JP2005062105A - Photodetection device and photodetection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a photodetection device and a photodetection method, capable of simply and inexpensively coping with the changes in a medium temperature, without impeding measuring work on a test sample. <P>SOLUTION: This photodetection device has a holding means 4 for holding the test sample and a known sample, a light source means 2 for irradiating the test sample and the known sample with light, a photodetection means 12 for detecting light from the test sample and the known sample, a means 13 for holding the relation between the physical properties and the temperature of the known sample acquired beforehand, and a means 13 for performing the correction operation of the physical properties of the test sample, relative to the temperature from the relation between the physical property and the temperature of the known sample. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a light detection apparatus and a light detection method for detecting scattered light emitted from a fine substance such as carrier particles labeled on a sample or fluorescence emitted from a fluorescent substance labeled on a sample. The present invention relates to a light detection apparatus and a light detection method for performing correlation analysis of light intensity fluctuations, correlation spectroscopy analysis (FCS), and the like.

  With recent advances in optical technology and data analysis technology, analysis methods have been developed that statistically analyze the intensity of light to obtain information on properties such as molecules. As such a technique, Fluorescence Correlation Spectroscopy is known.

  In fluorescence correlation spectroscopy, in the field of view of a confocal optical microscope, carrier particles such as proteins and colloidal particles labeled with a fluorescent substance are suspended in a solution, and this is irradiated with laser light to excite the fluorescent substance. . The intensity of the fluorescence emitted from the fluorescent material is a time-series signal that changes with time because the fluorescent molecules floating in the medium are in Brownian motion. On the other hand, the speed of the translational diffusion movement of the fluorescent molecule varies depending on a change in the apparent size of the fluorescent molecule due to a chemical reaction or a binding reaction or a change in the temperature of the medium. Therefore, the change in the speed of the diffusion motion due to the chemical reaction or binding reaction of this molecule in the cell is regarded as a statistical change in the time series signal of the fluorescence light intensity, and the fluorescence based on the Brownian motion of these fine substances. By analyzing the fluctuation of the intensity and obtaining the autocorrelation function, the number of target fine particles, the translational diffusion rate, and the like can be measured.

  This technique is discussed in, for example, Masataka Kaneshiro “Protein Nucleic Acid Enzyme” (1999) Vol. 44, No. 9, p1431-1437. Furthermore, there are explanations such as “Fluorescence correlation spectroscopy” R.Rigler, E.S.Elson (eds.) Springer (Berlin).

  As techniques for measuring the physical properties of a test sample by detecting light and performing correlation analysis of fluctuations in light intensity, the following are known.

Using a confocal optical microscope, a sample that is fluorescently labeled on the sample stage is irradiated with laser light, the intensity fluctuation of the fluorescence emitted from the sample is analyzed, and statistical properties such as the translational diffusion coefficient of fluorescent molecules are analyzed. A method and apparatus for obtaining an interaction between molecules (for example, Patent Document 1). A device that generates a confocal region in a sample containing a fluorescent material housed in a microplate, and irradiates this with laser light to excite the fluorescent material, and measures the intensity and lifetime of the fluorescence emitted from the sample (for example, Patent Document 2). A technique for condensing laser light with a lens and exciting a fluorescently labeled sample placed in a well of a microplate (for example, Patent Document 3).
JP-T-11-502608 US Pat. No. 6,071,748 Special table 2001-502062 gazette

  However, when performing the measurement by applying the above-described technique, there are still the following problems to be solved.

  In other words, if the temperature of the medium changes during the measurement or before and after the reaction, it appears to be the same state as when the speed of the diffusion movement of the fluorescent molecule occurs, so the influence of the temperature change of the medium from the measurement results. There is a problem that it becomes difficult to distinguish changes due to pure diffusion motion due to reaction.

  For example, it is pointed out that when the medium temperature changes by 2 ° C. at a temperature of about room temperature, the translational diffusion time of the fluorescent molecules changes by about 5%. For this reason, if the temperature of the medium changes during the measurement, there is a problem that the error of the measurement value increases or the measurement result itself changes greatly. In particular, when measuring the course of a reaction for a relatively long period of time, such as when measuring a protein binding reaction such as signal transduction that occurs inside or outside a living cell, the temperature change of the medium is meticulous. It is necessary to pay attention, which is a heavy burden of measurement work.

  Therefore, in order to cancel out such measurement errors, it is conceivable to measure the temperature of the medium being measured or to install a mechanism for controlling the medium temperature in the measuring device. Therefore, it is necessary that the operability of measurement of the test sample is not deteriorated and generation of extra costs can be suppressed. However, there is no known technique suitable for such purposes.

  The present invention has been made in view of such circumstances, and can provide a light detection device and a light detection that can easily and inexpensively respond to changes in the medium temperature without hindering measurement work related to a test sample. It aims to provide a method.

  According to a first aspect of the present invention, there is provided a photodetecting device according to the first aspect of the present invention, in which a physical property of a test sample is measured using light, a holding means for holding the test sample and a known sample, Light source means for irradiating light to a known sample, light detection means for detecting light from the test sample and the known sample, means for maintaining the relationship between the physical properties and temperature of the previously obtained known sample, and the known sample Means for correcting the physical property of the test sample with respect to the temperature based on the relationship between the physical property of the sample and the temperature.

  According to a third aspect of the present invention, there is provided the light detection apparatus according to the third aspect, further comprising a temperature control means for controlling the test sample temperature or a temperature in the vicinity of the test sample.

  According to a third aspect of the present invention, in the photodetector according to the first aspect of the present invention, the physical properties of a known sample acquired in advance are at least a translational diffusion coefficient, a rotational diffusion coefficient, a polarizability. Any one of the average particle densities.

  According to a fourth aspect of the present invention, there is provided a photodetection device according to the present invention, wherein the light is used to measure the physical properties of the test sample, the holding means for holding the test sample, and light to the test sample. Light source means for irradiating, light detecting means for detecting light from the test sample, temperature measuring means for measuring the temperature of the test sample or the vicinity of the test sample, and the temperature measured by the temperature measuring means And a means for correcting the physical property of the test sample with respect to the temperature.

  According to a fifth aspect of the present invention, there is provided the photodetection device according to the above-described invention, wherein the test sample or the temperature measuring means in the vicinity of the test sample is the test sample solution or the test sample. It further has means for storing a solution having the same composition as the solution in which the sample is dissolved or dispersed, and means for measuring the temperature of the stored solution.

  Further, the light detection device according to claim 6 of the present invention is the light detection device according to the above-described invention, wherein the physical property of the test sample is corrected and output as a value at a specified temperature, or Means for displaying.

  The light detection method according to claim 7 of the present invention is a measurement method for measuring physical properties of a test sample using light, irradiating the held test sample and a known sample with light, Detects light from the test sample and known sample, and corrects the physical property of the test sample with respect to the temperature based on the relationship between the physical property of the known sample and temperature and the measured value of the physical property of the known sample. To do.

  According to an eighth aspect of the present invention, in the light detection method of the present invention, the temperature of the test sample or the vicinity of the test sample is controlled.

  The light detection method according to claim 9 of the present invention is the light detection method according to the above-described invention, wherein at least the translational diffusion coefficient, the rotational diffusion coefficient, the polarizability, and the average particle as physical properties of the known sample. Measure any one of the densities.

  According to a tenth aspect of the present invention, in the method for measuring physical properties of a test sample using light, the held test sample is irradiated with light to measure the physical property of the test sample. The light is detected, the temperature of the test sample or the vicinity of the test sample is measured, and the physical property is corrected and calculated with respect to the temperature based on the measured temperature.

  According to the eleventh aspect of the present invention, there is provided the photodetection method according to the above-described invention, wherein the test sample or a temperature in the vicinity of the test sample is measured by a test sample solution or a test sample. A solution having the same composition as the solution in which is dissolved or dispersed is stored in a container, and the temperature of the stored solution is measured.

  The optical detection method according to claim 12 of the present invention is the optical detection method according to the invention described above, wherein a test sample, a known sample, or a solution in which the test sample is dissolved or dispersed is applied to the microplate. Contained.

  The light detection method according to claim 13 of the present invention is the light detection method according to the invention described above, wherein the physical property of the test sample is corrected and output as a value at a specified temperature, or indicate.

  According to the present invention, it is possible to easily and inexpensively respond to changes in the medium temperature without hindering the measurement work related to the test sample.

  Hereinafter, an embodiment of a light detection device according to the present invention will be described based on a fluorescence measurement device.

[First Embodiment]
FIG. 1 is a diagram showing a schematic configuration of a fluorescence measuring apparatus according to a first embodiment of the present invention, and FIG. 2 is a side view showing the fluorescence measuring apparatus according to the first embodiment of the present invention. is there. The configuration and operation of the fluorescence measuring apparatus will be described with reference to FIGS.

  The basic device configuration of the main part of the fluorescence measuring device is based on a confocal optical microscope.

  The fluorescence measuring apparatus includes a sample stage 1, a light source 2, an objective lens 3, a microplate 4, a well 5, a dichroic mirror 6, a bandpass filter 7, a reflecting prism 8, a lens 9, a pinhole 10, a lens 11, and a photodetector 12. And a computer 13 and an objective lens longitudinal axis adjusting mechanism 17.

  The microplate 4 is a reaction container for containing a sample, and generally a resin and glass are used. The well 5 is a hole for accommodating a sample provided in the microplate 4, and is arranged in a large number (38 holes or the like) in the microplate 4. The bottom surface of each well 5 is a window made of a material that transmits visible light such as glass (not shown).

  The sample stage 1 places and fixes a microplate 4 containing a sample. The position of the sample is adjusted by moving the sample stage 1 horizontally or vertically. The objective lens vertical axis adjustment mechanism 17 adjusts the position of the sample stage 1 in the vertical direction.

  As the light source 2, an argon laser (oscillation output 10 mW, wavelength: 488 nm) is used. The objective lens 3 is installed below the microplate 4 mounted on the sample stage 1, condenses the laser beam, and forms a condensing region (confocal region) in the well 5. As the objective lens, for example, a x40 water immersion objective lens (NA 0.9) is used. As a result, the confocal region obtained in the well 5 has a substantially cylindrical shape with a diameter of about 0.6 μm and a length of about 2 μm.

  The dichroic mirror 6 selectively transmits or reflects light according to the wavelength. The dichroic mirror 6 is manufactured such that the spectral characteristics of transmission and reflection are optimized by applying a multilayer coating to the slope of a prism-shaped glass block. A flat plate may be used for the dichroic mirror 6.

  The bandpass filter 7 transmits light having a specific range of wavelengths and removes noise components. The lens 9 and the pinhole 10 transmit light from the confocal region in the well 5 and remove background light from outside the confocal region.

  The photodetector 12 is arranged with the light receiving surface aligned with the focal position of the lens 11 and converts the signal light into an electric signal (photocurrent pulse). The photodetector 12 uses, for example, a weak photodetector such as an avalanche photodiode (abbreviation: APD) or a photomultiplier tube. The computer 13 is a processing device that executes operations such as correlation analysis.

  Next, the operation of the fluorescence measuring apparatus will be described.

  The laser light emitted from the light source 2 is reflected by the dichroic mirror 6 installed in the main body of the fluorescence measuring apparatus, enters the objective lens 3, and is condensed in the well 5 in the microplate 4 containing the sample. The condensing position of the laser light is 150 μm above the upper wall position of the well bottom in the horizontal direction (XY axis) and in the vertical direction (Z axis) in the vertical direction (Z axis).

  Since the condensed laser light excites fluorescent molecules in the sample, fluorescence is emitted from the fluorescent molecules. This fluorescence again passes through the objective lens 3 and then the dichroic mirror 6 and enters the bandpass filter 7. The band-pass filter 7 has a disk shape, the spectrum of transmitted light is adjusted in accordance with the fluorescence emission spectrum, and only the light in the wavelength region of the fluorescence emission spectrum that is signal light passes through. Thereby, scattered light generated in the sample container and noise light such as part of incident light reflected from the wall of the well 5 and returning to the incident optical path can be cut. Note that the noise light can be blocked because the wavelength of fluorescence and the wavelength of background light are different.

  The signal light that has passed through the bandpass filter 7 is reflected by the reflecting prism 8 and is condensed through the lens 9 onto the pinhole surface of the pinhole 10 disposed behind the lens 9. That is, the focal plane of the lens 9 and the opening surface of the pinhole 10 coincide. The pinhole diameter of the pinhole 10 is, for example, 50 μm in diameter. The pinhole 10 removes background light from outside the confocal region in the reaction vessel.

  The signal light that has passed through the pinhole 10 is condensed again by the lens 11. The signal light is converted into an electric signal (photocurrent pulse) by the photodetector 12 arranged with the light receiving surface aligned with the focal position of the lens 11, and after being subjected to amplification and waveform shaping, it becomes an ON-OFF voltage pulse. To the computer 13. This voltage pulse is stored in a memory (not shown) of the computer 13 and subsequently subjected to calculations such as correlation analysis. Measurement results such as an autocorrelation function or a cross-correlation function of the fluorescence intensity fluctuation obtained by this calculation are presented on the screen of the computer 13.

  Next, a method for correcting the temperature of the measured physical property value of the test sample will be described.

  A known sample is prepared in advance. Here, it is necessary that the known sample has at least the molecular size. It is more useful if the concentration of the molecule is known. Therefore, for example, Rhodamine Green (RhG) is used as a known sample. Rhodamine green has a peak wavelength of excitation light in the vicinity of 490 nm, and an emission wavelength has a peak in the vicinity of 530 nm.

  Next, a calibration curve showing the relationship between the medium temperature and the diffusion time for rhodamine green is obtained in advance. The following relational expression is established between the medium temperature and the diffusion time.

In general, the translational diffusion coefficient D (m ² / s) of a molecule that undergoes Brownian motion is expressed by the following Einstein-Stokes equation when the shape of the molecule is assumed to be spherical.

D = k _B T / (6πηr) (1)
k _B : Boltzmann constant (J / K)
T: Absolute temperature of medium (K)
r: radius of the molecule (m)
η: Medium viscosity coefficient (N · s / m ² )
The formula (1) is described in Masataka Kaneshiro “Protein Nucleic Acid Enzyme” Vol. 44, No. 9, (1999) p1431-1437.

  In the fluorescence correlation spectroscopy, when the diffusion time of the translational Brownian motion of the fluorescent dye molecule is τ, the following formula (2) is established (Reference 3).

τ = w ₀ ² / (4 · D) (2)
w ₀ : radius (m) of the narrowest part of the confocal region
Formula (2) is described in SRAragon and R. Pecora: J. Chem. Phys. Vol. 64, No. 4, (1971) p1971-1803.

  By substituting D in equation (1) into equation (2), equation (3) is obtained.

τ = (3π / 2 k _B ) · w ₀ ² · r · (η / T) (3)
The translational diffusion coefficient D of the fluorescent dye rhodamine green is 2.8 × 10 ⁻¹⁰ (m ² / s) as a measured value when the medium is water at 22 ° C. When this is used, the relationship (calibration curve) between the temperature of the medium and the diffusion time of rhodamine green molecules when the medium is water can be obtained. In addition, the measured value of the translational diffusion coefficient D of rhodamine green is R. Rigler, U. Mets, J. Widengren and P. Kask: Eur. Biophys. J., Vol. 22, (1993), p169-175. Has been described.

  FIG. 3 is a graph showing a calibration curve of the medium temperature and the diffusion time of rhodamine green molecules. The concentration of rhodamine green at this time is 10 nM. This calibration curve may be created by measurement. The created calibration curve is stored in the computer 13 in the form of data or a data table and used in the subsequent processing.

Next, rhodamine green, which is a known sample, is accommodated in one well 5 in the microplate 4 and irradiated with laser light to perform fluorescence correlation spectroscopy measurement. By this measurement, an autocorrelation function or a cross-correlation function of fluorescence intensity fluctuation can be obtained, and based on these correlation functions, a known sample diffusion time τ _A at the medium temperature can be obtained. By fitting the tau _A calibration curve shown in FIG. 3, the temperature T _A of the medium is determined.

Next, the sample to be measured is accommodated in another well 5 in the microplate 4, the sample stage 1 is moved, and the position is adjusted so that the incident laser light strikes the well 5. Then, the fluorescence correlation spectroscopy measurement is performed by irradiating laser light, and the diffusion time τ _B of the unknown sample (test sample) is obtained. By substituting the unknown sample obtained the temperature T _A of the diffusion time tau _B and medium (test sample) Equation (3), the resulting radius r of the unknown sample (test sample) before It can be determined by comparing with the diffusion time of a known sample. When the measurement takes a long time, the known sample diffusion time τ _A is obtained at appropriate time intervals, and the radius r of the unknown sample (test sample) may be obtained each time.

  Thus, the molecular size r of the unknown sample (test sample) can be obtained without measuring the temperature T of the medium. However, for the known sample and the unknown sample (test sample), the medium is the same substance (for example, water). If the medium is different between the known sample and the unknown sample (test sample), it is clear from Equation (3) that the viscosity coefficient of both needs to be obtained and the measured value corrected.

  By obtaining the radius r of an unknown sample (test sample), for example, the reaction of protein molecules in cells can be captured. This is because when a protein reacts and binds, the size of the binding molecule is apparently increased, resulting in a change in the diffusion time of the molecule.

[Second Embodiment]
FIG. 4 is a diagram showing a measurement state of the fluorescence measuring apparatus according to the second embodiment of the present invention. The second embodiment is different from the first embodiment in that the temperature of the medium is measured. Accordingly, the same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  In the second embodiment, a thermometer 14 such as a thermocouple is placed in the well 5 of the microplate 4 containing an unknown sample (test sample), and the temperature T of the medium is measured. Then, the viscosity coefficient η of the medium is obtained based on the measured temperature. The viscosity may be directly measured using a viscometer.

Next, fluorescence correlation spectroscopy is performed on the unknown sample (test sample) in the well 2 of the microplate 1 to determine the diffusion time τ _B of the unknown sample (test sample). Substituting the diffusion time τ _{B of} the unknown sample (test sample) thus obtained, the viscosity coefficient η of the medium, and the temperature T of the medium into Equation (3), the unknown sample (test sample) The size r of the molecule can be determined. In order to obtain the molecular size r of an unknown sample (test sample) at a certain reference temperature, the viscosity coefficient η of the medium at a certain reference temperature is obtained, and this and the value of the reference temperature are substituted into equation (3). Thus, the molecular size r of the unknown sample (test sample) can be obtained. Thus, the molecular size r of the unknown sample (test sample) can be obtained without using the calibration curve.

  Here, the temperature of the medium is most preferably measured by measuring the temperature of the test sample itself, but if it is in the vicinity of the container (well) in which the test sample is stored, the temperature difference from the test sample Since it is small, it is possible to set the temperature in the vicinity of the container in which the test sample is stored.

  The temperature in the vicinity of the container includes the temperature of the container itself, the temperature of the microplate 4 around the container, the temperature of the container around the container, the temperature of the medium accommodated in the container around the container, This corresponds to the temperature of the gap between the container and the adjacent container.

  Although it is possible to measure the temperature by inserting a temperature sensor such as a thermocouple or resistance temperature detector into the test sample itself, the test sample attached to the temperature sensor may be mixed into other test samples. So it needs to be washed thoroughly. Therefore, when a solution having the same composition as the solution in which the test sample is dispersed or dissolved is arranged at a plurality of positions on the microplate and the temperature sensor is inserted therein to measure the temperature, there is no need to clean the temperature sensor. Since it is possible to monitor the temperature at a plurality of positions, it is preferable because correction with temperature can be performed more precisely.

  Further, the temperature of the medium can be measured not only by a contact type temperature sensor such as a thermocouple but also by a non-contact type thermometer such as a radiation thermometer or a temperature image measuring device. When a large number of test samples are accommodated in the microplate, it is possible to monitor all the test samples at the same time with a thermometer, and it is possible to perform more precise correction with temperature. Furthermore, since it is a non-contact type, it is preferable because the temperature of the test sample can be measured without obstructing the observation.

  Further, in a microplate containing a large number of test samples, measurement may be performed by putting a plurality of temperature sensors in several wells in the microplate. At this time, if the measured values are different among a plurality of temperature sensors, the temperature of the medium in each well is estimated by calculation from the relationship between each measured temperature value and the spatial position of each well. It can be a temperature value.

[Third Embodiment]
FIG. 5 is a diagram showing a measurement state of the fluorescence measuring apparatus according to the third embodiment of the present invention. The third embodiment is different from the first embodiment in that a temperature controller 15 is attached to the sample stage 1. Accordingly, the same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  The temperature controller 15 is composed of a Peltier element, and controls the temperature by heating or cooling the microplate 1 from the surroundings.

First, as shown in FIG. 3, a calibration curve is created in advance for a known sample (for example, rhodamine green), and a set temperature is determined. For example, it is set to 20 ° C. Next, a known sample, for example, rhodamine green is accommodated in the well 5 in the microplate 4 and irradiated with laser light to perform fluorescence correlation spectroscopy measurement. By this measurement, the diffusion time τ _A of a known sample (rhodamine green) is obtained. By applying this τ _A to the calibration curve, the temperature of the medium is obtained.

  If the temperature of the medium is different from the set temperature, the computer 13 outputs a control signal corresponding to the temperature deviation to the temperature controller 15 so that the temperature of the medium becomes the set temperature.

When the temperature of the medium reaches the set temperature, the sample to be measured is accommodated in the well 5 of the microplate 4 and fluorescence correlation spectroscopy measurement is performed. By substituting the obtained diffusion time τ _B of the unknown sample (test sample) into the equation (3), the apparent radius r of the unknown sample (test sample) can be obtained. Thereby, the apparent radius r of the molecule of the unknown sample (test sample) can be obtained without measuring the temperature T of the medium. However, the medium is the same for the known sample and the unknown sample (test sample). When the medium is different between the known sample and the unknown sample (test sample), it is necessary to correct the viscosity coefficient.

[Fourth embodiment]
FIG. 6 is a diagram showing a measurement state of the fluorescence measuring apparatus according to the fourth embodiment of the present invention. In the fourth embodiment, one of the wells in the microplate 4 is used as a temperature reference well, a medium containing a known sample is accommodated in the well, and the temperature of the medium is monitored by the thermometer 14. This is different from the first embodiment. Accordingly, the same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  The temperature of the microplate 4 is adjusted by the temperature controller 15 based on the temperature of the medium in the temperature reference well measured by the thermometer 14. The temperature controller 15 has a gap only in a portion through which light passes or is a window of a glass plate having a high transmittance of visible light, and from the periphery of the microplate 4 to the inside of each well without disturbing the measurement. The temperature of the medium can be adjusted. As the temperature controller 15, in addition to the above-described Peltier element, a thermal heater may be used, or circulation type temperature control may be performed with temperature adjustment water.

  FIG. 7 is a diagram showing a change in diffusion time with and without temperature control. FIG. 7 shows the results of measuring the diffusion time of rhodamine green. In FIG. 7, ● represents a change in diffusion time when temperature control was not performed, and □ represents a change in diffusion time when temperature control was performed.

By carrying out the temperature control according to the present invention, almost constant diffusion time data is obtained despite measurement over a long period of time.

  In addition, when the measured value is temperature-corrected by the fluorescence measuring apparatus according to the present embodiment, it is desirable to correct the measured value to a value at a specified temperature. Since the viscosity coefficient of various media has been well examined for a value of 20 ° C., η in the equations (1) and (3) can be referred to immediately, so the specified temperature is preferably 20 ° C. Further, if the specified temperature is 27 ° C., it will be 300K (absolute temperature), so it is preferable to specify it at 27 ° C. because the calculation is easy.

  FIG. 8 is a diagram illustrating a result of correcting the measured value of the translational diffusion time to a value at a specified temperature.

  In this measurement, rhodamine green was used as the test sample. The temperature of the medium is measured by a temperature sensor, and the temperature is corrected by setting the specified temperature to 20 ° C.

  In this example, rhodamine green was used as the test sample, but even when detecting the reaction state between certain molecules, if it is a reaction system in which the size of the molecule after the reaction is known, as in this example, Using the equations (1) to (3), it is possible to perform output and display with correction at a specified temperature of 20 ° C. or 27 ° C. For example, when the measurement is performed at different times, the temperature of the test sample may be different. At this time, if the actually measured value is corrected to the value at the specified temperature according to the present invention, the comparison of the results becomes very easy.

  The present invention is not limited to the intensity fluctuation of the fluorescence emitted from the fluorescent substance, and is also applied to a method for performing the antigen-antibody reaction measurement by colloidal particles such as latex particles from the correlation analysis of the scattered light intensity fluctuation by the laser beam. can do.

  As described above, in this embodiment, the relationship between the medium temperature and the translational diffusion time by the known fluorescent molecules is obtained in advance, and the measured values such as the translational diffusion speed of the fluorescent molecules by fluorescence correlation spectroscopy of the unknown sample and the known Compare the translational diffusion time with the fluorescent molecule and apply it to the calibration curve showing the relationship between the translational diffusion time with the known fluorescent molecule and the temperature obtained in advance. The translation speed of the fluorescent molecule of the corresponding unknown sample is derived.

  Further, the estimated medium temperature is changed to the standard measurement temperature by the temperature controller, the measurement is suspended until the medium temperature reaches the standard measurement temperature, and the measurement is started after the temperature environment is adjusted.

  Therefore, changes in the physical properties of the sample in the medium accompanying changes in the temperature of the sample can be automatically corrected. Therefore, even if the temperature of the sample changes, the accuracy of the sample in the medium can be increased. Measurement of physical properties can be performed. In addition, if a calibration curve based on the measurement values of a known sample is used, temperature measurement is not required, and the physical properties of the sample can be easily measured regardless of the temperature change of the medium.

  In the above-described embodiment, the diffusion time, the viscosity coefficient, and the like are obtained as the physical properties of the test sample. However, the physical properties other than this include, for example, “translational diffusion coefficient, rotational diffusion”. The present invention can also be applied to the case of obtaining “coefficient, polarizability, average particle density”. These values are values that can be measured by various photodetection devices, and these values are related to reactions between molecules and molecular behavior.

  Regarding the relationship between the translational diffusion coefficient and the reaction between molecules, since a mass increases when a certain molecule is bonded, the translational diffusion coefficient becomes small as a result of the movement speed of the molecule due to Brownian motion being slowed. In addition, when a certain molecule is dissociated, the mass is reduced, and as a result, the moving speed of the molecule due to Brownian motion is increased, resulting in an increased translational diffusion coefficient.

  The relationship between the rotational diffusion coefficient and the reaction between molecules is that when a molecule is bound, the mass increases or the shape of the molecule changes, so the rotational Brownian motion changes (generally decreases) and the molecule rotates. As the speed changes, the rotational diffusion coefficient changes. Also, when a molecule is dissociated, the mass decreases or the shape of the molecule changes, so the rotational Brownian motion changes (generally increases) and the rotational speed of the molecule changes, so the rotational diffusion coefficient changes. To do.

  In the reaction between the polarizability and the molecule, when a certain molecule is bonded, the shape of the molecule and the electronic state of the molecule change, so the polarizability changes. Also, when a molecule is dissociated, the molecular shape and the electronic state of the molecule change, so the polarizability changes.

  In the reaction between the particle density and the molecule, when a certain molecule is bonded, the number of molecules is decreased, so that the particle density is lowered. Further, when a certain molecule is dissociated, the number of molecules increases, so that the particle density increases.

  And when measuring these physical properties, it is indispensable to correct the temperature as described in the above embodiment.

  Regarding the relationship between the translational diffusion coefficient and the temperature, the Brownian motion becomes intense as the temperature of the medium rises, so that the translational diffusion coefficient increases as a result of the increase in the moving speed of the molecules.

  Regarding the relationship between the rotational diffusion coefficient and the temperature, the rotational Brownian motion becomes more intense as the temperature of the medium rises, so that the rotational speed of the molecule increases, resulting in an increased rotational diffusion coefficient.

  Regarding the relationship between the polarizability and temperature, when the temperature of the medium rises, the vibration of atoms constituting the molecule becomes intense, so that the change in the shape of the molecule increases, resulting in an increase in the polarizability.

  Regarding the relationship between the particle density and the temperature, as the temperature of the medium increases, the vibration of the medium and molecules becomes intense, resulting in a decrease in the molecular density.

  Therefore, by measuring the relationship between these physical properties and temperature in advance and applying the present invention, it is possible to correct the temperature of the sample to be measured and perform accurate measurement.

  Note that the present invention is not limited to the above-described embodiment as it is, and can be embodied by modifying the constituent elements without departing from the scope of the invention in the implementation stage. Further, various inventions can be formed by appropriately combining a plurality of constituent elements disclosed in the embodiment. For example, some components may be deleted from all the components shown in the embodiment. Furthermore, you may combine suitably the component covering different embodiment.

The figure which shows schematic structure of the fluorescence measuring apparatus of 1st Embodiment which concerns on this invention. The side view which shows the fluorescence measuring apparatus of 1st Embodiment which concerns on this invention. The figure which shows the calibration curve of the diffusion time of medium temperature and rhodamine green molecule. The figure which shows the measurement state of the fluorescence measuring apparatus of 2nd Embodiment which concerns on this invention. The figure which shows the measurement state of the fluorescence measuring apparatus of 3rd Embodiment which concerns on this invention. The figure which shows the measurement state of the fluorescence measuring apparatus of 4th Embodiment which concerns on this invention. The figure which shows the diffusion time change by the presence or absence of temperature control. The figure which shows the result of having corrected the measured value of translational diffusion time to the value in regulation temperature.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Sample stage, 2 ... Light source, 3 ... Objective lens, 4 ... Microplate, 5 ... Well, 12 ... Photodetector, 13 ... Computer, 14 ... Thermometer, 15 ... Temperature controller.

Claims (13)

In an apparatus that measures the physical properties of a test sample using light,
Holding means for holding a test sample and a known sample;
A light source means for irradiating a test sample and a known sample with light;
A light detecting means for detecting light from the test sample and the known sample;
Means for maintaining the relationship between the physical properties and temperature of a previously obtained known sample;
And a means for correcting the physical property of the test sample with respect to the temperature based on the relationship between the physical property of the known sample and the temperature.   The photodetection device according to claim 1, further comprising temperature control means for controlling a test sample temperature or a temperature in the vicinity of the test sample. The photodetection device according to claim 1 or 2, wherein the physical properties of the known sample acquired in advance include at least one of a translational diffusion coefficient, a rotational diffusion coefficient, a polarizability, and an average particle density. . In an apparatus that measures the physical properties of a test sample using light,
Holding means for holding the test sample;
Light source means for irradiating the test sample with light;
A light detecting means for detecting light from the test sample;
Temperature measuring means for measuring the temperature of the test sample or the vicinity of the test sample;
And a means for correcting the physical property of the test sample with respect to the temperature based on the temperature measured by the temperature measuring means.   The test sample or the temperature measuring means in the vicinity of the test sample includes a test sample solution or a means for storing a solution having the same composition as the solution in which the test sample is dissolved or dispersed, and the temperature of the stored solution The light detection apparatus according to claim 4, further comprising means for measuring the light intensity.   6. The photodetecting device according to claim 1, further comprising means for correcting or outputting a physical property of the test sample as a value at a specified temperature. In a measurement method that measures the physical properties of a test sample using light,
Irradiate light to the held test sample and known sample,
Detects light from test samples and known samples,
A photodetection method comprising correcting a physical property of a test sample with respect to temperature based on a relationship between a physical property of the known sample and temperature and a measured value of the physical property of the known sample.   The light detection method according to claim 7, wherein the temperature of the test sample or the vicinity of the test sample is controlled.   The physical property of the known sample is measured by at least one of a translational diffusion coefficient, a rotational diffusion coefficient, a polarizability, and an average particle density, according to any one of claims 7 and 8. Light detection method. In a method for measuring the physical properties of a test sample using light,
Irradiate light to the held test sample,
Detects light from the test sample,
Measure the temperature of the test sample or the vicinity of the test sample,
A photodetection method characterized in that a physical property is corrected for temperature based on a measured temperature.   The temperature of the test sample or the vicinity of the test sample is measured by storing the test sample solution or a solution having the same composition as the solution in which the test sample is dissolved or dispersed in the container and measuring the temperature of the stored solution. The light detection method according to claim 10, wherein measurement is performed.   The light detection method according to claim 7, wherein a test sample, a known sample, or a solution in which the test sample is dissolved or dispersed is contained in a microplate.   The light detection method according to claim 7, wherein the physical property of the test sample is corrected and output or displayed as a value at a specified temperature.

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