JP2014059192A - Fluorescence microscopy device and method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To derive long distance information in comparison with a FRET method in which a distance is evaluated through a fluorescence measurement.SOLUTION: The fluorescence microscopy device comprises: (1) an electromagnetic system that is capable of synthesizing and applying a static magnetic field from three axial directions orthogonal to each other; (2) a fluorescence detection magnetic resonance measurement part that measures fluorescence detection magnetic resonance spectrum of a paramagnetic nanoparticle marked by a phosphor while changing an application angle of the static magnetic field generated by the electromagnetic system; and (3) a calculation part that calculates a distance between the phosphor and the paramagnetic nanoparticle from an angle dependency on a static magnetic field direction of fluorescence intensity of the fluorescence detection magnetic resonance spectrum.

Description

  The present invention belongs to a technique for evaluating long-range structural change information of biomolecules existing in cells in observation using a fluorescence microscope that visualizes physiological functions in a living cell on the spot. In particular, the fluorescence shows how far the two specific sites of the fluorescent molecule (donor) responsible for energy donation that modifies the biomolecule of interest and the fluorescent molecule (acceptor) responsible for energy reception exist in the structure. This technique belongs to a conventional technique for deriving distance information by using a principle of fluorescence resonance energy transfer (FRET) that is evaluated by intensity measurement.

  FRET's explanations include "Principles of Fluorescence Spectroscopy" (Springer), "Understanding bioimaging-various techniques for observing intracellular molecules and their principles" (Yodosha), and fluorescence using FRET The type of measurement method is introduced in “FRET imaging” (Nature biotechnology 21, 187 (2003)).

  The simplest apparatus configuration of the fluorescence microscope is shown in FIG. The light output from the excitation light source (1-1) is introduced into the microscope objective lens (1-3) through the dichroic mirror (1-2), converged to an appropriate beam diameter, and placed in the sample (1-4). Irradiate existing phosphor (1-5). Fluorescence from the phosphor (1-5) passes through the same optical path, and only the desired wavelength is selected through the dichroic mirror (1-2) and further the bandpass filter (1-6), and the fluorescence detector (1-7) It is measured by. The fluorescence intensity data measured by the fluorescence detector (1-7) is taken into the computer (1-8) and analyzed and displayed. If necessary, a control signal is transmitted to the microscope stage control circuit (1-9) to control the position of the microscope stage (1-10), and the microscope stage (1-4) on which the sample (1-4) is installed -10) is controlled.

The general method of fluorescence measurement using fluorescence resonance energy transfer (FRET) is from a donor generated when an energy donor (donor: D) and an energy acceptor (acceptor: A) are present at a short distance. Return to energy transfer to the acceptor. Energy transfer efficiency is as follows: (1) Donor fluorescence spectrum and acceptor excitation spectrum overlap, (2) Donor quantum efficiency, (3) Donor-acceptor transition moment symmetry, (4) Donor-acceptor distance: R Depends on 4 items, minus 6th power (R ^-6 ). A typical example of the observation method is shown in FIG. In the left figure, donors and acceptors are chemically modified on two different biomolecules and introduced into the sample (intermolecular FRET). When the fluorescence of the donor is observed by irradiating the excitation light, the donor emits fluorescence by the excitation light before the interaction between the biomolecules, and the fluorescence intensity from the acceptor is minimal. After the biomolecule interaction, the fluorescence energy from the donor moves to the acceptor by the FRET phenomenon, and the fluorescence intensity of the acceptor increases. On the other hand, in the right figure, the donor and acceptor are modified at two locations within one biomolecule and introduced into the sample (intramolecular FRET). The molecular structure changes due to the interaction with other molecules in the sample, and when the donor and acceptor come close, the interaction can be monitored by the FRET phenomenon. Such examples include measurement of fluorescent molecules having specific binding properties to a pathological site using antigen-antibody reaction, and are very useful for research and development of drug discovery and physiological activity.

On the other hand, an example of a phosphor whose fluorescence spectrum changes through interaction with a magnetic field is a quantum state NV center ( NVC ) formed when a nitrogen atom (N) present as an impurity in a diamond and a lattice void (V) are close to each other. enter, hereinafter referred to as NVC). This is an S = 1 spin quantum state having a valence of −1 due to the presence of two electron spins adjacent to each other. It is known that this NVC has a unique energy level with an energy gap corresponding to 2.87 GHz even when no static magnetic field is applied, and is capable of electron spin resonance. Furthermore, the electrons contributing to electron spin resonance also affect the fluorescence phenomenon emitted from NVC, and the fluorescence spectrum changes depending on the presence or absence of the magnetic resonance phenomenon and the magnetic field intensity at the NVC position.

  In a paper published in Nature 455,648 (2008), both electron spin resonance and fluorescence spectrum measurement techniques are introduced into a scanning probe microscope, and the location of a single NVC and nanomagnets are generated around the fluorescence spectrum measurement. The distribution of the static magnetic field was observed at room temperature and normal pressure. Furthermore, a nano-diamond having a single NVC was used as a probe as an atomic size magnetic sensor for detecting a static magnetic field, and the possibility of a microscope having a nanometer spatial resolution was shown. A similar paper using a single NVC was also published on page 644 of the same issue. In the latter paper, in order to verify the magnetic field detection sensitivity when using the NVC center as a magnetic sensor, the general-purpose spin echo method used in the magnetic resonance technique was introduced to demonstrate the magnetic field sensitivity of sub-micro Tesla. did.

  There are several examples of living body observation using nanodiamonds. In particular, nanodiamonds are considered to have low toxicity inside cells, and are easy to chemically modify. Therefore, they attract attention as phosphors that can be introduced into living bodies. In the example reported in PNAS 104, 727 (2007), 35 nm nanodiamond with NVC was introduced into Hela cells, and scanning image observation of fluorescence intensity was performed using a confocal laser microscope or fluorescence microscope. About the result, the authors conclude that "It is an ideal fluorescent probe that is not affected by the fading or irregular emission (blinking) of phosphors that have been a problem in the past."

JP, 2011-180570, A.

FRET is an energy transfer phenomenon based on short-range interactions and is limited to the range of 1-10 nm. In addition to the distance between the two phosphors, the spectra of the two phosphors need to overlap appropriately, and the symmetry of the transition moment derived from the binding form of the phosphors to the biomolecule in the cell ( There are some reasons that cannot be dealt with by calibration data at the time of chemical synthesis in a test tube, for example, FRET efficiency changes greatly depending on orientation factor (κ). Therefore, at present, with respect to the orientation factor: κ squared, the distance between the donor and the acceptor is always discussed as κ ² = 2/3 under the assumption that the donor and acceptor are rotating at random within the lifetime of fluorescence. To do.

  However, the situation where this assumption holds is rare, and it is clear that such motility is actually constrained in the fluorescent substance labeled with the biomolecule introduced into the cell. In addition, since the quantum efficiency of the donor phosphor may change depending on the pH value of the cell culture medium, care must be taken when evaluating the acceptor fluorescence intensity associated with FRET. In this way, the presence or absence of intermolecular interactions can be monitored by fluorescence measurement using the FRET phenomenon, but it is extremely difficult to analyze the molecular structure quantitatively including distance information.

  An object of the present invention is to perform spectrum analysis using a fluorescent substance whose photodetection magnetic resonance spectrum changes through interaction of a dipole magnetic field formed around magnetized paramagnetic fine particles and a synthetic magnetic field vector of an external magnetic field. Thus, it is possible to derive further long-distance information and further the absolute value of the distance as compared with the FRET method in which the distance is evaluated through fluorescence measurement.

In order to achieve this object, the fluorescence microscope of the present invention comprises:
(1) An electromagnet system capable of synthesizing and applying a static magnetic field from three orthogonal directions.
(2) a fluorescence detection magnetic resonance measurement unit for measuring a fluorescence detection magnetic resonance spectrum of paramagnetic fine particles labeled with a phosphor while changing the application direction of a static magnetic field generated by the electromagnet system;
(3) a calculation unit for obtaining the distance between the phosphor and the paramagnetic fine particles from the angle dependence of the fluorescence intensity of the fluorescence detection magnetic resonance spectrum with respect to the direction of the static magnetic field,
It is characterized by having.

  Further, the phosphor is a diamond having NVC.

  The paramagnetic fine particles are iron oxide fine particles.

Further, the fluorescence microscopic method of the present invention comprises:
(1) Fluorescence detection magnetic resonance of paramagnetic fine particles labeled with a phosphor using an electromagnet system capable of synthetically applying a static magnetic field from three orthogonal directions and changing the direction of application of the static magnetic field generated by the electromagnet system Measuring the spectrum;
(2) determining the distance between the phosphor and the paramagnetic fine particles from the angle dependence of the fluorescence intensity of the fluorescence detection magnetic resonance spectrum with respect to the static magnetic field direction;
It is characterized by comprising.

  Further, the phosphor is a diamond having NVC.

  The paramagnetic fine particles are iron oxide fine particles.

According to the fluorescence microscope according to the present invention,
(1) An electromagnet system capable of synthesizing and applying a static magnetic field from three orthogonal directions.
(2) a fluorescence detection magnetic resonance measurement unit for measuring a fluorescence detection magnetic resonance spectrum of paramagnetic fine particles labeled with a phosphor while changing the application direction of a static magnetic field generated by the electromagnet system;
(3) a calculation unit for obtaining the distance between the phosphor and the paramagnetic fine particles from the angle dependence of the fluorescence intensity of the fluorescence detection magnetic resonance spectrum with respect to the direction of the static magnetic field,
So that
Compared with the FRET method, where distance was evaluated through fluorescence measurement, it became possible to derive long-range information.

Further, according to the fluorescence microscopic method according to the present invention,
(1) Fluorescence detection magnetic resonance of paramagnetic fine particles labeled with a phosphor using an electromagnet system capable of synthetically applying a static magnetic field from three orthogonal directions and changing the direction of application of the static magnetic field generated by the electromagnet system Measuring the spectrum;
(2) determining the distance between the phosphor and the paramagnetic fine particles from the angle dependence of the fluorescence intensity of the fluorescence detection magnetic resonance spectrum with respect to the static magnetic field direction;
Because it consists of
Compared with the FRET method, where distance was evaluated through fluorescence measurement, it became possible to derive long-range information.

It is a figure which shows an example of the conventional fluorescence microscope apparatus. It is a figure which shows an example of a FRET phenomenon. It is a figure which shows an example of the paramagnetic nanoparticle labeled with fluorescent substance. It is a figure which shows an example of a fluorescence detection magnetic resonance spectrum. It is a theoretical explanatory drawing of the fluorescence detection magnetic resonance method. It is a figure which shows an example of the resonant frequency of the signal which appeared in the fluorescence detection magnetic resonance spectrum when the magnetic field direction was changed. It is a figure which shows an example of the resonant frequency of the signal which appeared in the fluorescence detection magnetic resonance spectrum when the magnetic field direction was changed. It is a perspective view which shows a 3 axis | shaft control electromagnet system. It is a block diagram which shows a 3 axis | shaft control electromagnet system. It is a figure which shows one Example of the fluorescence microscope apparatus concerning this invention. It is a figure which shows the example of the measurement result using this invention.

In this embodiment, the angle dependence of the spectrum of a fluorescent substance whose photodetection magnetic resonance spectrum changes by sensing a magnetic field is measured using an electromagnet system that can generate a static magnetic field in an arbitrary direction. To do. Since the signal position is influenced by the static magnetic field and the dipole magnetic field and shows a unique angle dependence, the fitting process is performed using the distance between the NVC and the paramagnetic substance as a parameter to derive the distance that minimizes the residual. Is described.
[Theoretical explanation]
A fluorescent substance whose photodetection magnetic resonance spectrum changes through the interaction between a paramagnetic substance having a nanometer size and a magnetic field is chemically modified into a molecular complex as shown in FIG. 3 using a chemical synthesis technique. In the case of the above-mentioned two-molecule FRET, it may be a molecular complex bonded to two molecules and then bonded to each other, or a complex bonded to one molecule like the above-mentioned one-molecule FRET. .

  In the above-mentioned NVC, which is an example, when a photodetection magnetic resonance spectrum associated with magnetic resonance is measured, a decrease in fluorescence intensity is observed when a high frequency electromagnetic wave having a frequency of 2.87 GHz is irradiated in a zero magnetic field environment. On the other hand, the presence of the magnetic field can be observed to divide the high frequency frequency position indicating the decrease into two and spread with the magnetic field strength (extracted from Fig. 4, Nature 455,648 (2008)). If this phenomenon is used, the magnetic field intensity can be measured through measurement of the fluorescence spectrum.

  In a molecular complex composed of a paramagnetic substance and NVC, the magnetic field created around the paramagnetic substance polarized by being supplied with a static magnetic field from the outside is a vector quantity called a dipole magnetic field. For example, materials such as iron oxide nanoparticles with superparamagnetic characteristics used in MRI contrast agents induce a large magnetization when a magnetic field is applied, and a vector magnetic field called a dipole magnetic field is generated around them. .

A magnetic particle is placed at the origin (0,0,0), and an external static magnetic field: a dipole magnetic field created by a vector H _{0 at a position} of polarization magnetization: vector M = χ · vector H separated by a distance vector: vector R Is represented by (Formula 1).

（式１）
（式１）を見ると明らかなように、双極子磁場は外部磁場ベクトルと距離ベクトルの内積を含む項や、２つの項のベクトル和で与えられる複雑な方向性を持ち、外部磁場の方位とその磁場強度に応じて、磁性粒子の双極子磁場の大きさ・方向・向きが変化する。

(Formula 1)
As is clear from (Equation 1), the dipole magnetic field has a complicated direction given by the term including the inner product of the external magnetic field vector and the distance vector, and the vector sum of the two terms. The magnitude, direction, and direction of the dipole magnetic field of the magnetic particles change according to the magnetic field strength.

  On the other hand, if the orientation of the external magnetic field and the NVC position can be defined in the same laboratory coordinate system in the laboratory coordinate system, it is easy to calculate the composite magnetic field vector at the NVC position. In FIG. 5, the polarization magnetization when a magnetic field is applied from the outside to the magnetic material placed at the origin is indicated by a black arrow (left figure), and the state of the dipole magnetic field generated at the place where the NVC is placed (Center view, right view, viewing angle is different, but the situation at the same magnetization orientation). Since an external magnetic field also exists at the position of NVC, the effective magnetic field felt by NVC is a composite vector of both.

In the calculation of the dipole magnetic field, since the magnetic particle is placed at the origin (0,0,0), the NVC position (x, y, z) is given, and the magnetic field strength: H and its orientation unit vector ( If e ₁ , e ₂ , e ₃ ) are known, the dipole magnetic field vector: vector H _dip is obtained using (Equation 1). The composite vector at the NVC position: vector H _total = vector H ₀ + vector H _dip is calculated by adding each component of the external magnetic field: vector H ₀ and vector H _dip .

  The optical detection magnetic resonance spectrum of NVC depends on the main axis of the NVC, the magnetic field orientation felt by NVC, and the magnetic field strength. The main points are described below.

NVC forms a spin triplet state with S = 1 and has a zero field splitting of D = 2.87 GHz. When a finite magnetic field: vector H _total exists, the Zeeman Hamiltonian shown in the second term of (Expression 2) is added.

（式２）
第一項のＤ＝2.87ＧＨｚに相当する磁場強度（約0.1テスラ相当）に比べてベクトルＨ_totalの強度が弱い場合には、ＮＶＣ主軸に沿ったゼロ磁場分裂による基本状態が第二項目のゼーマンハミルトニアンにより若干摂動を受けるが、その量はＮＶＣ主軸方位とベクトルＨ_totalとの成す角度に依存する（第二項目が内積であることに注意）。

(Formula 2)
When the strength of the vector H _total is weaker than the magnetic field strength corresponding to D = 2.87 GHz (corresponding to about 0.1 Tesla) in the first term, the basic state due to zero magnetic field split along the NVC main axis is the second item Zeeman Slightly perturbed by the Hamiltonian, the amount depends on the angle between the NVC principal axis orientation and the vector H _total (note that the second item is the inner product).

  The basic state along the NVC main axis, that is, the pure spin state represented by Sz = 0 + 1 and -1 degenerated with Sz = 0 is mixed with other states due to the presence of the magnetic field, and the state of Sz = ± 1 The degeneration of will be solved. As a result, the energy levels of the three spin states (Sz = 0, +1, −1) are determined through diagonalization of the 3 × 3 matrix shown below, and Sz = 0⇔Sz = + 1, An energy difference corresponding to a transition between levels of Sz = 0⇔Sz = 1 (this is called a resonance frequency in the magnetic resonance field) appears as a signal in the photodetection magnetic resonance spectrum.

（式３）
ＮＶＣが一個の場合には、Ｓｚ＝0⇔Ｓｚ＝+1と、Ｓｚ＝0⇔Ｓｚ＝-1の順位間遷移に相当する２つの共鳴周波数が出現するが、その周波数位置は上記の式２、式３からも明らかなようにベクトルＨ_totalの強度とＮＶＣ主軸との成す角度によって変化する。

(Formula 3)
When there is one NVC, two resonance frequencies corresponding to the transition between the ranks of Sz = 0⇔Sz = + 1 and Sz = 0⇔Sz = −1 appear, and the frequency position is expressed by the above equation 2 As is clear from Equation 3, the angle varies depending on the angle between the intensity of the vector H _total and the NVC main axis.

To summarize, the signal frequency position of the photodetection magnetic resonance spectrum is
(1) It depends on the NVC principal axis direction, the synthetic magnetic field vector direction, and the strength of the synthetic magnetic field.
(2) The composite magnetic field at the NVC position is given by the vector sum of the external magnetic field whose direction is known and the dipole magnetic field described in the previous section.

On the other hand, the dipole magnetic field depends on (1) the angle formed by the distance vector connecting the magnetic particle at the origin and NVC and the external magnetic field orientation (polarization magnetization orientation).
(2) The dipole strength also depends on the strength of the external magnetic field.

That is, the signal position (frequency) appearing in the ODMR spectrum is
(1) Value of D (2) External magnetic field orientation (θh, φh) and intensity H ₀ defined in the laboratory coordinate system
(3) Distance defined in the laboratory coordinate system: R, distance azimuth angle (Rθ, Rφ)
(4) NVC principal axis azimuth angles (NVCθ, NVCφ) defined in the laboratory coordinate system,
(5) Magnetization, which is a variable of the dipole magnetic field—this is equal to the multiplication of the external magnetic field (strength H ₀ and orientation (Hθ, Hφ)) and the magnetic susceptibility per particle: χ.
Depends on a total of 10 variables.

However, in actual experiments,
(1) The physical properties evaluation and synthesis methods of magnetic nanoparticles have been established recently, and it has become possible to produce extremely uniform spherical magnetic nanoparticles. You may think that you are given
(2) The value of D can be measured from a photodetection magnetic resonance spectrum in a zero magnetic field,
(3) The applied magnetic field strength and orientation are known in the laboratory coordinate system.

  Therefore, the photodetection magnetic resonance spectrum is fitted with the NVC principal axis azimuth angle that cannot be controlled by the experimenter and the five variables related to the distance vector connecting the magnetic particle and NVC.

  Therefore, a method of defining the rotation axis of the external magnetic field in an arbitrary direction and rotating the external magnetic field by 0 to 180 ° within the axial plane was adopted.

  For example, an upward magnetic field is applied in the X-axis direction in the Z-axis plane with the Z-axis vertically upward as the rotation axis (if the polar coordinate angle display is used (0 °, 0 °)). In this state, the optical detection magnetic resonance spectrum is measured, and the signal position is acquired (by peak fit or something). If there is only one NVC, two signal positions should be obtained.

  Next, an external magnetic field is applied in a direction rotated by an angle from the X axis in the Z axis plane, and the signal position is acquired in the same manner. When this operation is repeated, it is expected that the direction and intensity of the synthesized magnetic field vector felt by the NVC will change under the influence of the dipole magnetic field and show the angle dependence as shown in FIG.

  In the above, the magnetic field is rotated in the Z-axis plane, but if the external magnetic field is rotated around the X-axis (90 °, 0 °) and the Y-axis (90 °, 90 °) as the rotation axis, the main axis orientation of NVC Reflecting the direction of the distance vector, three types of measurement data having different angular dependencies as shown in FIG. 7 are obtained.

As described above, the photodetection magnetic resonance spectrum is sequentially measured while changing the external magnetic field direction, and the signal frequency position that appears as a decrease in fluorescence intensity is displayed using the magnetic field direction as a variable. Affected angular dependence data can be obtained.
[Example]
FIG. 8 shows a schematic diagram of three orthogonal electromagnets.

FIG. 9 shows a hardware block diagram for generating a magnetic field in the electromagnet.
In order to supply current to the three electromagnets, an electric circuit including a current power source and a control signal source is prepared. In response to a command from the computer, three independent control signal generators generate control signals and supply them to the corresponding current power sources.

  Hereinafter, a method for deriving a magnetic field component that each electromagnet takes charge of in order to generate a magnetic field in an arbitrary direction will be described.

  First, the position vector of the electromagnet is obtained. In the laboratory system, the electromagnet orientation is represented by the following unit vector.

（式４）
ここで、θは試料面から垂直方向に伸びたＺ軸からの開角、φは電磁石１を基準に取ったときのＺ面内での電磁石１から電磁石２への角度であり、φ＝120°である。Mag1vectorとMag2vectorが直交する条件を考慮すると、両者の内積がゼロである必要があるので、

(Formula 4)
Here, θ is an open angle from the Z-axis extending vertically from the sample surface, φ is an angle from the electromagnet 1 to the electromagnet 2 in the Z plane when the electromagnet 1 is taken as a reference, and φ = 120 °. Considering the condition that Mag1vector and Mag2vector are orthogonal, the inner product of both needs to be zero.

これによりθ≒54.7°と求まる。したがって、各電磁石の方位ベクトルは以下定数となる。

As a result, θ≈54.7 ° is obtained. Therefore, the orientation vector of each electromagnet is a constant below.

次に、希望する静磁場の方向を、以下の実験室系単位ベクトルで定義する。

Next, the desired direction of the static magnetic field is defined by the following laboratory system unit vector.

（式５）
静磁場強度Ｈ₀を上記方向に発生させたい場合には、Ｈ₀の各電磁石方向への射影成分を各電磁石によって発生させれば良い。

(Formula 5)
When it is desired to generate the static magnetic field strength H ₀ in the above direction, the projection component of H _{0 in} the direction of each electromagnet may be generated by each electromagnet.

（式６）
以下に、実験室系で定義される（θh、φh）の組み合わせとして代表的な例、（0,0）、（90,0）、（90,90）、（90,180）、（90,270）、（180,0）時の各電磁石の供給磁場強度成分を示す。

(Formula 6)
The following are typical examples of (θh, φh) combinations defined in the laboratory system: (0,0), (90,0), (90,90), (90,180), (90,270), ( 180,0) shows the magnetic field strength component of each electromagnet.

任意の方向に回転軸を設定し、その周りを回転する磁場を発生したい場合には、以下の行列演算を実施することで、磁場方位ベクトル：ベクトルＨを求めることができる。

When a rotation axis is set in an arbitrary direction and a magnetic field rotating around the rotation axis is desired, a magnetic field direction vector: vector H can be obtained by performing the following matrix calculation.

  First, a normal coordinate transformation matrix is defined as follows.

（式７）
それぞれの式は行列で書くと以下と同じである。

(Formula 7)
Each formula is the same as the following when written as a matrix.

上記行列式を使って、Ｘ軸上にあった単位ベクトルv＝｛1,0,0｝をＺ軸周りに任意の角度：αを回転させ、実験室系鉛直Ｚ軸方向からの角度をθとしてＹ軸周りにθ、再度実験室系鉛直Ｚ軸周りに角度φを回転させると、

Using the above determinant, the unit vector v = {1,0,0} on the X-axis is rotated around the Z-axis by an arbitrary angle: α, and the angle from the laboratory system vertical Z-axis direction is θ As θ around the Y axis and again rotating the angle φ around the laboratory vertical Z axis,

（式８）
となる。即ち、実験室系で定義された｛θ、φ｝方向を方位軸としてその面内で回転する磁場方位ベクトル：ベクトルＨは、上記式に代入することで得られる。各電磁石が担当する磁場強度は、前出の計算に習って、磁場強度：Ｈ₀を乗じたベクトルＨの各電磁石への射影成分を計算すればよい。

(Formula 8)
It becomes. That is, a magnetic field azimuth vector: vector H that rotates in the plane with the {θ, φ} direction defined in the laboratory system as the azimuth axis is obtained by substituting into the above equation. The magnetic field intensity that each electromagnet takes charge of may be obtained by calculating the projection component to each electromagnet of the vector H multiplied by the magnetic field intensity: H _{0 according} to the above calculation.

  A procedure for measuring and collecting the angular dependence of the photodetection magnetic resonance spectrum using the orthogonal triaxial electromagnet system will be described with reference to FIG.

  (1) The direction {θ, φ} for rotating the magnetic field is set. Further, a rotation angle in the rotation axis plane: α is defined and set to 0 °.

  (2) Using the equation (8), a command is sent from the computer (10-8) of FIG. 9 to FIG. 10 to the control signal unit so as to supply the magnetic field carried by the three orthogonal electromagnets.

  (3) The control signal section is usually a digital-analog circuit, and generates a reference signal to the next power supply section (10-17 in FIG. 10).

  (4) The power supply unit (10-17 in FIG. 10) supplies a current to the electromagnet unit (10-16 in FIG. 10) according to the gain defined in the power supply unit with respect to the input reference voltage value.

  (5) Each electromagnet (10-16 in FIG. 10) generates a magnetic field in charge according to the applied current.

  An apparatus for measuring a photodetection magnetic resonance spectrum will be described.

  FIG. 10 shows an example of the apparatus configuration, which is an extension of the configuration of the general fluorescence microscope apparatus shown in FIG. The light output from the excitation light source (10-1) is introduced into the microscope objective lens (10-3) through the dichroic mirror (10-2), converged to an appropriate beam diameter, and irradiated onto the sample (10-4). Is done. In the sample, magnetic nanoparticles (10-11) already prepared by the user and a phosphor (NVC) (10-5) whose fluorescence spectrum changes by sensing a magnetic field form a molecular complex by intermolecular interaction. Suppose that the distance between the two is close. The fluorescence from NVC (10-5) passes through the same optical path as the excitation light, and only the desired wavelength is selected through a dichroic mirror (10-2) and further a bandpass filter (10-6), and a high-sensitivity cooled CCD ( The fluorescence intensity is measured as a two-dimensional image using 10-7) or the like, and is taken into the computer (10-8). In order to accurately control the position of the microscope stage, a feedback circuit via a microscope stage control circuit (10-9) may be prepared.

  On the other hand, in order to generate an electron spin magnetic resonance phenomenon with respect to the NVC (10-5) in the sample, high frequency magnetic field irradiation is performed. A high frequency for generating magnetic resonance is output from a high frequency transmitter (10-12). The high frequency output signal is amplified by a high frequency amplifier (10-13) and introduced into a high frequency coil (10-14) installed close to the sample, and a high frequency magnetic field is applied to the in-sample phosphor (10-5). Irradiated. In order to measure the amount of decrease in fluorescence intensity associated with magnetic resonance, two-dimensional image acquisition of fluorescence intensity is repeated while updating the frequency of the high-frequency magnetic field. The time schedule of the update of the high frequency frequency and the acquisition of the fluorescence intensity image may be carried out by a computer (10-8) for controlling the apparatus, or a pulse circuit (10-15 for giving a master clock and an instruction to start the operation of each apparatus ) May be prepared.

  The fluorescence intensity image data set in the environment with the frequency of the high frequency magnetic field set is taken into the computer (10-8). In the computer, the maximum value of fluorescent luminescent spots selected in advance for intensity measurement or the integrated value or average value over the area surrounding the luminescent spots is measured and stored in the memory. This operation is repeated with the frequency of the high-frequency magnetic field as a parameter, and the fluorescence intensity spectrum accompanying magnetic resonance is measured. These are implemented by the measurement control unit of the computer (10-8).

  (6) A signal position is calculated with respect to the photodetection magnetic resonance spectrum measured under a given magnetic field using an automatic fitting tool using a least square method or the like.

  (7) Update the magnetic field angle of the computer (10-8) and repeat the same work from step 2.

  (8) When the calculation of the photodetection magnetic resonance spectrum and further the signal position within the set rotation angle range is completed, the same magnetic field rotation with respect to the next rotation axis is performed, and the signal calculation is performed.

  For example, when {0, 0}, {90, 0}, {90, 90} are defined as rotation axes, as an example, the angle dependency as shown in the left diagram of FIG. 11 may be acquired. This is because the molecular complex in which paramagnetic fine particles and diamond containing NVC are bonded cannot be oriented, so the angular dependence of the signal from NV to the magnetic field orientation defined in the laboratory coordinate system is always an arbitrary phase difference. By being observed.

  The right diagram in FIG. 11 is an example in which the distance is calculated using the nonlinear least squares algorithm using the equations (1) and (2).

Claims (6)

(1) An electromagnet system capable of synthesizing and applying a static magnetic field from three orthogonal directions.
(2) a fluorescence detection magnetic resonance measurement unit for measuring a fluorescence detection magnetic resonance spectrum of paramagnetic fine particles labeled with a phosphor while changing the application direction of a static magnetic field generated by the electromagnet system;
(3) a calculation unit for obtaining the distance between the phosphor and the paramagnetic fine particles from the angle dependence of the fluorescence intensity of the fluorescence detection magnetic resonance spectrum with respect to the direction of the static magnetic field,
A fluorescence microscopic device comprising: 2. The fluorescence microscope according to claim 1, wherein the phosphor is diamond fine particles having an NV center. 2. The fluorescence microscope according to claim 1, wherein the paramagnetic fine particles are iron oxide fine particles. (1) Fluorescence detection magnetic resonance of paramagnetic fine particles labeled with a phosphor using an electromagnet system capable of synthetically applying a static magnetic field from three orthogonal directions and changing the direction of application of the static magnetic field generated by the electromagnet system Measuring the spectrum;
(2) determining the distance between the phosphor and the paramagnetic fine particles from the angle dependence of the fluorescence intensity of the fluorescence detection magnetic resonance spectrum with respect to the static magnetic field direction;
A fluorescence microscopic method characterized by comprising: 5. The fluorescence microscopic method according to claim 4, wherein the phosphor is diamond having an NV center. The fluorescence microscopic method according to claim 4, wherein the paramagnetic fine particles are iron oxide fine particles.

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