constituents or contaminants other than the molecule to be observed in the sample of the single-molecule fluorescence analysis technique as described above should be eliminated by removing those impurities from the sample or purifying the molecule to be observed. Can do. However, extracting a specific molecule from a sample or removing other than a specific molecule requires time, labor and a large amount of sample, and single molecule fluorescence that can be measured in a short amount of time. The advantage of analysis technology will be lost. In the case of a biological sample, the target substance may be denatured during the process of extracting / purifying or removing the substance, and eventually the target measurement may not be performed.

  Thus, one of the main problems of the present invention is to remove impurities other than the molecule to be detected contained in the sample that cause the accuracy and reliability of the detection / observation results to be impaired in the single molecule fluorescence analysis technique. Without eliminating the background light, it is possible to eliminate or relatively reduce the background light due to the presence of these contaminants.

  Another object of the present invention is to use a biological sample or a sample derived from a living body as a sample in the detection and observation of molecular properties, reactions, interactions, etc. using the single-molecule fluorescence analysis technique as described above. When using a sample with relatively strong autofluorescence such as serum, plasma, etc., the effect of autofluorescence is eliminated in the measurement result, and the accuracy and reliability of the measurement result are further improved.

  According to the present invention, in a sample containing a large amount of contaminants having autofluorescence, particularly in a biological sample solution, a fluorescent or phosphorescent label having a longer emission lifetime than the autofluorescence of contaminants is added to a substance to be observed. At the time of optical measurement, after the sample is irradiated with excitation light, the autofluorescence of the contaminants disappears, and then the intensity of the fluorescence or phosphorescence at the fluorescence single molecule level is measured accurately by measuring the emitted light of the sample. Provided is a fluorescence or phosphorescence spectroscopic method or an apparatus therefor that allows good or reliable detection.

  According to one aspect of the present invention, in a method for measuring and analyzing fluorescence or phosphorescence emitted from a sample containing impurities having autofluorescence using an optical system of a confocal optical microscope, A flow path for circulating a sample configured to pass the sample through the focal region of the objective lens of the focus optical microscope, and excitation light to the sample upstream of the focal region of the objective lens in the flow channel Excitation light irradiation means for irradiating is provided. After that, the process of circulating the sample in the flow path and the process of measuring the intensity of fluorescence or phosphorescence from the sample that irradiates the sample with excitation light upstream from the focal region and reaches the focal region are executed. Then, after a predetermined time has elapsed since the sample was irradiated with excitation light, fluorescence or phosphorescence from the sample is measured. In such a method of the present invention, the sample has a longer emission lifetime than the autofluorescence of contaminants, particularly for a substance (hereinafter referred to as “test substance”) whose fluorescence or phosphorescence is to be measured in the sample. A luminescent material having a state in which a luminescent material is added is prepared. In addition, the test substance is in a state labeled with a fluorescent substance or a phosphorescent substance, such as having a fluorescent molecular group or a phosphorescent molecular group that has a longer emission lifetime than the autofluorescence of contaminants. In some cases, the test substance may be used as it is. However, the test substance may be labeled by an arbitrary method, for example, by adding a fluorescent substance or phosphorescent substance having a long fluorescence lifetime and having a long fluorescence lifetime. The fluorescent material having a long fluorescence lifetime may be a rare earth complex. The intensity of the measured fluorescence or phosphorescence is analyzed using fluorescence correlation spectroscopy (FCS), fluorescence depolarization spectroscopy (FDS), fluorescence intensity performed using an optical system of a confocal microscope. It may be performed by a technique according to either a distribution analysis method (FIDA) or a fluorescence cross-correlation spectroscopy (FCCS).

  According to the configuration of one aspect of the present invention described above, the sample containing the test substance is irradiated with the excitation light, and then flowed to the focal region of the downstream objective lens in the flow path. That is, after elapse of a predetermined time after the excitation of the sample, fluorescence or phosphorescence emitted from the sample at that time is measured. As described above, the sample used in the method of the present invention is prepared so that the emission lifetime of the test substance is longer than the autofluorescence of the contaminants. Therefore, as in the above configuration, the measurement of the emitted light from the test substance is delayed for a predetermined time from the irradiation of the excitation light. Therefore, it is expected that the emitted light from the test substance is measured with higher accuracy than before. Note that the time from the excitation of the sample to the measurement of the emitted light may be appropriately determined according to the lifetime of the autofluorescence of the contaminants in the sample used and the emission lifetime of the label of the test substance. This may be done by adjusting the flow rate of the sample passed through the flow path. Specifically, the time (predetermined time) from the excitation of the sample to the measurement of the radiated light is 100 μsec or more, considering the luminescent materials (fluorescent materials and phosphorescent materials) that can be used up to now. More preferably, it is 1 ms or longer, but the longer the emission lifetime of the label of the test substance, the longer the time from the excitation of the sample to the measurement of the emitted light may be extended. Should be understood.

  The sample used in the method of the present invention is serum, plasma or other living body used in research in the fields of biological science, medicine or pharmacy, clinical diagnosis of diseases or screening of physiologically active substances. It may be a solution sample obtained.

  In the method of the present invention described above, in any optical analyzer having an optical system of a confocal microscope, a flow path including the focal region of the objective lens, means for circulating a sample in the flow path, and a flow path This is achieved by an apparatus provided with excitation light irradiation means for irradiating the sample with excitation light upstream from the focal region. Therefore, as another aspect of the present invention, according to the present invention, a sample containing a contaminant having autofluorescence and having a longer emission lifetime in the test substance in the sample than the autofluorescence of the contaminant or An optical analyzer for measuring and analyzing the intensity of fluorescence or phosphorescence from a sample to which a phosphorescent material has been added, having a confocal optical microscope optical system and a sample in the focal region of the objective lens A light detection means for detecting fluorescence or phosphorescence emitted from the sample, a sample distribution means including a flow path for flowing the sample including the focal area of the objective lens, and a sample upstream of the focal area in the flow path Excitation light irradiating means for irradiating excitation light to the light source, and fluorescence or phosphorus emitted from the sample and detected by the light detecting means when the sample flowing in the flow channel reaches the focal region after being irradiated with excitation light. Analyzing light intensity Apparatus is provided which comprises a means. According to such an apparatus, as described in relation to the above-described method of the present invention, a sample containing a test substance by a luminescent substance having a longer luminescence lifetime than the autofluorescence of the contaminants is obtained by exciting light. After irradiation, the light is flowed to the focal region of the downstream objective lens in the flow path. At this stage, fluorescence or phosphorescence emitted from the sample is measured in a state in which the autofluorescence of impurities is relatively reduced. The

  The spectroscopic method using the optical system of the conventional confocal microscope can surely detect and analyze fluorescence or phosphorescence at the fluorescence single molecule level, but usually, when the sample is relatively “clean”. Only when a solution sample obtained from a living body such as serum or plasma was used as it was as a test sample, a highly accurate result was not obtained. One reason for this is that, as already mentioned, a large number of contaminants are mixed in the solution sample obtained from the living body, and no matter how much background light is prevented by the optical system of the confocal microscope, This is because it is impossible to block light emission from impurities in the focal region. The above-described method and apparatus of the present invention provides a light-emitting substance having a longer emission lifetime than the autofluorescence of impurities as an observation sample, and detects blind fluorescence or scattered light outside the observation region. In the optical system of the confocal microscope that blocks light from entering the detector, the timing of excitation of the sample and detection of the emitted light are shifted to detect the light from the target analyte within the observation area. By using the technique of relatively reducing the autofluorescence of foreign substances, it is possible to apply the optical analysis method using the optical system of the confocal microscope even for samples containing foreign substances that emit autofluorescence. It can be said.

  In the prior art, for example, in Patent Document 4, in a normal epi-illumination type fluorescence analyzer, for the purpose of blocking the incidence of blind fluorescence or scattered light outside the observation region to the photodetector. There are examples of spatially shifting the excitation position of the sample and the detection position of the emitted light, but in the case of the present invention, the optical system of the confocal microscope is used for blocking or removing blind fluorescence and scattered light outside the observation region. Has already been achieved. The characteristic configuration of the present invention is to shift the timing of excitation of the sample and detection of the emitted light in order to remove unnecessary light from the focal region in the optical system of the confocal microscope (temporal). In an epi-illumination fluorescence analyzer, spatially shifting the excitation position of the sample and the detection position of the emitted light to remove blind fluorescence and scattered light outside the observation area It should be understood that not only the structure but also the purpose is different.

  As described above, the optical analysis method of the present invention makes it possible to detect and analyze good fluorescence / phosphorescence at a single fluorescence level even in the case of solution samples obtained from serum, plasma or other living organisms. The labor or cost for removing contaminants from the sample or purifying the analyte to be observed is reduced, and the field of biological science, medicine or pharmacy for single-molecule fluorescence analysis technology such as FCS, FIDA, FCCS, etc. It is expected that the scope of application in research, clinical diagnosis of diseases, screening of bioactive substances, etc. will be expanded.

  Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

  The present invention will now be described in detail with reference to a few preferred embodiments with reference to the accompanying drawings.

The principle of light detection As described in the “Disclosure of the Invention” section, when light emission from a specific substance (test substance) to which a luminescent label is added in a sample is to be measured, By irradiating the sample with excitation light, the contaminants in the sample emit autofluorescence, and thus there is a case where luminescence from the test substance cannot be detected. In particular, biological samples such as serum and plasma that will be used for observation of interactions such as biomolecules and clinical diagnosis of diseases are substances that emit autofluorescence such as proteins, carbohydrates, and lipids. Therefore, when optical analysis is performed using a sample derived from such a living body, good measurement cannot often be performed due to autofluorescence of impurities. This situation is the same even if a confocal microscope optical system is used for light detection.

  Therefore, in the photoanalysis method and apparatus of the present invention, a substance having a longer emission lifetime than a normal fluorescent substance such as a fluorescent substance or a phosphorescent substance having a long fluorescent lifetime is added to the test substance as a luminescent label. In addition, after a predetermined time has elapsed from the irradiation of the sample with the excitation light, the autofluorescence of the contaminants disappears, and then light emission from the test substance is detected. For this purpose, in the optical analyzer, means for separating the part for irradiating the sample with excitation light and the part for detecting the radiated light from the sample and moving the sample from the excitation light irradiation part to the radiant light detection part Is provided.

  FIG. 1 schematically shows the principle of optical analysis in the above-described method and apparatus of the present invention. Referring to the figure, in the present invention, in the figure, a sample containing contaminants that emit autofluorescence and a test substance to which a luminescent label is added is sequentially moved from the left (a) to the right. To (c). In this state, first, the sample is irradiated with excitation light in (a), and the contaminants emit autofluorescence together with the test substance (in this state, light emission and impurities from the test substance). Can not be distinguished from autofluorescence.) Thereafter, while the sample is moved to the positions (b) and (c), the excitation light disappears, so the autofluorescence of the contaminants disappears, but the emission lifetime of the test substance is relatively Since it is longer than the autofluorescence, the light emission continues even at the position (c). Thus, by arranging the means for detecting the emitted light at the position (c), it becomes possible to detect only the emitted light from the test substance.

  Typically, the luminescent label added to the test substance may be a fluorescent substance or a phosphorescent substance having a long fluorescence lifetime as mentioned above. For example, a complex such as rare earth, europium, terbium, samarium, dysprosium may be used as the long-lived fluorescent dye. In particular, a europium complex having a long fluorescence lifetime is preferably used. As the phosphorescent dye, Ir (ppy) 3, which is an iridium compound used in an organic EL element, or an organic compound such as coronene or pyrene may be used. The addition of the luminescent label to the test substance may be performed by any technique that is usually performed in this field.

Apparatus Configuration and Photodetection / Analysis Method FIG. 2A schematically shows a configuration of an optical analysis apparatus 10 used in a preferred embodiment of the photodetection / analysis method of the present invention. Referring to the figure, in the optical analysis apparatus 10, the sample solution is circulated including the light detection means having the optical system of the confocal optical microscope and the focal region S of the objective lens 18 of the confocal optical microscope. Therefore, a sample distribution means having a flow path 12 and excitation light irradiation means for irradiating the excitation light λex in a region upstream of the focal region S of the objective lens 18 in the flow path 12 are provided. .

  In short, the configuration of the light detection means may be one in which the excitation light irradiation means for exciting the sample is omitted in the optical system of a normal confocal optical microscope (existing normal confocal light). When a microscope is used, the optical system and light source for the excitation light irradiation are not used.) As shown in the figure, in the light detection means, the radiated light (fluorescence / phosphorescence) λem from the sample in the focal region S of the objective lens 18 is focused on the pinhole 22 by the condenser lens 20. Then, the light is incident on the light receiver APD via the relay lens 24 and the mirror 26. Here, since light other than the focal region S of the objective lens 18 is blocked by the pinhole 22, the light that can reach the light receiver APD is only radiation light from the sample in the focal region S (both common). Principle of Focus Microscope) Thus, the focus area S becomes the light observation area in the optical analysis of this embodiment. The size of the focal region S is defined by the numerical aperture of the objective lens, the size of the pinhole 22 and the detection wavelength (usually 1 to 2 μm in the optical axis direction of the objective lens and the diameter in the direction perpendicular to the optical axis). About 1 μm). In addition, in the detection light, it is not necessary in principle to remove light such as blind fluorescence, scattered light, and reflected light by irradiating each part of the apparatus with excitation light, but stray light of excitation light, etc. In order to more reliably block the light, it is desirable that a band pass filter 28 is provided so that only the wavelength to be detected is incident on the light receiver APD. The light receiver APD may be any light receiving element or light detection device that is usually used for light detection by a confocal optical microscope, and may be, for example, an unbalanced photodiode. In the light receiver APD, an electric signal corresponding to the intensity of the received light is generated and transmitted to the signal analyzer 50 in a normal manner.

  The sample observed in the focal region S of the objective lens 18 of the light detection means is supplied by the sample distribution means in the optical analyzer of the present invention. As illustrated in FIG. 2A, the sample flow means has a flow path 12 installed so as to include the focal region S and a pump P for pumping the sample to the focal region S. In the operation of the sample circulation means, the pump P sucks the solution from the solution tank 12b and sends it out to the flow path 12 in the direction along the arrow F in the figure. A sample inlet 12a is provided at an arbitrary position of the flow path 12 from the pump P to the focal region S, from which the sample is introduced into the flow path 12 and is focused on the flow of the solution from the pump P. It is sent to the area S (the solution and the sample that have passed through the focal area S may then be discarded or collected). Typically, the material of the flow path 12 may be glass, particularly non-fluorescent glass such as synthetic quartz.

  The means for performing the irradiation of the excitation light on the sample prior to the detection of the radiation light from the sample in the focal region S is located in the flow channel 12 upstream of the sample flow from the focal region S. Configured to irradiate the site. Typically, as schematically shown in the figure, the light from the light source is guided to irradiate a part of the flow path 12 by an objective lens (or condenser) 16a carried on the gantry 16. It is burned. FIG. 2B is a schematic view of the positional relationship between the focal region S and the excitation light irradiation region λex in the flow channel 12 as viewed from the optical axis direction of the objective lens 18. For the irradiation of the excitation light, the region λex may be determined by adjusting the position, size and contour of the field stop using epi-illumination (Kohler illumination or the like) normally performed in this field. The position is variable in the direction of arrows X1 and X2.) Typically, a laser L is used as the light source, and the laser light may be incident on the objective lens 16a through the optical fiber 14, as shown in the figure. What is normally used as an excitation light source of a phosphorescent substance may be adopted.

  As described above, after the apparatus is configured, when light detection and analysis are performed, first, the sample is added or introduced from the sample inlet 12a by placing the solution on the flow path 12 by the pump P. The The test substance and impurities in the sample are excited when passing through the excitation light irradiation region λex and start to emit light. Then, when riding on the flow of the solution and passing through the focal region S, it is expected that the autofluorescence of the contaminants is extinguished. Thus, only the emitted light from the test substance is detected by light. The light passes through each part of the means and is received by the light receiver APD, and the light receiver generates an electrical signal corresponding to the received light intensity and transmits it to the signal analyzer 50. The signal analysis device 50 records the emitted light intensity sequentially or in time series, and performs optical analysis according to various spectroscopic analysis methods such as FCS, FDS, FIDA, or FCCS based on the recorded light intensity.

ここで、Ｎは、焦点領域中の平均蛍光粒子数、ｔｄは、拡散のみにより粒子が領域を通過する場合（流れがない時）の粒子の領域内での平均滞在時間、ｔｆは、拡散がないとした場合に流れにより粒子が領域を通過する場合の粒子の領域内での平均滞在時間、ｒ／ｚは、焦点領域の縦横比（光軸に垂直方向の幅／光軸方向の幅）である。従って、蛍光強度データから算出される自己相関関数Ｇ（ｔ）に上記の式をフィッテングし、ｔｄが算出される。そして、ｔｄから蛍光粒子の拡散定数、分子間相互作用の有無、結合解離状態等のＦＣＳで測定可能な情報が得られることとなる。その他の分析方法についての被検物質が一方向に流れる試料中に於いて拡散しつつ焦点領域を通過することを考慮した解析処理は、当業者に於いて任意に構成されてよい。 In the spectroscopic analysis method as described above, in the analysis of the acquired fluorescence intensity, it is considered that the analyte passes through the focal region while diffusing in the sample flowing in one direction. . For example, in the analysis of the fluorescence intensity when using FCS, the autocorrelation function of the fluctuation of the fluorescence intensity of the fluorescent particles diffusing in a unidirectional flow can theoretically be given by the following equation. (For example, refer nonpatent literature 4.)

Here, N is the average number of fluorescent particles in the focal region, td is the average residence time in the region of the particles when the particles pass through the region only by diffusion (no flow), and tf is the diffusion If not, the average residence time in the region of the particle when the particle passes through the region by flow, r / z is the aspect ratio of the focal region (width perpendicular to the optical axis / width in the optical axis direction) It is. Therefore, the above equation is fitted to the autocorrelation function G (t) calculated from the fluorescence intensity data, and td is calculated. Then, information that can be measured by FCS such as the diffusion constant of the fluorescent particles, the presence or absence of intermolecular interaction, and the bond dissociation state can be obtained from td. Analytical processing in consideration of passing through the focal region while diffusing in the sample flowing in one direction with respect to other analysis methods may be arbitrarily configured by those skilled in the art.

  In addition, for clarity of explanation, although not shown, it should be understood that the number of wavelength bands of excitation light, the number of wavelengths, the number of light receivers, and the wavelength may be appropriately set according to the analysis method to be performed. It is. When an analysis method using a plurality of optical signals such as FDS and FCCS is executed, a plurality of light receivers are provided, and an optical system for dividing the radiation light into each light receiver is in a normal mode. It may be provided. Similarly, in the FCCS, when it is necessary to excite a sample in a plurality of excitation light wavelength bands, an optical system for appropriately mixing light from a plurality of light sources may be provided.

  It should be understood that the distance between the focal region S and the excitation light irradiation region λex and the flow rate of the sample may be appropriately set depending on the emission lifetime of the luminescent label of the test substance. For example, when the distance between the focal region S and the excitation light irradiation region λex is 1 mm and the flow velocity is 2 m / s, the required time from the excitation light irradiation region λex to the focal region S is set to 500 μsec. For example, when a sample is serum and a specific protein in the sample is a test substance, the fluorescence lifetime of autofluorescence in the serum is several nanoseconds when the test substance is labeled with pyrene. Therefore, although the sample is extinguished when passing through the focal region, pyrene has a long emission lifetime of 930 μsec, so that the emitted light is detected when it passes through the focal region S.

FIG. 1 schematically shows the state of molecules in a sample for explaining the principle of light detection according to the embodiment. In the figure, the contaminants indicated by broken lines indicate a state in which the fluorescence is extinguished. FIG. 2A is a schematic diagram of the configuration of the light detection / analysis apparatus according to the preferred embodiment of the present invention, and FIG. 2B shows the relationship between the focal region (observation region) and the excitation light irradiation region. Is shown from the direction of the optical axis of the objective lens 18. The dimensions of each component are significantly different from the actual ones for clarity of configuration.

Explanation of symbols

DESCRIPTION OF SYMBOLS 12 ... Flow path 12a ... Sample inlet 16 ... Objective lens for excitation light 18 ... Objective lens for light detection 22 ... Pinhole 50 ... Signal analyzer L ... Laser P ... Pump APD ... Light receiver S ... Focus area (observation area)
λex: Excitation light irradiation region λem: Synchrotron radiation

Claims (7)

A method of measuring and analyzing fluorescence or phosphorescence emitted from a sample containing impurities having autofluorescence using an optical system of a confocal optical microscope,
Providing a flow path for circulating the sample configured to pass the sample through a focal region of the objective lens of the confocal optical microscope;
Providing excitation light irradiation means for irradiating the sample with excitation light upstream of the focal region in the flow path;
A step of preparing a sample in which a luminescent substance having a luminescence lifetime longer than the autofluorescence is added to a test substance in the sample;
A process of circulating the sample in the flow path;
Irradiating the sample with the excitation light upstream from the focal region and measuring the intensity of fluorescence or phosphorescence from the sample that has reached the focal region, and the sample is irradiated with the excitation light. A method comprising measuring and analyzing fluorescence or phosphorescence intensity from a sample after a predetermined time has elapsed since irradiation.   2. The method of claim 1, wherein the predetermined time is 100 [mu] s or longer.   3. The method according to claim 1, wherein the analysis of the fluorescence or phosphorescence intensity is performed by fluorescence correlation spectroscopy, fluorescence depolarization, fluorescence intensity distribution analysis, or fluorescence cross correlation spectroscopy. And how to.   2. The method of claim 1, wherein the sample is serum, plasma or a solution sample obtained from a living body.   4. The method according to claim 1, wherein the test substance in the sample is labeled with a fluorescent substance or a phosphorescent substance having a long fluorescence lifetime.   6. The method according to claim 5, wherein the fluorescent material having a long fluorescence lifetime is a rare earth complex. Measures the intensity of fluorescence or phosphorescence from a sample containing contaminants having autofluorescence, and a sample in which a fluorescent or phosphorescent substance having a longer emission lifetime than the autofluorescence is added to the test substance in the sample An optical analyzer for analyzing
A light detecting means having an optical system of a confocal optical microscope for detecting fluorescence or phosphorescence emitted from a sample in a focal region of the objective lens of the confocal optical microscope;
Sample distribution means including a flow path for distributing the sample including the focal region of the objective lens;
Excitation light irradiation means for irradiating the sample with excitation light upstream of the focal region in the flow path;
Analyzes the intensity of fluorescence or phosphorescence emitted from the sample and detected by the light detection means when the sample flowing through the channel reaches the focal region after being irradiated with the excitation light. Means.

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