Magnetic microscope

Abstract

To provide a magnetic microscope that can observe the vitality of individual IPS cells, because the vitality of individual cells cannot be observed, although a growth process and motility can be observed using optical microscopes, making it difficult to properly manage the growth process.SOLUTION: The present invention is a magnetic microscope that makes it possible to miniaturize conventional GSR sensors, precisely arrange them on a magnetic sensor element grid substrate 61, create a magnetic sensor grid, and arrange this under a petri dish 62 during cell observation to measure a magnetic field emitted by the cells, calculate the distribution of current elements flowing within the cells, and display this as an image on a PC screen. By combining the magnetic microscope with an optical microscope 64a, it is possible to easily observe both the mechanical movement and shape change of cells and the activation level of each cell in real time.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] The present invention relates to a magnetic microscope that observes the vitality of an entire cell aggregate by measuring the magnetic field emanating from within the cell aggregate and the electric current fragments that generate the magnetic field, while at the same time observing the structure and movement of the cells using a transmission or incident light optical microscope used in cell research. [Background technology]

[0002] When observing the growth of cells such as iPS cells, in addition to observing the morphological changes and movement of the cell body using an optical microscope, it is also necessary to observe the vitality of the entire cell aggregate by observing the electrical current fragments flowing within the cell aggregate.

[0003] However, the size of a cell is about 20 μm, and the size of the aggregate is less than 5 mm. By measuring the magnetic field emitted by the current flowing inside such a tiny cell, the vitality of the cell can be predicted from the strength and direction of the current fragment. However, there is currently no known magnetic microscope that can measure the current fragment flowing inside such a tiny cell. Here, a magnetic microscope can be defined as a device that detects tiny magnetic fields emitted from tiny cells, amplifies their size by a magnification of 100 to 1,000 times in accordance with the shape of the cells, and observes the distribution of magnetic fields or the distribution of electric current fragments that are the source of magnetic fields (Figure 1).

[0004] Incidentally, a cell evaluation device using a magnetic sensor is disclosed in Patent Document 1. Figure 17 of the document describes that amorphous wires with a diameter of 20 μm are arranged in a checkerboard structure at intervals of 80 μm, high-frequency alternating current is passed through both ends of each wire, and the impedance of the amorphous wire is measured from the voltage at both ends, making it possible to measure the magnetic field Hx or Hy at the intersection of the wires. However, the magnetic impedance sensor measures the average value of the magnetic field associated with the flowing wire when a voltage is applied to both ends of the wire and an AC current is passed through it, and it is theoretically clear that it cannot measure the magnetic field at the intersection point alone. In fact, no papers supporting this invention have been published, and no products are on the market.

[0005] The inventor seems to have misunderstood the technology of a grid sensor in which an element is placed at the (i,j)th position of a grid and a switch is operated so that an AC current flows from the i-th wire to the j-th wire, driving only the (i,j)-th element, although this type of sensor is widely known. In other words, a magnetic grid sensor in which magnetic sensors are placed in a grid pattern with intervals of 2 mm or less has not yet been developed.

[0006] The magnetic field emitted from a cell is thought to be about 10 nT or less directly above the cell. Patent Document 1 explains that it is 1 nT or less, but the measurement position of the sensor is 900 μm away from the cell body, whereas the present invention assumes a distance of 300 μm or less, and since the generated magnetic field is inversely proportional to the square of the distance, the views of both documents can be said to be consistent.

[0007] Considering the size of the cell, the size of the magnetic sensor element to detect the magnetic field needs to be about 10 μm to 500 μm. Currently known GSR sensors have a detection power of about 15 nT when the length is 500 μm, and no small, highly sensitive magnetic sensor that meets the above requirements is known. Moreover, the manufacturing technology for densely arranging a large number of these magnetic sensor elements in a grid on the X-axis and Y-axis has not been established.

[0008] There is a need to develop a small, highly sensitive magnetic sensor and a magnetic sensor grid using the sensor, and to develop a magnetic microscope. However, no prior art documents related to magnetic microscopes could be found by searching using j-platpat. [Prior art documents] [Patent documents]

[0009] Patent No. 5526384 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention measures the magnetic field emitted from cells of 20 μm size, and measures the vitality of cells by observing the minute magnetic field generated within the cells and the electric current fragments that are the basis of the magnetic field.

[0011] The inventor came up with the idea that if a small GSR sensor was developed and multiple elements were arranged along multiple rows of grid lines in a checkerboard pattern along the X and Y axes, it might be possible to invent a magnetic microscope capable of observing cells approximately 20 μm in size.

[0012] Specifically, the idea was that if a magnetic sensor grid with numerous magnetic sensors arranged in a checkerboard pattern along the X and Y axes were attached to the underside of the petri dish being observed with the optical microscope, the micromagnetic field would be measured, and a current element would be calculated from the measured value, making it possible to measure the micromagnetic field, current element, and cell activity at the cellular level in real time. After examining the feasibility of this, the following six problems were identified.

[0013] The technological development issues for this purpose are: The first challenge is to develop a magnetic sensor with a detection power of about 1 pT to 10 nT and a size of 10 μm to 2 mm or less. Specifically, the goal is to miniaturize and improve the performance of current GSR sensors, but the detection power of a magnetic sensor is proportional to its size, and there is a trade-off between the two, making it difficult to improve both characteristics simultaneously.

[0014] The second challenge is to develop technology to arrange 100 to 4 million magnetic sensor elements in a grid along the X and Y axes on an ASIC board. The number of elements should be selected based on the magnification of 40 to 1,000 times and the measurement area of ​​about 10 mm to 50 mm in diameter.

[0015] The third challenge is to develop a program that measures the magnetic field emitted from the current element using a magnetic sensor element grid, calculates contour maps of the current element distribution and magnetic field distribution flowing through the cell aggregate, and displays an image on a screen.

[0016] The fourth challenge is to develop a magnetic microscope with a magnification of 1,000, using a magnetic sensor grid arranged at an ultra-high density, with an image pixel size of about 20 μm and a magnetic field distribution resolution of about 1 μm.

[0017] The fifth challenge is to eliminate the need for a magnetic shielding box, which is used to eliminate external magnetic fields.

[0018] The sixth challenge is to make it possible to simultaneously observe cells using an incident-light or transmitted-light optical microscope and a magnetic microscope above a petri dish containing cells. [Means for solving the problem]

[0019] For the first issue, a GSR sensor was adopted as the magnetic sensor. By setting the coil pitch to 0.1 μm to 3 μm and installing multiple magnetic wires as necessary, the number of coil turns was set to 150 to 2,000, and the detection power was set to 1 pT to 50 pT. This problem can be solved by making the GSR sensor small and highly sensitive. Figure 2 shows a GSR sensor element.

[0020] The GSR sensor is described in detail in Japanese Patent Publication No. 5839527 by the present inventor, and is cited in the present invention. The GSR sensor is an ultra-sensitive micro-magnetic sensor that includes a GSR element that is composed of a conductive magnetic wire for detecting a magnetic field on a substrate, a detection coil formed of a circumferential coil wound around the magnetic wire, two electrodes for passing a current through the magnetic wire, and wiring that connects two electrodes for detecting the coil voltage, as well as a means for passing a pulse current having a frequency of GHz through the magnetic wire and an electronic circuit that detects the coil voltage generated when the pulse current is passed and converts the coil voltage into an external magnetic field H.

[0021] The second problem was solved by forming a large number of elements on an ASIC substrate, attaching magnetic wires in a grid pattern along the X-axis and Y-axis, and using a technology to bake coil wiring and electrode wiring onto the wires. The ASIC and the large number of elements were connected using via holes. Multiple ASICs may be used to correspond to each element, or an ASIC that has a channel switching function and controls a large number of elements may be used. The magnetic sensor element grid is shown in Figures 3 to 5. FIG. 3 is a top view of the magnetic sensor element grid, FIG. 4 (FIGS. 4A, 4B, and 4C) is a plan view of the structure of a unit element of the magnetic sensor element grid, and FIG. 5 (FIGS. 5A, 5B, and 5C) is a cross-sectional view of the magnetic sensor element grid.

[0022] The magnetic field measurements are taken at the numerous intersections of the X-axis and Y-axis elements, Hz in Fig. 4A(a-1), Hx in Fig. 4A(a-2), and Hy in Fig. 4A(a-3), and these values ​​are used as the magnetic field of the grid to calculate the magnetic field distribution and current element distribution. In other words, Hz = (Hz1 + Hz2 + Hz3 + Hz4) / 4, Hx = (Hx1 + Hx2 + Hx3 + Hx4) / 4, and Hy = (Hy1 + Hy2 + Hy3 + Hy4) / .

[0023] The top surface of the covering portion of the magnetic sensor element grid substrate is flattened to a flatness of 5 μm or less. In addition, the outside of the bottom surface of the cell observation petri dish, which comes into direct contact with the flat surface of the top of the covering portion of the magnetic sensor element grid substrate with a flatness of 5 μm or less, is also flattened to a flatness of 5 μm or less. This ensures that the flat surfaces that come into contact with each other are in close contact with each other. Furthermore, the distance from the cells to be observed to the magnetic sensor element is kept small, at 300 μm or less, to facilitate measurement of the minute magnetic field emitted by the cells. The shorter the distance from the cells to be observed to the magnetic sensor element, the better.

[0024] For the third task, before the observation, the magnetic field at the magnetic sensor element grid position (i, j) is measured to obtain the initial magnetic field measurement value mH ij (→)(b) is calculated and the value is displayed as the magnetic field distribution corresponding to the grid size on the X-axis and Y-axis plane.

[0025] Next, after placing the cells in the petri dish, the magnetic field at the magnetic sensor element grid position (i, j) is measured to obtain the magnetic field measurement value mH ij (→)(a) is found, and mH ij (→) Subtract (b) to obtain the measured value. That is, mH ij (→)=mH ij (→)(a)-mH ij The result is (→)(b).

[0026] This value is considered to be the magnetic field emitted from the current element Ids in the cell aggregate, and is measured by the magnetic sensor at the (i,j)th position of the magnetic sensor element grid and expressed as mH ij (→) and mH ij Create a contour plot of the absolute value, mH ij (→) from the peak of the current element Ids position P(x,y,z) and mH ij From the spread of the peak of (→), the length of the current fragment Ids(→) is assumed, and it is assumed that the current fragment Ids exists within the cell aggregate. If there are n peak locations, a calculation model is created assuming that there are n current fragments Ids.

[0027] Next, the kth current element I k ds k (→) Position P k (x k ,y k ,z k ) and the magnetic field measurement position Gij(x ij ,y ij , 0), and the current element position P k (x k ,y k ,z k ) and the measurement position Gij(x ij ,y ij ,0) is Rijk(→), The kth current element I k ds k The magnetic field that (→) creates at the measurement position G(i,j) is H ijk (→) =1 / 4πRijk 3 ×I k dsk The theoretical value tH at the measurement position G(i,j) of the magnetic sensor element grid can be calculated by adding up the magnetic fields. ij (→) is tH ij (→)=ΣtH ijk Let (→). (Σ adds k from 1 to n.)

[0028] The error between the two is e ij (→)=mH ij (→)-tH ij (→), and the error function E = Σ(e ij ) 2 Create a I k For the direction of (→), the inclination angle is φ k Let the angle with respect to the X-axis be θ k Then, the error function is partially differentiated using the Gauss-Newton method to derive 7n simultaneous equations, I k Absolute value and ds k and θ k and φ k and X k , Y k , Z k The goal is to derive 7n simultaneous equations consisting of the above equations, determine the 7n unknowns from these equations, calculate the numerous current elements flowing through the cell aggregate, and use the results to create a distribution map of the current elements and display it on the screen.

[0029] Here, we have decided to affix (→) to the vector representation of each vector physical quantity. FIG. 6 shows a magnetic field distribution map obtained from magnetic field measurements taken with a magnetic microscope, and FIG. 7 shows a flowchart of a calculation program for obtaining the current element. It should be noted that the present invention is directed to the configuration of a magnetic microscope, and the method of calculating the current element vector from the error function is not limited to the above method.

[0030] For the fourth issue, in the unit element of the magnetic sensor element grid, the coil pitch is set to 0.1 μm, the element length is 10 μm, the number of magnetic wires is 2, and the number of coil windings is 180, resulting in a pixel of the sensor grid of 10 μm x 10 μm. The detection power of the magnetic sensor is ensured to be 5 pT. If a magnetic field distribution map is created from these measured values, the pixel of the magnetic field distribution map will be about 1 μm, and a magnetic microscope with a magnification of 1000 times can be obtained.

[0031] Regarding the fifth issue, a method can be used that first measures the magnetic field in the microscope's measurement environment using a magnetic sensor element grid, determines the magnetic field average and magnetic field distribution, and then corrects the values ​​when cells are measured using a magnetic microscope, making the magnetic shield unnecessary. Moreover, the external magnetic field environment can also be eliminated by combining two sets of magnetic sensor element grids and making them function as a differential element.

[0032] For the sixth task, we devised a structure that allows both measurements to be performed simultaneously by placing an optical microscope and / or a CCD camera above the petri dish containing the cells, and placing a magnetic microscope directly below the magnetic sensor element grid substrate below the petri dish. In the case of a transmission optical microscope, the material of the magnetic sensor element grid substrate is made transparent to allow light to pass through from below the microscope.

[0033] Needless to say, the petri dish is fixed on the magnetic microscope base, and both are fixed on the microscope observation stand so that they do not move during observation. The fixing method may be any of mechanical methods such as clips, or chemical methods such as adhesives.

[0034] Next, the current element distribution measured by the magnetic microscope is superimposed on the shape diagram of the cell observed by the optical microscope, and the image is displayed on a screen, which makes it easy to observe both the mechanical movement and shape change of the cells, as well as the activation level of each cell, in real time. The combined images of the optical microscope and magnetic microscope are shown in Figures 8 and 9, and the results of each observation (the magnetic microscope is an image) are shown in Figure 10.

[0035] In order to make the spatial resolution of the magnetic field equal to the resolution of an optical microscope, the magnetic field at the intermediate position between the discretely measured magnetic field values ​​is calculated by functionally approximating the magnetic field distribution using an interpolation method. This interpolation method can obtain a spatial resolution of the magnetic field of about 1 / 20 of the grid interval. It was confirmed that the position accuracy of the current element was theoretically about 1 / 20 of the grid interval using a program that calculates the position of the current element.

[0036] By combining these six solutions, the magnetic microscope was invented. It should be noted that the present invention is intended for small, highly sensitive magnetic sensors and is not limited to GSR sensors, as is apparent from the configuration. Effect of the Invention

[0037] This invention makes it possible to observe the vitality of cells. Moreover, by integrating it with an optical microscope, it becomes possible to simultaneously measure the shape, movement, and vitality of individual cells in real time, and it is expected to become a basic tool for cell research. [Brief description of the drawings]

[0038] [Figure 1] An image of a magnetic microscope. (a) is a perspective view, and (b) is a side view. [Diagram 2] FIG. 2 is a diagram showing a GSR sensor element. [Diagram 3] FIG. 2 is a plan view of a magnetic sensor element grid. [Figure 4A] FIG. 2 is a plan view of a grid unit of a magnetic sensor element, showing a single-axis element consisting of a Z-axis element, an X-axis element, or a Y-axis element. [Figure 4B]FIG. 2 is a plan view of a grid unit of a magnetic sensor element, showing a two-axis element consisting of an X-axis element and a Y-axis element. [Figure 4C] FIG. 2 is a plan view of a grid unit of a magnetic sensor element, showing a three-axis element consisting of an X-axis element and a Y-axis element arranged on an element base. [Figure 5A] 1A is a cross-sectional view of the z-axis type, x-axis type, and y-axis type of one-axis element grid along line A1-A2. 1B is a diagram of a combination with a petri dish and a cell solution. [Figure 5B] 1 is a cross-sectional view of the two-element grid taken along line A1-A2, and is a diagram showing a combination of the grid with a petri dish and a cell solution. [Figure 5C] 1 is a cross-sectional view of the three-element grid taken along line A1-A2, and is a diagram showing a combination of the grid with a petri dish and a cell solution. [Figure 6] FIG. 1 is a magnetic field distribution diagram obtained from magnetic field measurements taken with a magnetic microscope. [Figure 7] FIG. 13 is a flowchart showing a calculation program for determining a current element. [Figure 8] This is an image diagram of an incident-light optical microscope and a magnetic microscope combined. [Figure 9] This is an image diagram of a transmission optical microscope and a magnetic microscope integrated together. [Figure 10] FIG. 1 is an image of the observation results obtained using a magnetic microscope. BEST MODE FOR CARRYING OUT THEINVENTION

[0039] The first embodiment of the present invention is as follows. A magnetic microscope comprising a petri dish for cell observation, a magnetic sensor element grid substrate, a signal processing circuit, and a display device, The petri dish can have a cell solution placed on its upper surface and a magnetic sensor element grid substrate placed on its bottom surface, The petri dish has a bottom thickness of 300 μm or less, and the flatness of the outer part of the bottom surface is flattened to 5 μm or less; the magnetic sensor element grid substrate includes a magnetic sensor element grid and a magnetic sensor element grid circuit disposed on a substrate, a covering portion for protecting the magnetic sensor element grid and the magnetic sensor element grid circuit, and an external connection terminal; The upper surface of the covering portion is flattened to a flatness of 5 μm or less, the petri dish is fixed to an upper surface of the covering portion of the magnetic sensor element grid substrate, and the magnetic sensor element grid substrate is fixed to a cell observation stand so as not to move during observation; The signal circuit detects a grid voltage corresponding to a micromagnetic field generated by a current element flowing in a cell by the magnetic sensor element grid, and converts the grid voltage into a value of a grid magnetic field; the display device arranges magnetic sensor elements at intersections of a checkerboard pattern along an X-axis and a Y-axis on the substrate in the magnetic sensor element grid, and measures a magnetic field at the intersections; A magnetic microscope characterized in that the absolute value of the grid magnetic field is displayed as a contour map.

[0040] In addition, the magnetic sensor element of the magnetic microscope is It is characterized by being composed of a single-axis element that measures one of the following magnetic fields: Hz magnetic field, Hx magnetic field, or Hy magnetic field.

[0041] In addition, the magnetic sensor element of the magnetic microscope is It is characterized by being composed of a two-axis element that measures the Hx magnetic field and the Hy magnetic field.

[0042] In addition, the magnetic sensor element of the magnetic microscope is It is characterized by being composed of a three-axis element that measures the Hx magnetic field, Hy magnetic field and Hz magnetic field.

[0043] In addition, the magnetic microscope The present invention is characterized by comprising a program for calculating the current element flowing within a cell from the value of the grid magnetic field, and a device for displaying that value as an image on a screen.

[0044] In addition, the magnetic microscope Before the observation, measure the magnetic field at the sensor grid position (i,j) to obtain the initial magnetic field measurement mH ij (→)(b) is calculated and the value is displayed as the magnetic field distribution corresponding to the grid size on the X-axis and Y-axis plane.

[0045] Next, after placing a cell in the petri dish, measure the magnetic field at the sensor grid position (i, j) to obtain the magnetic field measurement value mH ij (→)(a) is found, and mH ij (→) Subtract (b) to get the measured value mH ij (→). That is, the measured value mH ij (→)=mH ij (→)(a)-mH ij The result is (→)(b).

[0046] This measurement value mH ij (→) is considered to be the magnetic field emitted from the current element in the cell aggregate, and the measured value is expressed as mH ij (→) and mH ij Create a contour plot of the absolute values, mH ij Assuming the position P(x,y,z) of the current element Ids from the peak of the absolute value of (→), ij A length ds of the current element Ids is assumed from the spread of the peak of the absolute value of (→), and a calculation model is created in which n current elements Ids exist within the cell aggregate.

[0047] Next, the kth current element I k ds k Position P k (x k ,y k ,z k ) and the magnetic field measurement position G ij (x ij ,y ij , 0) as the current element position P k (x k ,y k ,z k ) and magnetic field measurement position G ij (x ij ,y ij ,0) and the distance Rijk Then, kth current element I k ds k The magnetic field created by is H ijk (→)=1 / 4πR ijk 3 ×I k ds k (→)×R ijk (→), the magnetic fields 1 to n are added together to obtain the measurement position G of the magnetic sensor element grid. ij The theoretical value of the magnetic field created at position (i,j) is tH ij (→)=ΣH ijk (→) and The error between the two is e ij (→)=mH ij (→)-tH ij (→) and Error function E=Σ(e ij ) 2 Create a.

[0048] I k For the direction of (→), the inclination angle is φ k Let the angle with respect to the X-axis be θ k year, From this error function, I k and ds k and θ k and φ k and X k , Y k , Z k Find the 7n simultaneous equations related to, and find the unknowns from those equations. It calculates n current elements flowing through a cell aggregate, and uses the results to create a distribution map of the current elements, which is then displayed on the screen.

[0049] In addition, the magnetic microscope The method is characterized by first measuring the magnetic field in the measurement environment of the microscope with a magnetic sensor element grid, determining the magnetic field average and magnetic field distribution, and correcting the values ​​when cells are measured in real time with the magnetic microscope, or by combining two sets of magnetic sensor element grids to function as a differential element, thereby removing the external magnetic field environment.

[0050] A second embodiment of the present invention is as follows. The magnetic microscope is A magnetic sensor element grid is placed below the surface of the petri dish substrate for cell observation, and an incident-light optical microscope and / or a CCD camera are placed above, allowing the shape and movement of cells to be observed optically and magnetically at the same time.

[0051] In addition, the magnetic microscope A magnetic sensor element grid is placed below the surface of the cell observation petri dish substrate, with the magnetic sensor element grid made of a transparent material, and a transmission optical microscope and / or a CCD camera are placed above, allowing the shape and movement of cells to be observed optically and magnetically at the same time.

[0052] Hereinafter, an embodiment of the present invention will be described in detail with reference to FIGS. <Magnetic microscope> The positional relationship between the grid substrate of the magnetic sensor elements that constitute the magnetic microscope, the cells (cell aggregates) of the specimen, and the petri dish is shown in Figure 1. The magnetic microscope is also composed of a signal processing circuit that converts the magnetic field signals detected by the magnetic sensor elements, a display device, etc. In the magnetic sensor element grid substrate 10, a large number of magnetic sensor elements 101 are arranged on a grid substrate (substrate) to form a magnetic sensor element grid. A cell (cell aggregate) 12 is placed on a petri dish (for cell observation) 11, and the petri dish 11 is placed on a magnetic sensor element grid substrate 10 as shown in FIG. The bottom surface 111 of the petri dish 11 and the top surface 102 of the covering portion of the magnetic sensor element grid substrate 10 are both flat surfaces with a flatness of 5 μm or less, and the two flat surfaces are in close contact with each other. The positional relationship between the two can be arbitrarily selected as an optimal combination taking into consideration the size and volume of the cell (cell aggregate) to be observed, the detection capability of the magnetic sensor element grid, and further the combination with an optical microscope.

[0053] <Magnetic sensor> A GSR sensor is used as the magnetic sensor. The basic structure of the GSR element is shown in Fig. 2, and the structure of the magnetic sensor element grid is shown and explained in Figs. The diameter of the magnetic wire 22 is 1 μm to 10 μm. The coil pitch of the detection coil 23 is 0.1 μm to 3 μm, and the coil width is 3 μm to 30 μm. The number of magnetic wires is 1 to 8 as required. It should be noted that the magnetic sensor constituting the magnetic microscope is not limited to the GSR sensor, so long as it can exhibit the characteristics required for a magnetic microscope in terms of size and performance.

[0054] The size of unit elements (magnetic sensor elements) 32 of the magnetic sensor element grid 3 formed and arranged on a substrate (grid substrate) 31 is 10 μm to 1 mm. When the size of the unit element 32 is 10 μm, it is preferable that the coil pitch is 0.1 μm, the coil width is 3 μm, the number of magnetic wires is two, and the number of coil turns is 150. On the other hand, when the size of the unit element 32 is 1 mm, it is preferable that the coil pitch is 3 μm, the coil width is 30 μm, the number of magnetic wires is 1 to 8, and the number of coil turns is 2400.

[0055] Here, the GSR sensor is an ultra-sensitive micro-magnetic sensor that includes a GSR element 2 that is composed of a conductive magnetic field detection magnetic wire 22 on a substrate 21, a detection coil 23 formed by a circular coil wound around the magnetic wire, and wiring 26, 29 that connects two electrodes 24 for passing current through the wire and two electrodes 28 for detecting the coil voltage, as shown in Figure 2, and includes a means for passing a pulse current having a frequency of GHz through the magnetic wire, and an electronic circuit that detects the coil voltage generated when the pulse current is passed through the magnetic wire and converts the coil voltage into an external magnetic field H.

[0056] <Magnetic sensor element grid (grid)> The magnetic sensor element grid 3, as shown in FIG. 3, is configured by arranging a large number of unit elements (magnetic sensor elements) 32 on a grid substrate 31. The size of the magnetic sensor element grid 3 is 5 mm square to 20 mm square, or 5 mm diameter to 20 mm diameter. The size of the unit element 32 is 10 μm to 1 mm. The number of unit elements 32 is a minimum of 5×5, or 25 pixels, to a maximum of 2000×2000, or 4 million pixels.

[0057] As shown in FIG. 4, the unit elements 32 are of three types: a one-axis element type (FIG. 4A), a two-axis element type (FIG. 4B), and a three-axis element type (FIG. 4C). 5 (5A, 5B, and 5C) show cross-sectional views of the three types taken along line A1-A2 in Fig. 3. The cross-sectional views show a cross-section of a magnetic sensor element grid placed on a petri dish.

[0058] First, the type of one-axis element (FIG. 4A) is a type in which the GSR sensor element 321 is arranged symmetrically on the grid substrate 31 with respect to the intersection point 321o of the X-axis and Y-axis. (a-1) A one-axis element type in which the Z-axis elements 321z are arranged one by one in the Z-axis direction; (a-2) A single-axis element type in which the X-axis elements 321x are arranged one by one in the X-axis direction; (a-3) A type of single-axis element in which the Y-axis elements 321y are arranged one each in the Y-axis direction There is.

[0059] These cross-sectional views (FIG. 5A) are shown together with the cell fluid (cell body) 12 and a petri dish 40 carrying the cell fluid 12. The same applies below. (a-1) One-axis element grid-z-axis type (4(41)) is made up of a GSR element 411, an ASIC 412, an electrode 413 and a covering part 415 for protecting a grid wiring 414, and is placed under a petri dish 40. (a-2) The one-axis element grid-x-axis type (4(42)) comprises a GSR element 421, an ASIC 422, an electrode 423 and a covering portion 425 for protecting the grid wiring 424, and is placed under the petri dish 40. (a-3) One-axis element grid-y-axis type (4(43)) comprises a GSR element 431, an ASIC 432, an electrode 433 and a covering part 435 for protecting a grid wiring 434, and is placed under a petri dish 40.

[0060] Next, the two-axis element type (FIG. 4B) is a type in which the GSR sensor element 321 is attached to the grid substrate 31 along the intersection 321o of the X-axis and Y-axis. (b) A two-axis element type in which two X-axis elements 321x in the horizontal direction and two Y-axis elements 321y in the vertical direction are symmetrically arranged with the origin 321o at the center. There is.

[0061] The cross-sectional view of this biaxial element type (4(44)) is shown in FIG. 5B, and it comprises a GSR element 441, an ASIC 442, an electrode 443, and a covering 445 for protecting a grid wiring 444, and is placed under a petri dish 40.

[0062] Finally, the type of three-axis element (FIG. 4C) is a three-axis type in which a magnetic field vector sensor is arranged on grid substrate 31 along intersection 321o of the X-axis and Y-axis. This three-axis element type has four GSR elements or on-ASIC type GSR sensors arranged in four-fold symmetry and mirror symmetry on the inclined surface 331 of a base 330 formed from a square pyramid, an octagonal pyramid, or an irregular octahedral pyramid, so that the inclination direction coincides with the magnetic wire 321w of the GSR element. The magnetic fields in the X-axis and Y-axis directions are measured from two X-axis elements 321x and two Y-axis elements 321y, and the magnetic field in the Z-axis direction is found by calculation. The on-ASIC type GSR sensor was invented by the inventors and is disclosed in a patent publication (Patent Publication No. 7062216). Please refer to the description in this patent publication for details. The magnetic field vector sensor was invented by the inventors and is disclosed in a patent publication (Patent Publication No. 7215702). Please refer to the description in this patent publication for details.

[0063] The cross-sectional view of this triaxial element type (4(45)) is shown in FIG. 5C, and it comprises a GSR element 451, an ASIC 452, an electrode 453, and a covering 455 for protecting a grid wiring 454, and is placed under a petri dish 40. The magnetic field in the Z-axis direction is obtained by calculation, and element 451z is a calculated element.

[0064] <Positional relationship between magnetic sensor element grid and cells> The position of the magnetic sensor element grid (magnetic sensor element grid substrate) 10 is preferably such that the thickness of the petri dish 11 is 300 μm or less, the covering portion of the upper surface of the magnetic sensor element grid 10 is flattened to a flatness of 5 μm or less, this flat surface is directly pressed against the back side of the bottom surface of the petri dish which is flattened to a flatness of 5 μm or less, and the distance from the cell to the measurement portion of the magnetic sensor element 101 is 300 μm or less. Since the magnetic field generated by the cell is inversely proportional to the square of the distance, it is necessary to make this distance as small as possible. The flat surfaces of both are flattened to a flatness of less than μm, so that they come into close contact with each other, making the distance between the cells in the dish and the magnetic sensor element uniform.

[0065] <Measurement of magnetic field components at the intersections of unit elements> (A) Measurement of magnetic field components in a uniaxial element (a-1) From the four data Hz1, Hz2, Hz3, and Hz4 measured by the four unit elements 321z centered on the intersection 321o, the magnetic field component Hz at the intersection can be calculated from Hz = (Hz1 + Hz2 + Hz3 + Hz4) / 4. (a-2) From the four pieces of data Hx1, Hx2, Hx3, and Hx4 measured by the four unit elements 321x centered on the intersection 321o, Hx, which is the magnetic field component of the intersection, can be calculated from Hx=(Hx1+Hx2+Hx3+Hx4) / 4. (a-3) From the four data Hy1, Hy2, Hy3, and Hy4 measured by the four unit elements 321y centered on the intersection 321o, Hy, which is the magnetic field component of the intersection, can be calculated from Hy=(Hy1+Hy2+Hy3+Hy4) / 4.

[0066] (B) Measurement of magnetic field components in a two-axis element (b) From the four pieces of data Hx1, Hx2, Hy1, and Hy2 measured with two unit elements 321x oriented in the X-axis direction and two unit elements 321y oriented in the Y-axis direction centered on the intersection 321o, the magnetic field components Hx and Hy at the intersection can be calculated from Hx=(Hx1+Hx2) / 2, Hy=(Hy1+Hy2) / 2.

[0067] (C) Measurement of magnetic field components in a triaxial element (c) In the magnetic field vector sensor, the tilt angle of the base is θ, and from four pieces of data Hx1, Hx2, Hy1, and Hy2 measured with two unit elements 321x oriented in the X-axis direction and two unit elements 321y oriented in the Y-axis direction centered on the intersection 321o, the magnetic field components Hx and Hy at the intersection 321o can be calculated from Hx = (1 / 2 cosθ) (Hx1 - Hx2) and Hy = (1 / 2 sinθ) (Hy1 - Hy2). The Hz in the Z-axis direction can be calculated from Hz = (1 / 4 sinθ) (Hx1 + Hx2 + Hy1 + Hy2).

[0068] To eliminate the influence of the external magnetic field, the environmental magnetic field can be measured before the measurement, and then the environmental magnetic field can be subtracted from the measured value when the cells are placed in the petri dish for observation. Alternatively, two sets of magnetic sensor element grids can be arranged differentially and the difference between the two measured values ​​can be calculated.

[0069] The magnetic sensor element grid is such that the intersection position of the unit element 32 is the i-th position in the X-axis direction and the j-th position in the Y-axis direction is P ij Let us express it as P ij The measured value at H ij As, H ij It also has an electronic circuit that transfers the signal to a current element distribution calculation program. In addition, the position on the grid substrate of each element (elements in the X-axis, Y-axis, and Z-axis directions) constituting the unit element 32 may be designated as (i, j), and the measurement values ​​of each element may be directly used as grid magnetic field measurement values ​​to calculate the magnetic field distribution and current element distribution.

[0070] <Program to calculate current element distribution> The program for calculating the current element distribution measures the magnetic field emitted from the current element in the cell aggregate with a magnetic sensor at the (i,j)th position of the magnetic sensor element grid, and converts the measured value into mH ij Let us assume that.

[0071] In practice, the magnetic field is affected by the environmental magnetic field, so before observation, the magnetic field at the magnetic sensor element grid position (i, j) is measured to obtain the initial magnetic field measurement value mH ij (→)(b) is calculated and the value is displayed as the magnetic field distribution corresponding to the grid size on the X-axis and Y-axis plane.

[0072] Next, after placing the cells in the petri dish, the magnetic field at the magnetic sensor element grid position (i, j) is measured to obtain the magnetic field measurement value mH ij (→)(a) is found, and mH ij (→)Subtract (b) to obtain the measured value. That is, mH ij (→)=mH ij (→)(a)-mH ij The result is (→)(b). This value is considered to be the magnetic field emitted from the current element in the cell aggregate, and is measured by the magnetic sensor at the (i,j)th position of the magnetic sensor element grid and expressed as mH ij Let's say (→).

[0073] mH ij Create a contour plot of the absolute value, identify the n peak positions, and ij Position P of the current element from the peak of the absolute value k (x k ,y k ,z k ) and the spread of the peak of the current element ds k Assuming the length of (→), the position P k (x k ,y k ,z k ) to current element I k ds kAssume that (→) exists. If there are n peak locations (one or more), create a calculation model assuming that there are n current segments.

[0074] Next, the kth current element I k ds k Position P k (x k ,y k ,z k ) and the magnetic field measurement position G ij (x ij ,y ij ,o) as the current element position P k (x k ,y k ,z k ) and measurement position G ij (x ij ,y ij ,o) and the distance R ijk Then, kth current element I k ds k The magnetic field created by is H ijk (→)=1 / 4πR ijk 3 ×I k ds k (→)×R ijk (→) equation, the magnetic fields created by n current elements are added together to create the theoretical value tH at the (i,j)th position of the magnetic sensor element grid. ij (→)=ΣtH ijk Let's say (→).

[0075] The error between the two is e ij =mH ij (→)-tH ij (→), and the error function E = Σ(e ij ) 2 Create a I k The direction of the inclination angle is φ k Let the angle with respect to the X-axis be θ k Then, the error function is partially differentiated using the Gauss-Newton method to derive 7n simultaneous equations, I k and ds k and θ k and φk and X k , Y k , Z k A set of 7n simultaneous equations consisting of the above equations is then determined, and the 7n unknowns are then obtained from these equations. The numerous current elements flowing through the cell aggregate are then calculated, and the results are used to create a distribution map of the current elements, which is then displayed on the screen as a magnetic field distribution map.

[0076] The program procedure is as follows (Figure 7). Note that vector display is (→). 1st step(101); Before the observation, measure the magnetic field at the sensor grid position (i,j) to obtain the initial magnetic field measurement mH ij Find (→)(b). However, the position of each element is specified in advance using the sensor grid coordinate system O-XYZ.

[0077] Second step (102); After placing the cell in the dish, measure the magnetic field at the sensor grid position (i, j) to obtain the magnetic field measurement value mH ij (→)(a) is found, and mH ij (→) Subtract (b) to get the measured value mH ij (→), calculate the magnetic field distribution, mH ij (→) If there are k peaks in the absolute value distribution, the peak position P k (X k , Y k , Z k ) to current element I k ds k Assume that there exists ds k The length is determined from the spread of the mountain, with the peak position as the center.

[0078] Figure 6 shows an example of a magnetic field distribution map. The cell aggregate 5 is composed of five cells (n=5), namely, cells 51 to 55, and the contour lines 511 to 551 of the respective cells are illustrated to show a contour map. 1 ds 1 ~Current element I 5 ds5 and its position is position P 1 ~Position P 5 So it is. This is transferred to the current element distribution calculation program.

[0079] Third step (103); Peak position P k Current element I k ds k Assuming that there exists P k Located at n pieces The current element Ids creates the position G of each magnetic sensor element grid ij (X ij ,Y ij The theoretical magnetic field strength at the kth current element is calculated by the formula tH ijk (→)=1 / 4πR ijk 3 ×I k ds k (→)×R ijk (→) can be obtained. Since there are n current segments from 1, G ij (X ij ,Y ij The theoretical magnetic field strength at the position tH ij =Σ 1 / 4πR ijk 3 ×I k ds k ×R ijk The sum of n numbers is calculated as follows.

[0080] Fourth step (104); Position I of the kth current element k ds k and magnetic field measurement position G ij (X ij ,Y ij ) distance vector R ijk (→) and the inclination of the current element with respect to the Z axis is φ k The inclination with respect to the X-axis is θ k Then, tH ij is the current intensity I k and the length of the current piece ds k and position X ijk , Y ijk , Zijk and azimuth angle θ k , φ k It is a function of .

[0081] 5th step(105); Calculate the measurement error. e ij (→) = mH ij (→)-tH ij (→)

[0082] 6th step(106); Calculate the sum of squares of the errors. E = Σe ij 2

[0083] 7th step(107); Current intensity I at which the sum of squared errors is minimized using the Gauss-Newton method k and the length ds to the current element k and X k、 Y k , Z k and azimuth angle θ k , φ k Calculate.

[0084] 8th step(108); The size of each current element I k ds k and position X k , Y k , Z k The current element distribution is calculated from the value of and displayed as an image on the PC screen. Or the current intensity I k and position X k , Y k , Z k The current distribution is calculated from the values ​​of and displayed as an image on the PC screen.

[0085] The center position of the current fragment has a positional accuracy of 1 / 10 of the pixel size or less, and the current intensity can be calculated with an accuracy of ±10% or less.

[0086] In addition, the distribution of each current element is restricted by the law of conservation of current, and it is confirmed whether there is any contradiction with the law of conservation of current.

[0087] In the second embodiment of the present invention, as shown in Figures 8 and 9, a magnetic sensor element grid 61 is placed below the substrate surface of a petri dish 62, and an optical microscope 64 (64a, 64b) is placed above it, so that the movement of cells 63 can be observed optically and simultaneously magnetically.

[0088] The current element distribution measured by the magnetic microscope is superimposed on a shape diagram of the cell 63 observed and photographed by the optical microscope 64, and the image is displayed on a screen. This allows for easy observation of both the mechanical movement and shape change of cells (Figure 10(a)) and the activation level of each cell 71 (Figure 10(b)) in real time. EXAMPLES

[0089] [Example 1] In the first embodiment of the present invention, the design elements are as follows: A GSR sensor is used as the magnetic sensor. The structure of the GSR element is shown in Figure 2. The length of the magnetic wire 22 was 0.95 mm, the wire diameter was 10 μm, the coil pitch was 3 μm, the coil width was 30 μm, and the number of magnetic wires was 4. The number of coil turns was 1,200. The size of the unit element of the sensor grid was 1 mm. As a result, the magnetic detection power of the unit element was 1 pT.

[0090] The GSR sensor used here is an ultra-sensitive micromagnetic sensor consisting of a GSR element consisting of a conductive magnetic wire on a substrate for detecting a magnetic field, a detection coil formed by a circular coil wound around the magnetic wire, two electrodes for passing current through the wire, and wiring connecting two electrodes for detecting the coil voltage, as shown in Figure 2, and also consisting of a means for passing a pulse current having a frequency of 1.2 GHz through the magnetic wire, and an electronic circuit that detects the coil voltage generated when the pulse current is passed through it and converts the coil voltage into an external magnetic field H.

[0091] The magnetic sensor element grid is an arrangement of many unit elements as shown in Figure 3. The unit elements are made up of two-axis elements, and as shown in Figure 4B(b), two GSR sensor elements are arranged on the grid substrate along the X-axis and two along the Y-axis, allowing the magnetic field (Hx, Hy) at the intersection of the X-axis element and the Y-axis element to be measured. The size of the grid sensor is 20 mm square. The number of elements in the grid is 361 pixels. The grid substrate is made of a flat insulating material or an ASIC substrate coated with an insulating film. The grid substrate consists of the top surface on which the elements are placed, an electronic circuit surface that processes signals, and a strong material that ensures mechanical strength. The GSR elements are placed on the ASIC substrate surface, which consists of the electronic circuit and strong material, and the two are connected by via holes. Furthermore, the grid substrate is protected by a coating, and the top surface of the coating is flattened to a flatness of 5 μm or less.

[0092] The bottom of the dish had a thickness of 160 μm, and the outside of the bottom was flattened to a flatness of 3 μm. The flat surface of the covering part of the magnetic sensor element grid substrate, which had a flatness of 3 μm, was directly pressed against the back side (outside of the bottom) of the dish, making the distance from the cell to the measurement part of the magnetic sensor element from the cell liquid surface 240 μm.

[0093] From the four data points Hx1, Hx2, Hy1, and Hy2 measured by the unit element, the magnetic field components Hx and Hy at the intersection were calculated from Hx = (Hx1 + Hx2) / 2 and Hy = (Hy1 + Hy2) / 2. Before the measurement, the environmental magnetic field was first measured, and when the cells were placed in the dish and observed, the environmental magnetic field was subtracted from the measured value to prevent the influence of the external magnetic field.

[0094] The magnetic sensor element grid is a grid of sensor elements, whose intersection points are the i-th position in the X-axis direction and the j-th position in the Y-axis direction. ij and P ij The measured value at H ij (→) and the magnetic field measurement distribution was obtained. This measurement value H ij (→) was transferred via an electronic circuit to a current element distribution calculation program. Furthermore, for the magnetic field between the grids, an approximation curve was created by the interpolation method, and a magnetic field spatial distribution ability of about 1 / 20 of the grid spacing was obtained.

[0095] The program for calculating the current element distribution measures the magnetic field emitted from the current element in the cell aggregate with the magnetic sensor at the (i, J)th position of the magnetic sensor grid, and converts the measured value into mH ij (→), the theoretical value tH generated by the kth current element ij (→) The magnetic field created by each current element is tH ijk (→) =1 / 4πR ijk 3 ×I k ds k (→)×R ijk (→), so we added up the magnetic fields created by n current elements. The error between the two is e ij (→)=mH ij (→)-tH ij (→), and the error function E = Σ(e ij ) 2 The direction of I is defined as the inclination angle φ with respect to the XY axis plane, and the angle with respect to the X axis is defined as θ.

[0096] When there are six current elements (i.e., n=6), by partially differentiating the error function using the Gauss-Newton method, 42 simultaneous equations can be derived and then the solution can be found.

[0097] The program procedure is as follows (Figure 7). Vector display is (→). The first step (101) is to measure the magnetic field at a sensor grid location (i,j) before observation to obtain an initial magnetic field measurement mH ij Find (→)(b). However, the position of each element is specified in advance using the sensor grid coordinate system O-XYZ.

[0098] In the second step (102), after placing the cell in the petri dish, the magnetic field at the magnetic sensor element grid position (i, j) is measured to obtain a magnetic field measurement value mH ij (→)(a) is found, and mH ij (→) Subtract (b) to get the magnetic field measurement value mH ij (→), calculate the magnetic field distribution, mH ij (→) If there are n peaks in the absolute value distribution, the kth peak position P k (x k , y k , z k ) to current element I k ds k Assume that the length of ds is determined from the peak position and the spread of the peak. Transfer this to the current element distribution calculation program.

[0099] The third step (103) is to determine the peak position P k Current element Ids k Assuming that there exists P k The theoretical magnetic field strength at each magnetic sensor element grid position P(i,j) generated by n current elements Ids (k ranges from 1 to n) at the position is calculated by the formula tH ij =1 / 4πR ij 3 ×Ids×R ij Since there are n current elements, the theoretical magnetic field strength at the position of P(i,j) is tH ij =Σ 1 / 4πR ijk 3 ×I k ds k ×R ijk The sum of n numbers is calculated as follows.

[0100] In the fourth step (104), the k-th current element position I k ds k and magnetic field measurement position G k Distance vector R to ijk (→) and the inclination of the current element with respect to the Z axis is φ k The inclination with respect to the X-axis is θ k Then, the theoretical magnetic field strength tH ij is the current intensity I k and the length of the current piece ds k and position X ijk , Y ijk , Z ijk and azimuth angle θ k , φ k It is a function of .

[0101] The fifth step (105) is to calculate the measurement error. e ij (→) = mH ij (→)-tH ij (→)

[0102] The sixth step (106) is to find the sum of the squares of the errors. E = Σe ij 2

[0103] The seventh step (107) is to find the I k and ds k and X k、 Y k , Z k and azimuth angle θ k , φ k Calculate.

[0104] The eighth step (108) is to determine the magnitude I k and ds k and position X k , Y k , Z k and azimuth angle θ k , φ k From the value of , the current element vector distribution or the current intensity of the current element is calculated and displayed as an image on the PC screen. The center position, intensity, and direction of the current segment have a positional accuracy of less than 1 / 10 of the pixel size, and the current intensity and direction can be calculated with an accuracy of less than ±10%. Furthermore, the distribution of each current element was consistent with the law of conservation of current.

[0105] The size of the sensor grid was 20 mm square, and the number of grid elements was 400 pixels. The magnetic field spatial resolution of the magnetic microscope and the positional accuracy of the center position of the current element were 50 μm, which corresponds to the accuracy of a 20x optical microscope. The intensity and direction of the current were estimated to be accurate to within ±10% as a result of simulation calculations using a current element calculation program.

[0106] [Example 2] In Example 1, the GSR sensor had a magnetic wire length of 10 μm, a wire diameter of 8 μm, a coil pitch of 0.1 μm, a coil width of 20 μm, and two wires. The size of the unit element of the sensor grid was 0.10 mm. The number of coil turns was 200. As a result, the magnetic detection power of the unit element was 100 pT.

[0107] The size of the sensor grid was 10 mm square, and the number of elements in the grid was 2500 pixels.

[0108] The magnetic field spatial resolution of the magnetic microscope and the positional accuracy of the center position of the current element are 10 μm. The accuracy of the current was predicted to be within ±10%.

[0109] [Example 3] In Example 1, the GSR sensor had a magnetic wire length of 10 μm, a wire diameter of 2 μm, a coil pitch of 0.2 μm, a coil width of 4.5 μm, and a number of wires of 2. The size of the unit element of the sensor grid was 0.011 mm. The number of coil turns was 100. As a result, the magnetic detection power of the unit element was 500 pT.

[0110] The size of the sensor grid was 5 mm square, and the number of elements in the grid was 250,000 pixels.

[0111] The magnetic field spatial resolution of the magnetic microscope and the positional accuracy of the center position of the current element are 1 μm. This corresponds to the accuracy of a 1000x optical microscope. The intensity and direction of the current were estimated to be within ±5% accuracy as a result of simulation calculations using a current element calculation program.

[0112] [Example 4] In Example 1, the GSR sensor had a magnetic wire length of 2 mm, a wire diameter of 10 μm, a coil pitch of 3 μm, a coil width of 40 μm, and a number of wires of 4. The size of the unit element of the sensor grid was 0.011 mm. The number of coil turns was 2000. As a result, the magnetic detection power of the unit element was 5 pT.

[0113] The size of the sensor grid was 50 mm square, and the number of elements in the grid was 625 pixels.

[0114] The magnetic field spatial resolution of the magnetic microscope and the positional accuracy of the center position of the current element are 50 μm. This enabled us to achieve the same accuracy as a 10x optical microscope. The intensity and direction of the current could be estimated to an accuracy of ±10% or less as a result of simulation calculations using a current element calculation program.

[0115] [Example 5] The magnetic microscope of Examples 1 to 4 is combined with an incident-light optical microscope. As shown in Figure 8, a magnetic sensor element grid substrate 61 was placed below the substrate surface of a petri dish 62, and an incident-light optical microscope 64a was placed above it, allowing the movement of cells 63 to be optically observed and photographed with a CCD camera 65, while simultaneously being magnetically observed. The current element distribution measured with a magnetic microscope was superimposed on a diagram of the cell shape observed with the optical microscope, and the image was displayed on a screen. This made it easy to observe both the mechanical movement and shape change of the cells, as well as the activation level of each cell, in real time (Figure 10).

[0116] [Example 6] The magnetic microscope of Examples 1 to 4 is combined with a transmission optical microscope. As shown in Figure 9, a magnetic sensor element grid substrate 61 was placed below the substrate surface of a petri dish 62, and a transmission optical microscope 64b was placed above it. The magnetic sensor grid substrate was made of transparent glass, which allowed light to pass through the cells. The movement of the cells 52 was optically observed and photographed with a CCD camera 65, and was simultaneously observed magnetically. The current element distribution measured with a magnetic microscope was superimposed on the shape diagram of the cells observed with the optical microscope, and the image was displayed on a screen. This made it easy to observe both the mechanical movement and shape change of the cells, as well as the activation level of each cell, in real time (Figure 10). [Industrial Applicability]

[0117] This invention makes it possible to easily observe both the mechanical movement and shape change of cells, as well as the activation level of each cell, in real time, and allows for more accurate observation of the growth of cells, especially IPS cells. Furthermore, by separating the petri dish from the magnetic sensor element grid substrate, an economical magnetic microscope has been made possible. [Explanation of symbols]

[0118] 10: magnetic sensor element grid substrate (magnetic sensor element grid); 101: magnetic sensor element, 102: covering portion (covering portion of magnetic sensor element grid substrate), 11: Petri dish, 111: Petri dish bottom (outer bottom of the petri dish), 12: Cells (cell aggregates, cell fluid) 2: Magnetic sensor element (GSR sensor element) 21: substrate, 22: magnetic wire, 23: detection coil, 24: wire terminal, 25: wire electrode, 26: wiring, 27: coil terminal, 28: coil electrode, 29: wiring

[0119] 3: Magnetic sensor element grid (plan view of grid unit element) 31: Grid substrate (substrate) 32: Grid unit element (unit element) 321x: magnetic sensor element (GSR sensor element) in the X-axis direction, 321y: magnetic sensor element (GSR sensor element) in the Y-axis direction, 321z: magnetic sensor element (GSR sensor element) in the Z-axis direction, 321o: origin (origin of unit element, origin of magnetic field vector sensor), 321w: magnetic wire 33: Element pedestal 330: pedestal, 331: trapezoidal slope, 332: ridgeline, 333: upper surface

[0120] 4: Element grid (cross-section of element grid, petri dish and cell solution) 4(41): 1-axis element grid - Z-axis type 40: Petri dish, 411: GSR element, 412: ASIC, 413: electrode, 414: grid wiring (wiring), 415: coating portion 4(42): 1-axis element grid - X-axis type 40: Petri dish, 421: GSR element, 422: ASIC, 423: electrode, 424: grid wiring (wiring), 425: coating part 4(43): 1-axis element grid - Y-axis type 40: Petri dish, 431: GSR element, 432: ASIC, 433: electrode, 434: grid wiring (wiring), 435: coating part 4(44): 2-axis element grid 40: Petri dish, 441: GSR element, 442: ASIC, 443: electrode, 444: grid wiring (wiring), 445: coating part 4(45): 3-axis element grid 40: Petri dish, 451: GSR element, 452: ASIC, 453: electrode, 454: grid wiring (wiring), 455: coating part

[0121] 5:Cell aggregate 51: cell, 511: contour line, 512: current element 52: cell, 521: contour line, 522: current element 53: cell, 531: contour line, 532: current element 54: cell, 541: contour line, 542: current element 55: Cell, 551: Contour line, 552: Current element

[0122] 6: Integrated system of magnetic microscope and optical microscope 61: magnetic sensor element grid substrate (magnetic microscope), 611: magnetic sensor element (GSR sensor element), 612: transparent grid substrate 62: Petri dish, 63:Cell aggregate, 64a: incident light optical microscope, 64b: transmitted light microscope 65: CCD camera 66: Light source for transmitted light microscope 7:Cell aggregate 71: Intense exercise area

Claims

1. A magnetic microscope comprising a petri dish for cell observation, a magnetic sensor element grid substrate, a signal processing circuit, and a display device, The petri dish can have a cell solution placed on its upper surface and the magnetic sensor element grid substrate placed on its bottom surface, The petri dish has a bottom thickness of 300 μm or less, and the flatness of the outer portion of the bottom surface is flattened to 5 μm or less; the magnetic sensor element grid substrate includes a magnetic sensor element grid and a magnetic sensor element grid circuit disposed on a substrate, a covering portion for protecting the magnetic sensor element grid and the magnetic sensor element grid circuit, and an external connection terminal; The upper surface of the covering portion is flattened to a flatness of 5 μm or less, the petri dish is fixed to an upper surface of the covering portion of the magnetic sensor element grid substrate, and the magnetic sensor element grid substrate is fixed to a cell observation stand so as not to move during observation; The signal circuit detects a grid voltage corresponding to a micromagnetic field generated by a current element flowing in a cell by the magnetic sensor element grid, and converts the grid voltage into a value of a grid magnetic field; the display device arranges magnetic sensor elements at intersections of a checkerboard pattern along an X-axis and a Y-axis on the substrate in the magnetic sensor element grid, and measures a magnetic field at the intersections; A magnetic microscope characterized in that the absolute value of the grid magnetic field is displayed as a contour map.

2. In claim 1, The magnetic microscope according to the present invention, wherein the magnetic sensor element is a one-axis element for measuring one of a Hz magnetic field, a Hx magnetic field, and a Hy magnetic field.

3. In claim 1, The magnetic microscope according to the present invention, wherein the magnetic sensor element is a two-axis element for measuring a Hx magnetic field and a Hy magnetic field.

4. In claim 1, The magnetic microscope according to the present invention, wherein the magnetic sensor element is a three-axis element for measuring a Hx magnetic field, a Hy magnetic field, and a Hz magnetic field.

5. In any one of claims 1 to 4, A magnetic microscope comprising a program for calculating the current element flowing within a cell from the value of the grid magnetic field, and a display device for displaying the value calculated by the program on a screen as an image.

6. In claim 5, The magnetic field generated from the current element in the cell is measured by a magnetic sensor at the (i, j)th position of the magnetic sensor element grid, and the measured value is expressed in mH ｉｊ (→) and mH ｉｊ Create a contour plot of the absolute values, The mH ｉｊ Assuming the position P(x, y, z) of the current element Ids from the peak of (→), ｉｊ A calculation model is created in which the length of the current element Ids is assumed from the spread of the peak of the peak (→) and n current elements Ids are present in the cell aggregate. Next, the k-th current element I ｋ ds ｋ Position P ｋ (x ｋ , y ｋ , z ｋ ) and the magnetic field measurement position G ｉｊ (x ｉｊ , y ｉｊ , 0) and the current element position P ｋ (x ｋ , y ｋ , z ｋ ) and the magnetic field measurement position G ｉｊ (x ｉｊ , y ｉｊ , 0) and the distance R ｉｊｋ Then, The kth current element I ｋ ds ｋ The magnetic fields generated on the sensor grid substrate are H ｋ (→) = 1 / 4πR ｋ ３ ×I ｋ ds ｋ (→) × R ｋ (→), these magnetic fields are added together to obtain the theoretical value tH at the measurement position G(i,j) of the magnetic sensor element grid. ｉｊ (→) and The error between the two is e ｉｊ (→) = mH ｉｊ (→) -tH ｉｊ (→) and Error function E = Σ(e ｉｊ ) ２ Create a I ｋ For the direction of (→), the inclination angle is φ ｋ The angle with respect to the X-axis is θ ｋ year, From this error function, I ｋ and ds ｋ and θ ｋ and φ ｋ and X ｋ , Y ｋ , Z ｋ Find the 7n simultaneous equations related to the equations, and find the unknowns from the equations. A magnetic microscope characterized in that n current elements Ids flowing through the cell aggregate are calculated, and a distribution map of the current elements Ids is created using the results and displayed on a screen.

7. In claim 6, A magnetic microscope characterized by removing the external magnetic field environment by first measuring the magnetic field in the measurement environment of the microscope with the magnetic sensor element grid, determining the magnetic field average and magnetic field distribution, and correcting the values ​​when cells are measured in real time with the magnetic microscope, or by combining two sets of the magnetic sensor element grids to function as a differential element.

8. In claim 6, This magnetic microscope is characterized in that the magnetic sensor element grid is placed below the surface of the cell observation petri dish substrate, and an incident-light optical microscope and / or a CCD camera is placed above it, allowing the shape and movement of cells to be observed optically and magnetically at the same time.

9. In claim 6, This magnetic microscope is characterized in that the magnetic sensor element grid, which has a transparent material for the magnetic sensor element grid substrate, is placed below the surface of the cell observation petri dish substrate, and a transmission optical microscope and / or a CCD camera are placed above, allowing the shape and movement of cells to be observed optically and magnetically at the same time.

Images