JP2011002415A - Fluorescence correlation spectroscopic device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescence correlation spectroscopic device capable of improving an S/N ratio in measurement and improving measurement throughput by enlarging a measurement region by changing the excitation of fluorescence and the size of a detection region in an aqueous solution or moving the excitation of fluorescence and a detection region in the aqueous solution during measurement and positively increasing the number of particles involved in the measurement within a limited time period.SOLUTION: The fluorescence correlation spectroscopic device comprises a laser light source 2; an optical system which forms a confocal region by collecting a laser beam from the laser light source to an aqueous solution being a measurement sample; an optical system for collecting fluorescence from the aqueous solution; and a photo-detector 16 for detecting collected fluorescence and is constituted as to enlarge a measurement region by changing the excitation of fluorescence and the size of a detection region in the aqueous solution or moving the excitation of fluorescence and the detection region in the aqueous solution during the measurement.

Description

  The present invention relates to a photodetection device and a photodetection method for detecting fluorescence emitted mainly from fine substances such as fluorescent dye molecules or fluorescent proteins labeled on a sample, and in particular, a correlation analysis of these light intensity fluctuations. It is related with the fluorescence correlation spectroscopy apparatus which performs.

  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 (hereinafter referred to as “FCS”) is known.

In FCS, in a 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 changes depending on a change in the 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 intensity fluctuation and obtaining the autocorrelation function, the number of target fine particles, the translational diffusion time, and the like can be measured. Translational diffusion time provides insight into the “size” of the molecule.

  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).

  The following techniques are known as techniques for measuring the physical properties of a test sample by detecting light and performing correlation analysis of fluctuations in light intensity. For example, Patent Document 1 uses a confocal optical microscope to irradiate a fluorescently labeled sample with a laser beam on a sample stage, analyze the intensity fluctuation of the fluorescence emitted from the sample, and translate the diffusion diffusion coefficient of the fluorescent molecule. A method and an apparatus for determining the statistical properties of molecules and interactions between molecules are shown.

  In Patent Document 2, a confocal region is generated in a sample containing a fluorescent material housed in a microplate, and the fluorescent material is excited by irradiating the confocal region with the laser beam. A device for measuring such as is shown. Further, Patent Document 3 discloses a technology for condensing laser light with a lens and exciting a fluorescently labeled sample placed in a well of a microplate.

JP-T-11-502608 US Pat. No. 6,071,748 Special table 2001-502062 gazette

  However, in order to evaluate numerically with high accuracy, it is necessary to measure and average a large number of particles, and the fluorescence from the particles jumping into that region is measured by Brownian motion with the detection region fixed. As long as this is done, there is a drawback that the number of particles for measurement cannot be expected.

  In principle, the larger the molecule, the slower the movement, so the larger the molecule, the more molecules are involved in the measurement during the measurement time.

  Furthermore, molecules that may take various shapes in solution, such as string-like DNA and peptide chains, need to be measured as sufficiently averaged by measuring as many states as possible. . However, in view of the above circumstances, if the measurement time is lengthened, the throughput is deteriorated.

  On the other hand, depending on the characteristics of the fluorescent dye, it is affected by fading (it does not emit fluorescence even when excited). Since the molecule that jumps in by Brownian motion is measured, only the molecules in the region that can diffuse during the measurement time can be involved, so the molecule once measured is excited again and enters the measurement region There is also a possibility that it may be affected by fading.

  The present invention has been made in view of the above problems, and changes the size of the excitation and detection areas of fluorescence in an aqueous solution, or moves the excitation and detection areas of fluorescence in an aqueous solution during measurement. The purpose is to provide a fluorescence correlation spectrometer that expands the measurement area, actively increases the number of particles involved in measurement within a limited time, improves the S / N ratio of measurement, and improves the measurement throughput It is said.

  In order to achieve the above object, a fluorescence correlation spectroscopic device according to the present invention includes a laser light source, an optical system for condensing the laser light from the laser light source into an aqueous solution as a measurement sample, and forming a confocal region, An optical system for collecting the fluorescence from the aqueous solution; and a photodetector for detecting the collected fluorescence, and further comprising means for exciting the fluorescence and changing the size of the detection region in the aqueous solution. It is a feature.

  In the fluorescence correlation spectroscopic apparatus of the present invention, it is preferable to provide a mechanism for changing the magnification of the objective lens in order to change the size of the excitation and detection areas of the fluorescence in the aqueous solution.

  In the fluorescence correlation spectroscopic device of the present invention, it is preferable to change the diameter of the incident beam to the objective lens in order to change the excitation of fluorescence and the size of the detection region in the aqueous solution.

  In the fluorescence correlation spectroscopy apparatus of the present invention, it is preferable that the fluorescence excitation and the size of the detection region can be selected depending on the size or concentration of the molecule to be measured.

  Alternatively, the fluorescence correlation spectroscopic device of the present invention includes a laser light source, an optical system for condensing the laser light from the laser light source onto an aqueous solution as a measurement sample to form a confocal region, and collecting fluorescence from the aqueous solution. It has an optical system that emits light and a photodetector that detects the condensed fluorescence, and includes means for moving the excitation of the fluorescence and the position of the detection region during measurement in the aqueous solution.

  In addition, the fluorescence correlation spectroscopic device of the present invention preferably includes a rotation mechanism around the optical axis in order to move the excitation of fluorescence and the position of the detection region in the aqueous solution during measurement.

  Further, in the fluorescence correlation spectroscopy apparatus of the present invention, it is preferable to have a mechanism capable of performing a raster scan like a scanning microscope in order to move the excitation of fluorescence and the position of the detection region during measurement in an aqueous solution. .

  In the fluorescence correlation spectroscopic apparatus of the present invention, it is preferable that the excitation of fluorescence and the movement state of the detection region can be selected depending on the size or concentration of the molecule to be measured.

  Furthermore, in the fluorescence correlation spectroscopy apparatus of the present invention, the theoretical formula for calculating necessary data in fluorescence correlation spectroscopy can be changed according to the excitation of fluorescence and the size of the detection region or the moving state. preferable.

  According to the fluorescence correlation spectroscopy apparatus of the present invention, the detection area in the solution is changed or moved during FCS measurement to expand the measurement area, and the number of particles involved in the measurement is actively increased within a limited time. Thus, the SN of measurement can be improved, and the measurement throughput can be improved.

It is the schematic which shows the basic composition of a fluorescence correlation spectroscopy apparatus. It is explanatory drawing which shows the difference in the confocal volume by the change of an objective lens, (a) shows the case of a high magnification objective lens, (b) shows the case of a low magnification objective lens. It is a representation figure of the size of a confocal volume. It is explanatory drawing of the change of the beam diameter by a beam expander. It is explanatory drawing of an eccentric mirror. It is explanatory drawing of a normal reflective mirror. It is explanatory drawing which shows rotation of the beam by an eccentric mirror.

Prior to the description of the embodiments, the outline and effects of the present invention will be described.
The fluorescence correlation spectroscopic device of the present invention includes a laser light source, an optical system for condensing the laser light from the laser light source onto an aqueous solution as a measurement sample to form a confocal region, and an optical system for collecting fluorescence from the aqueous solution And a light detector for detecting the condensed fluorescence, and is configured to include means for exciting the fluorescence and changing the size of the detection region in the aqueous solution. As a result, the excitation of fluorescence and the size of the detection region in the aqueous solution serving as the measurement sample can be changed, and the number of particles involved in the measurement is actively increased within a limited time to improve the SN ratio of measurement. be able to.

  In addition, the fluorescence correlation spectroscopic device of the present invention preferably includes a mechanism for changing the magnification of the objective lens in order to change the size of the excitation and detection areas of the fluorescence in the aqueous solution. Thereby, the excitation of the fluorescence and the size of the detection region can be selected depending on the size or concentration of the molecule to be measured.

  In the fluorescence correlation spectroscopic apparatus of the present invention, it is preferable that the diameter of the incident beam on the objective lens can be changed in order to change the excitation of fluorescence and the size of the detection region in the aqueous solution. Thereby, the excitation of the fluorescence and the size of the detection region can be selected depending on the size or concentration of the molecule to be measured.

  In the fluorescence correlation spectroscopic apparatus of the present invention, preferably, the excitation of fluorescence and the size of the detection region can be selected depending on the size or concentration of the molecule to be measured. The change of the incident beam diameter by the exchange of the objective lens or the beam expander can be changed by application software depending on the size or concentration of the molecule to be measured.

  Alternatively, the fluorescence correlation spectroscopic device of the present invention condenses the fluorescence from the laser light source, the optical system for condensing the laser light from the laser light source onto the aqueous solution as the measurement sample and forming a confocal region. It has an optical system and a photodetector for detecting the condensed fluorescence, and is configured to include means for moving the excitation of the fluorescence and the position of the detection region during measurement in the aqueous solution. As a result, the measurement area can be expanded by moving the excitation area and detection area of the fluorescence in the aqueous solution that becomes the measurement sample during measurement, and actively increasing the number of particles involved in the measurement within a limited time. The S / N ratio can be improved.

  The fluorescence correlation spectroscopic device of the present invention preferably includes a rotation mechanism centered on the optical axis in order to move the excitation of fluorescence and the position of the detection region in the aqueous solution during measurement. Thereby, the excitation of the fluorescence and the position of the detection region can be selected depending on the size or concentration of the molecule to be measured.

  In addition, the fluorescence correlation spectroscopic apparatus of the present invention preferably includes a mechanism capable of performing raster scanning like a scanning microscope in order to move the excitation of fluorescence and the position of the detection region in an aqueous solution during measurement. Thereby, the excitation of the fluorescence and the position of the detection region can be selected depending on the size or concentration of the molecule to be measured.

  In the fluorescence correlation spectroscopic apparatus of the present invention, preferably, the excitation of fluorescence and the movement state of the detection region can be selected according to the size or concentration of the molecule to be measured. The scanning of the incident beam can be changed by application software depending on the size or concentration of the molecule to be measured.

  Furthermore, in the fluorescence correlation spectroscopy apparatus of the present invention, preferably, in the fluorescence correlation spectroscopy, the theoretical formula for calculating necessary data is changed according to the excitation of the fluorescence and the size of the detection region or the moving state. It can be so. By appropriately selecting the autocorrelation function, it is possible to obtain an appropriate result according to the size of the target molecule and the size of the velocity.

  Next, before describing the embodiment of the present invention, the basic configuration of the fluorescence correlation spectroscopy apparatus will be described with reference to FIG. The fluorescence correlation spectroscopy apparatus of the present invention is a partial improvement of the fluorescence correlation spectroscopy apparatus whose basic configuration is shown in FIG. 1, as described in the following examples. In addition, the basic part of the fluorescence correlation spectroscopic apparatus uses a configuration of a confocal laser microscope as described below.

  The fluorescence correlation spectroscopic device includes a light source 2 that emits laser light, and a collimator 4 that constitutes an optical system that forms a confocal region by condensing the laser light from the light source 2 onto an aqueous solution of a microplate 7 serving as a measurement sample. , Dichroic mirror 5, reflecting mirror 10, reflecting mirror 6, objective lens 1, condenser lens 12, pinhole 13, barrier filter 14, and optical filter that collects fluorescence from the aqueous solution, A photodetector 16 to be detected, a correlation calculator 17 that inputs data of the photodetector 16 and calculates autocorrelation with respect to time, and a computer 18 are provided.

  The microplate 7 is a reaction container for storing an aqueous solution serving as a measurement sample, and is generally formed of a resin or glass transparent material. The well 20 is a hole for accommodating an aqueous solution to be a measurement sample provided in the microplate 7, and many wells 20 are arranged on the microplate 7. The microplate 7 is mounted and fixed on an XY stage (not shown).

  The light source 2 uses a laser light source. However, as shown in the figure, a plurality of laser light sources may be prepared so that they can be switched and used, or only one may be used. The objective lens 1 is installed below the microplate 7 and condenses laser light to form a condensing region (confocal region) in the aqueous solution.

  The dichroic mirror 5 selectively transmits or reflects light according to the wavelength. The dichroic mirror 5 is fabricated so that the spectral characteristics of transmission and reflection are optimized by applying a multilayer coating to the slope of a prism-shaped glass block. The dichroic mirror 5 may be a flat plate as shown in FIG.

  The barrier filter 14 functions as a band-pass filter that allows only light in the fluorescence wavelength range to pass, and a part of the laser light from the light source 2 is mixed with the fluorescence emitted from the sample and received by the photodetector 16. There is a role to prevent. The condensing lens 12 and the pinhole 13 transmit light from the confocal region in the aqueous solution and remove background light from outside the confocal region.

  The photodetector 16 is arranged so that the light receiving surface thereof is aligned with the focal position of the lens 21 provided between the barrier filter 14 and the photodetector 16, and converts the signal light into an electric signal (photocurrent pulse). Although the correlation calculator 17 and the computer 18 are shown as separate units in FIG. 1, they actually function as a processing unit that executes calculations such as correlation analysis together.

Next, the operation of the above-described fluorescence correlation spectroscopy apparatus will be described.
Laser light is emitted from a light source 2 that emits laser light for exciting fluorescent dye molecules used for measurement, and propagated by a single mode fiber 3.

  From the exit end of the single mode fiber 3, the laser beam is emitted as divergent light at a fixed angle due to the NA specific to the single mode fiber 3. The divergent light is converted into parallel light by the collimator 4, reflected by the dichroic mirror 5, reflected by the reflection mirror 10 and the reflection mirror 6, and incident on the objective lens 1.

  Above the objective lens 1, a microplate 7 containing an aqueous solution to be a measurement sample is disposed. The aqueous solution of the sample is put in a well 20 formed on the microplate 7, and an amount of about 1 μL (microliter) to several tens of μL is usually added.

In aqueous solution, the laser beam is focused and forms an excitation region. The molecule to be measured in the aqueous solution is labeled with a fluorescent dye molecule, and the molecule to be measured is freely diffused in the aqueous solution by Brownian motion.
Once this molecule enters the excitation region, the labeled fluorescent dye molecule is excited and fluorescence is emitted. The emitted fluorescence is again collected by the objective lens 1.

  The fluorescence passes through the dichroic mirror 5, is collected by the condenser lens 12, and passes through the pinhole 13 disposed at a position conjugate to the focal position of the objective lens 1. The volume of the detection region is determined by the pinhole 13 and is usually called a confocal volume, and the size is about 1 fL (femtoliter).

  Subsequently, the fluorescence is transmitted through the barrier filter 14, and only a component in a necessary wavelength band is selected. After entering the multimode fiber 15, the fluorescence is detected by the photodetector 16. The output of the photodetector 16 is normally a time-series photon pulse train and is input to the correlation calculator 17 where autocorrelation with respect to time is performed.

  The autocorrelation data of the fluorescence intensity is fitted using the following autocorrelation function (formula 1) obtained theoretically in advance, and the number of molecules present on average in the diffusion time and the confocal volume of molecules. Etc. are calculated. Usually, since the concentration of the aqueous solution used for the measurement is about 1 nM (nanomolar), the average number of molecules present in the confocal volume is about one. These calculations are executed by the computer 18.

G (t) = 1 + 1 / N [1 + t / τ _D ] ⁻¹ [1+ (W _O / W _Z ) ² t / τD] ^−1/2
... (Formula 1)
τD: Molecular diffusion time N: Number of molecules present in the confocal volume on average W _O : Radial radius of the confocal volume W _Z : Long axis of the confocal volume

  Next, an embodiment of the fluorescence correlation spectroscopy apparatus of the present invention will be described. Since the basic configuration is the same as that shown in FIG. 1, different parts will be described with reference to FIG. In this embodiment, the excitation of fluorescence and the size of the detection region are changed.

Laser light for exciting fluorescent dye molecules is incident on the objective lens 1 as parallel light. By replacing the objective lens 1 with an objective lens 8 having a different magnification, an aqueous solution as shown in FIG. The size of the confocal volume inside can be changed.
At this time, although not shown in the drawing, the size of the opening of the pinhole 13 is also changed to a size suitable for the size of the confocal volume.

  For example, when the concentration of the aqueous solution to be measured is thin due to the requirement for reaction conditions between the molecules to be measured (<1 nM), the number of molecules present on average in the confocal volume is 0.1 or less. . In such a case, for most of the measurement time, the molecule is not involved in the measurement. At this time, if the objective lens 8 has a low magnification, the size of the confocal volume becomes large, and molecules always exist in the confocal volume during the measurement time.

  Next, a description will be given of another example in which fluorescence is excited and the size of the detection region is changed. Since the basic configuration is the same as that shown in FIG. 1, different parts will be described with reference to FIG.

  By inserting the beam expander 9 into the parallel light emitted from the collimator 4 and changing the beam diameter, the size of the confocal volume in the aqueous solution can be changed as shown in FIG. In this case as well, although not shown in the figure, the size of the opening of the pinhole 13 is also changed to a size suitable for the size of the confocal volume.

  It should be noted that the replacement of the objective lens 1 from the objective lens 1 in Example 1 and Example 2 to the objective lens 8 or the insertion of the beam expander 9 is performed when the measurer knows in advance that the number of molecules in the solution is small. It is assumed that it can be changed by application software before. Alternatively, once the measurement is performed, when the number of molecules N is small, the application software can be set so that it can be automatically changed.

  Next, another embodiment of the fluorescence correlation spectroscopy apparatus of the present invention will be described. Since the basic configuration is the same as that shown in FIG. 1, different portions will be described with reference to FIGS. In this embodiment, the excitation of fluorescence and the position of the detection region are moved during measurement.

  Laser light for exciting fluorescent dye molecules is incident on the objective lens 1 as parallel light, and the reflection mirror 6 shown in FIG. 6 used here is provided with an angle as shown in FIG. The volume of the confocal volume involved in the measurement can be increased by using the eccentric mirror 19 as shown in FIG. 5 and rotating the motor 11 with the motor 11 attached thereto as shown in FIG.

  Usually, a state in which photometry is performed at one point on the optical axis sweeps a donut-shaped volume around the optical axis. Actually, when an incident beam is decentered by giving an angle of several minutes to a 40 × objective lens, the volume involved in excitation becomes 1 fL to 100 fL.

  Next, another example of excitation of fluorescence and movement of the position of the detection region during measurement will be described. Since the basic configuration is the same as that shown in FIG. 1, only different parts will be described.

  Although this embodiment is not shown in the figure, the volume can also be gained by performing measurement while performing scanning like a normal scanning laser microscope, for example, raster scanning in a uniform region in an aqueous solution.

  In the case of the third and fourth embodiments, since the volume of the confocal volume does not change at the moment, the size of the opening of the pinhole 13 may be left as it is. Further, the position of the pinhole 13 may be left as it is because a conjugate position is stored.

  In addition, the scanning of the incident beam to the objective lens 1 of the third and fourth embodiments is performed when the measurer knows in advance that the number of molecules in the solution is small, or the molecules are large and the diffusion rate is slow. If it is known, it can be changed by application software before measurement. Alternatively, once the measurement is performed, if the number of molecules N is small or the diffusion time is long, the application software can be set so that it can be automatically changed.

  Next, an example will be described in which the theoretical formula for calculating necessary data in fluorescence correlation spectroscopy is changed according to the excitation of fluorescence and the size of the detection region or the movement state.

  Depending on the relative velocity (V) of the confocal volume moving in the aqueous solution, the autocorrelation function (Equation 1) may deviate. In fact, as an application of the FCS technology, a steady velocity field distribution in a capillary can be analyzed by flowing a molecule with a known diffusion state in a fine capillary and performing FCS measurement in the capillary (Rigler: Ana. Chem. Vol 72, No 14, 2000, 3260-3265). At this time, the autocorrelation function (Equation 1) takes into account the velocity (V) of the solution containing the molecule and can be expressed as the following equation (Equation 2).

G (t) = 1 + 1 / N [1 + t / τ _D ] ⁻¹ [1+ (W _O / W _Z ) ² t / τ _D ] ^−1/2 exp {− [1 + t / τ _D ] ⁻¹ [1+ (W _O / W _Z ) ² t / τ _D ] ^−1/2 (V / W _O · t) ² } (Formula 2)

  Also in this embodiment, depending on the size of the target molecule or the speed, the exp term in Equation 2 cannot be ignored. In this case, instead of (Equation 1), (Equation 2) is used for fitting. Use it.

  Since the present invention can increase the number of molecules involved during measurement and improve the throughput, it is useful for a fluorescence analyzer.

DESCRIPTION OF SYMBOLS 1 Objective lens 2 Light source 3 Single mode fiber 4 Collimator 5 Dichroic mirror 6 Reflection mirror 7 Microplate 8 Objective lens (low magnification)
9 Beam expander 10 Reflecting mirror 11 Motor 12 Condensing lens 13 Pinhole 14 Barrier filter 15 Multimode fiber 16 Photo detector 17 Correlation calculator 18 Computer 19 Eccentric mirror 20 Well 21 Lens

Claims (9)

A laser light source;
An optical system for condensing the laser light from the laser light source into an aqueous solution to be a measurement sample to form a confocal region;
An optical system for collecting fluorescence from the aqueous solution;
A photodetector for detecting the condensed fluorescence;
A fluorescence correlation spectroscopic device comprising means for changing the size of excitation and detection areas of fluorescence in the aqueous solution.   2. The fluorescence correlation spectroscopic apparatus according to claim 1, further comprising a mechanism for changing the magnification of the objective lens in order to change the size of the excitation and detection area of the fluorescence in the aqueous solution.   2. The fluorescence correlation spectroscopic apparatus according to claim 1, wherein the diameter of the incident beam to the objective lens is changed in order to change the size of the excitation and detection areas of the fluorescence in the aqueous solution.   2. The fluorescence correlation spectroscopic apparatus according to claim 1, wherein the size of the excitation and detection region of fluorescence can be selected depending on the size or concentration of the molecule to be measured. A laser light source;
An optical system for condensing the laser light from the laser light source into an aqueous solution as a measurement sample to form a confocal region;
An optical system for collecting fluorescence from the aqueous solution;
A photodetector for detecting the condensed fluorescence;
A fluorescence correlation spectroscopic apparatus comprising means for moving fluorescence excitation and detection region positions in the aqueous solution during measurement.   The fluorescence correlation spectroscopic apparatus according to claim 5, further comprising a rotation mechanism centered on the optical axis in order to move the position of the excitation and detection region of the fluorescence in the aqueous solution during the measurement.   6. The fluorescence correlation spectroscopic apparatus according to claim 5, further comprising a mechanism capable of performing a raster scan such as a scanning microscope in order to move fluorescence excitation and detection region positions in an aqueous solution during measurement. .   6. The fluorescence correlation spectroscopic apparatus according to claim 5, wherein the excitation state of the fluorescence and the movement state of the detection region can be selected depending on the size or concentration of the molecule to be measured.   6. The theoretical formula for calculating necessary data in fluorescence correlation spectroscopy can be changed according to the excitation of fluorescence and the size of the detection region or the movement state. Fluorescence correlation spectrometer.

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