WO2016071985A1 - Procédé d'analyse de particules luminescentes - Google Patents

Procédé d'analyse de particules luminescentes Download PDF

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Publication number
WO2016071985A1
WO2016071985A1 PCT/JP2014/079433 JP2014079433W WO2016071985A1 WO 2016071985 A1 WO2016071985 A1 WO 2016071985A1 JP 2014079433 W JP2014079433 W JP 2014079433W WO 2016071985 A1 WO2016071985 A1 WO 2016071985A1
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WIPO (PCT)
Prior art keywords
luminescent
light
luminescent particles
observation area
particles
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PCT/JP2014/079433
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English (en)
Japanese (ja)
Inventor
田邊　哲也
山口　光城
克典 小江
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オリンパス株式会社
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Priority to PCT/JP2014/079433 priority Critical patent/WO2016071985A1/fr
Publication of WO2016071985A1 publication Critical patent/WO2016071985A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited

Definitions

  • the present invention relates to a method for analyzing luminescent particles.
  • Biomolecules to be analyzed include luminescent particles and fluorescent particles.
  • Luminescent luminance differs between the luminescent particles and the fluorescent particles.
  • the fluorescent dye emits photons of, for example, about 30 to 110 [kHz] when excited by being irradiated with light.
  • particles (luciferin) that emit light using luciferase as a typical luminescent particle as an enzyme emit photons of about 7 [Hz] (4.2 ⁇ 1024 [photons / s / mol]).
  • the brightness per unit time of the luminescent particles is about 1/4000 to 1/16000 times that of fluorescent molecules excited by irradiation with light.
  • a luminometer (lumino counter) is known as an apparatus for detecting luminescent or fluorescent particles (see Non-Patent Document 1).
  • methods for analyzing the diffusion characteristics of fluorescent molecules at the level of one particle methods described in Non-Patent Documents 2 and 3 are known.
  • Non-Patent Documents 2 and 3 are methods for analyzing the diffusion characteristics of fluorescent particles at a single particle level with high sensitivity. When these methods are applied to luminescent particles, the concentration of the luminescent particles is changed. It was difficult to analyze with high sensitivity. In these methods, the number of photons obtained while one fluorescent particle stays in the observation region is about several to several tens. On the other hand, when a luminescent particle is observed, one or less photons can be observed. Because it becomes.
  • One embodiment of the present invention includes a step of preparing a sample including luminescent particles and a confocal microscope in which the volume of an observation region is set to be equal to or larger than the volume of a sphere having a radius of 30 [ ⁇ m].
  • a luminescent particle analysis method comprising: a step of calculating a value; and a step of deriving a value indicating the concentration of the luminescent particle based on the calculated index value.
  • the present invention it is possible to provide a luminescent particle analysis method capable of performing analysis on luminescent particles with high accuracy.
  • FIG. 1 is a configuration diagram of a luminescent particle analysis system 1 for carrying out the luminescent particle analysis method.
  • the luminescent particle analysis system 1 is a combination of an optical system of a confocal microscope and a photodetector.
  • the measurement container 10 the objective lens 20, the optical scanning unit 30, the barrier filter 40, and the light collection.
  • a lens 50, an optical fiber 60, an optical sensor 70, and a control / analysis device 80 are provided.
  • the measurement container 10 is formed with the glass bottom plate 12 as the bottom.
  • a measurement sample F is accommodated in the measurement container 10.
  • the measurement sample F is a liquid in which luminescent particles are dispersed or dissolved. The luminescent particles will be described later.
  • the objective lens 20 converts light emitted from the luminescent particles in the measurement sample F into parallel light, and guides the parallel light to the optical scanning unit 30.
  • the objective lens 20 is a water immersion objective lens, an oil immersion objective lens, a silicone immersion objective lens, a dry (air immersion) objective lens, or the like.
  • the light detection area (observation area OA) of the objective lens 20 may be moved in the vertical direction by moving the objective lens 20 in the vertical direction (Z direction in the figure). Good.
  • the light scanning unit 30 reflects the light guided from the objective lens 20 toward the barrier filter 40.
  • the barrier filter 40 allows only light having a specific frequency component of light reflected by the optical scanning unit 30 to pass therethrough and guides only the light having the specific frequency component to the incident surface of the condenser lens 50.
  • the condensing lens 50 collects the light that has passed through the barrier filter 40 and causes the collected light to enter the incident surface of the optical fiber 60.
  • the optical fiber 60 causes the light incident on the incident surface to enter the optical sensor 70.
  • the optical sensor 70 converts the incident light into an electrical signal, and outputs the converted electrical signal to the control / analysis device 80.
  • the optical sensor 70 is preferably capable of detecting weak light from one luminescent particle. For example, an ultrasensitive optical sensor that can be used for photon counting is used as the optical sensor 70.
  • the incident surface of the optical fiber 60 is arranged at a position conjugate with the focal position of the objective lens 20. Thereby, only the light emitted from within the observation area OA enters the optical fiber 60, and the light from other than the observation area OA is blocked.
  • the observation area OA is an elliptical sphere that is long in the depth direction when viewed from the objective lens 20 side (from the ⁇ Z direction).
  • the observation area OA may have a shape that can be regarded as a sphere. Details of the observation area OA will be described later.
  • the luminescent particle analysis system 1 periodically moves the observation region OA in the measurement sample F by driving the optical scanning unit 30, for example.
  • the optical scanning unit 30 includes, for example, a mirror deflector similar to a galvanometer mirror device provided in a laser scanning microscope.
  • the optical scanning unit 30 changes the direction of the mirror deflector under the control of the control / analysis device 80, and accordingly moves the observation area OA in, for example, the horizontal direction (in the direction on the XY plane in the drawing).
  • the movement trajectory of the observation area OA may be arbitrarily selected from a circle, an ellipse, a rectangle, a straight line, a curve, or a combination thereof (so that various movement patterns can be selected in the program executed by the control / analysis device 80). It may be) In the following description of the present embodiment, it is assumed that the movement locus of the observation area OA is circular.
  • the measurement container 10 may be moved in the horizontal direction.
  • the control / analysis device 80 is a computer device including a storage unit 90 such as a CPU (Central Processing Unit), a RAM (Random Access Memory), a ROM (Read Only Memory), a HDD (Hard Disk Drive), and a flash memory. .
  • the control / analysis apparatus 80 includes a drive control unit 81, an information collection unit 82, an autocorrelation calculation unit 83, a fitting unit 84, and an analysis unit 85 as functional configurations.
  • the drive control unit 81 drives the optical scanning unit 30 to control the position of the observation area OA.
  • the information collecting unit 82 samples the output value (light intensity) of the optical sensor 70 in time series.
  • the autocorrelation calculation unit 83 calculates an autocorrelation function based on the output value sampled by the information collection unit 82.
  • the fitting unit 84 obtains the average number N of luminescent particles by fitting the autocorrelation function calculated by the autocorrelation calculating unit 83 using a predetermined formula.
  • the analysis unit 85 calculates the brightness CPP (Count per particle) per particle based on the light intensity, the average number of luminescent particles, and the like.
  • These functional units are, for example, software functional units that function when the CPU executes a program. Some or all of these functional units may be hardware functional units such as LSI (Large Scale Integration) and ASIC (Application Specific Integrated Circuit).
  • LSI Large Scale Integration
  • ASIC Application Specific Integrated Circuit
  • FIG. 2 is a flowchart illustrating an example of a processing flow according to the luminescent particle analysis method.
  • a measurement solution containing a luminescent enzyme such as luciferase is prepared (step S100).
  • a luminescent substrate is added to the measurement solution to obtain a measurement sample F (step S102).
  • FIG. 3 is a view of the movement path of the observation area OA as viewed from the objective lens 20 side.
  • the observation area OA is substantially circular when viewed from the objective lens 20 side, and moves in a circular orbit to form a donut-shaped periodic observation area POA.
  • the observation area OA has a size that does not deviate due to Brownian motion and includes several light emitting particles, and considers the need to capture the faint light of the light emitting particles, and observes one light emitting particle for a long time.
  • the applicant of the present application has stated that the minimum size is a volume of a sphere having a radius of 30 [ ⁇ m], that is, ⁇ (4/3) ⁇ ⁇ ⁇ 30 [ ⁇ m] 3 ⁇ 113097 [ ⁇ m]. 3 ⁇ was obtained.
  • the observation region OA having such a size is moved with a dilute luminescent particle solution having only a few luminescent particles in the observation region OA, the number of the luminescent particles in the observation region OA changes, and the observation region OA is changed.
  • the intensity of the light detected from the light also varies depending on the number of the luminescent particles.
  • the information collection unit 82 samples the output value of the optical sensor 70 until the scanning period in which the observation area OA is moved and the measurement sample F is scanned ends.
  • Time series data in which the integrated value of the output values measured within the sampling period is associated with the measurement time (or data input time) is stored in the storage unit 90 (step S106).
  • the autocorrelation calculation unit 83 calculates an autocorrelation function with the original time series data while shifting the measurement time with respect to the time series data (step S108).
  • FIG. 4 is a bar graph schematically showing the contents of time series data.
  • the white bars indicate the output value (hereinafter referred to as light intensity F (t)) of the light sensor by the luminescent particles to be measured, and the black bars are due to noise of the avalanche photodiode of the light sensor 70 or the like.
  • the light intensity F (t) is shown.
  • the light intensity F (t) due to the luminescent particles appears at a period substantially coincident with the period T for moving the observation area OA, whereas the light intensity F (t) due to noise generally appears at random timing.
  • FIG. 5 is a diagram illustrating the transition of the autocorrelation function g (dt). In the figure, dt is a shift time.
  • the autocorrelation function g (dt) is expressed by, for example, Expression (1).
  • g (dt) ⁇ F (t) ⁇ F (dt) ⁇ F 2 ⁇ / F 2 (1)
  • the autocorrelation function g (dt) when periodically scanned is expressed by Expression (2).
  • N is the average number of luminescent particles present in the observation area OA throughout the scanning period, that is, a value indicating the concentration of the luminescent particles
  • D is a diffusion constant
  • T 0 is the scanning period
  • r is the radius of the observation area OA (here, the observation area OA can be regarded as a sphere, but in the case of an ellipse, the radius of the circle of the maximum section cut along the XY plane).
  • g (dt) (1 / N) ⁇ ⁇ 1+ (4 ⁇ k ⁇ D ⁇ T 0 / w 2 ) ⁇ ⁇ 1 (2)
  • the fitting unit 84 derives the average number N of luminescent particles by fitting the autocorrelation function g (dt) calculated by the autocorrelation calculating unit 83 into the equation (2) (step S112).
  • the fitting unit 84 can derive the average number N of luminescent particles that does not depend on the particle diffusion characteristics by obtaining the reciprocal of the Y-intercept of the autocorrelation function. Further, the fitting unit 84 calculates the diffusion constant D by substituting the derived N into the equation (2).
  • the analysis unit 85 divides the total value ⁇ F (t) of the light intensity F (t) by the observation time T and the average number N of luminescent particles in accordance with the equation (3), so Brightness CPP is calculated (step S114).
  • CPP ⁇ F (t) / (T ⁇ N) (3)
  • step S106 and step S108 does not need to be performed continuously, for example, the process which moves observation area OA and stores time series data in the memory
  • the processing from step S108 onward may be performed collectively on a plurality of sets of time-series data at a later date.
  • control / analyzer 80 determines the average number of luminescent particles based on the elapsed time from the time when the luminescent substrate is added to the solution containing the luminescent enzyme to the time when the measurement of the light intensity F (t) is started. N or the like may be corrected. It is known that the luminance of bioluminescence decays from the reaction start time, and typically decays as shown in FIG. FIG. 6 is a diagram illustrating an example of attenuation of luminance of bioluminescence. The decay rate varies depending on the luminescent substrate and luminescent particles used, but by measuring in advance, the concentration of the luminescent particles can be corrected using the time elapsed from the start of the reaction.
  • the control / analysis device 80 maintains a map in which the elapsed time and the light attenuation rate are associated with each other, and corrects the average number N of luminescent particles by multiplying the correction coefficient having the opposite relationship. .
  • the applicant of the present application performed a simulation to confirm the effect of the embodiment described above.
  • the luminescent particles use luciferase
  • a solution with a luciferase concentration of 5 [am] is prepared
  • the measurement bin time (sampling period) is 100 [ ⁇ s]
  • the radius of the observation area OA is 0.4 [mm].
  • a measurement for 900 seconds was assumed at a scanning speed of 0.25 [mm / s] (scanning cycle 2 [s]).
  • FIG. 7 is a diagram showing the transition of the light intensity F (t) obtained as a simulation result.
  • the autocorrelation function g (dt) is obtained. As shown in FIG. 7, the shift times 2 [s], 4 [s], 6 equal to an integral multiple of the scanning period are obtained. It was found that the peak of the autocorrelation function g (dt) occurred in [s], and the luminescent particles could be detected with high accuracy.
  • FIG. 8 is a diagram showing a transition of the autocorrelation function g (dt) obtained as a simulation result.
  • the observation region in the sample containing the luminescent particles using the confocal microscope in which the volume of the observation region OA is set to be equal to or larger than the volume of a sphere having a radius of 30 [ ⁇ m].
  • the light intensity F (t) emitted from the observation area OA is measured while periodically moving the OA, and the autocorrelation function g (dt) is obtained while shifting the measurement time for the obtained light intensity F (t).
  • the value N indicating the concentration of the luminescent particles, the diffusion constant D, and the brightness CPP per one particle are obtained. It can be carried out.
  • Non-Patent Document 2 the method described in Non-Patent Document 2 is to analyze the fluctuation of the signal by periodically observing the same coordinates, but in order to increase the number of photons that can be observed, If the length is long (scanning is performed at a low speed), it becomes impossible to perform analysis with high sensitivity due to Raman scattering in the solution. Therefore, the configuration of the luminescent particle analysis system 1 in which the volume of the observation area OA is equal to or larger than the volume of a sphere having a radius of 30 [ ⁇ m] cannot be applied when observing fluorescent particles.
  • the intensity of Raman scattering of water is 8 [kHz] when the radius of the confocal optical system is 2 [ ⁇ m] in the apparatus having the configuration shown in Japanese Patent No. 5250152. From this, the Raman scattering intensity per molecule of water is 8.9 [Hz / molecule]. Further, the light emission amount of one molecule of the fluorescent dye is about 200-500 [kHz]. Therefore, when the fluorescence is observed by particle measurement as in the present application, the Raman scattering as the background light and the signal intensity of the fluorescent dye coincide with each other when the radius of the observation region is about 2 [ ⁇ m] (see FIG. 9).
  • FIG. 9 is a diagram illustrating a correspondence relationship between the fluorescence luminance and the observation region.
  • the radius of the observation region is about 20 [ ⁇ m]
  • the Raman scattering intensity of the background light and the signal intensity derived from the fluorescent dye coincide with each other, and 30 [ ⁇ m] or more. In such a large observation area, fluorescence measurement cannot be established.
  • the present invention can be used in industries such as chemistry, medical equipment, and computer software.
  • Luminescent particle analysis system 10 Measuring container 20 Objective lens 30 Optical scanning unit 40 Barrier filter 50 Condensing lens 60 Optical fiber 70 Optical sensor 80 Control / analysis apparatus 81 Drive control part 82 Information collection part 83 Autocorrelation calculation part 84 Fitting part 85 Analysis unit 90 Storage unit OA Observation area

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention concerne un procédé d'analyse de particules luminescentes qui comporte une étape d'utilisation d'un microscope confocal ayant un volume de région d'observation égal ou supérieur au volume d'une sphère ayant un rayon de 30 μm pour mesurer l'intensité d'une lumière émise à partir de la région d'observation tout en déplaçant périodiquement la région d'observation dans un échantillon comprenant des particules luminescentes, une étape de calcul d'une valeur d'indice indiquant l'autocorrélation de l'intensité lumineuse obtenue dans l'étape de mesure tout en décalant le temps de mesure, et une étape d'extraction d'une valeur indiquant la densité des particules luminescentes sur la base de la valeur d'indice calculée.
PCT/JP2014/079433 2014-11-06 2014-11-06 Procédé d'analyse de particules luminescentes WO2016071985A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0670797A (ja) * 1988-07-27 1994-03-15 Univ Wayne State 化学発光反応を利用する酵素の測定法
WO2013031439A1 (fr) * 2011-08-26 2013-03-07 オリンパス株式会社 Analyseur optique utilisant la détection de particules à émission de lumière, procédé d'analyse optique, et programme informatique utilisé pour analyse optique
JP2014149196A (ja) * 2013-01-31 2014-08-21 Olympus Corp 単一発光粒子検出を用いた光分析装置、光分析方法及び光分析用コンピュータプログラム
JP2014199179A (ja) * 2011-08-08 2014-10-23 オリンパス株式会社 共焦点顕微鏡又は多光子顕微鏡の光学系を用いた光分析装置及び光分析方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0670797A (ja) * 1988-07-27 1994-03-15 Univ Wayne State 化学発光反応を利用する酵素の測定法
JP2014199179A (ja) * 2011-08-08 2014-10-23 オリンパス株式会社 共焦点顕微鏡又は多光子顕微鏡の光学系を用いた光分析装置及び光分析方法
WO2013031439A1 (fr) * 2011-08-26 2013-03-07 オリンパス株式会社 Analyseur optique utilisant la détection de particules à émission de lumière, procédé d'analyse optique, et programme informatique utilisé pour analyse optique
JP2014149196A (ja) * 2013-01-31 2014-08-21 Olympus Corp 単一発光粒子検出を用いた光分析装置、光分析方法及び光分析用コンピュータプログラム

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