Substance estimation device, total reflection illumination microscope, substance estimation method, and program

Abstract

To provide a substance estimation device which can highly accurately estimate an image of a substance captured by a capturing body, and provide a total reflection illumination microscope, a substance estimation method and a program.SOLUTION: A substance estimation device comprises: an acquisition unit which acquires a photographed image of a composite body; and a calculation unit which calculates an estimation image of a substance being any of transfer factor, transmitter substance, transfer carrier and antigen captured by a capturing body on the basis of the photographed image of the composite body acquired by the acquisition unit. The calculation unit calculates the estimation image of the substance captured by the capturing body by executing deconvolution to the photographed image of the composite body. The substance captured by the capturing body is the substance obtained by capturing the substance emitted from a substance emission source in the liquid solution including a phosphor by the capturing body arranged on a bottom portion of the liquid solution. The composite body is formed by coupling the substance captured by the capturing body to the phosphor in the liquid solution.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a substance estimation device, a total internal reflection illumination microscope, a substance estimation method, and a program.

Background Art

[0002] Conventionally, there has been no method for directly observing cell-cell interactions through the exchange of soluble proteins (such as cytokines). The present inventors have successfully developed a system for real-time measurement of cytokine secretion from a single cell in a microwell (the single-cell secretion real-time measurement method) (see Non-Patent Document 1). In this method, an illumination method using an evanescent field by total internal reflection is utilized based on a fluorescence immunoassay. The present inventors have also established a method for further developing the single-cell secretion real-time measurement method. In this method, cytokines secreted from cells cultured in a microwell are captured by a capture antibody on the bottom surface of a glass well. The culture solution contains a fluorescence detection antibody, which binds to the captured cytokine to form a sandwich immune complex. In this method, only the fluorescent complex fixed to the bottom surface is detected without detecting free fluorescent antibodies in the solution. Specifically, only the surface of the solid layer is illuminated with an evanescent field using a total internal reflection illumination microscope, and only the sandwich immune complex composed of the capture antibody, antigen (cytokine), and fluorescence detection antibody on the solid layer is made to fluoresce, thereby detecting cytokines secreted in real time.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] By the way, in the above-described method, the cytokine captured by the capture antibody (that is, the cytokine at the stage before becoming the sandwich immune complex) does not fluoresce, and thus cannot be detected by the total internal reflection illumination microscope. That is, in the above-described method, an image of the cytokine captured by the capture antibody (that is, the cytokine at the stage before becoming the sandwich immune complex) cannot be obtained.

Means for Solving the Problems

[0005] From the time (capture time) when the cytokine secreted from the cells cultured in the microwell is captured by the capture antibody on the bottom surface of the glass well, until the time (detection time) when the fluorescence detection antibody contained in the culture solution binds to the cytokine captured by the capture antibody to form a sandwich immune complex and the sandwich immune complex is detected by the total internal reflection illumination microscope due to its fluorescence, there is a predetermined time delay. The present inventors assumed that the relationship between the cytokine captured by the capture antibody (which cannot be detected by the microscope) and the sandwich immune complex (which can be detected by the microscope) corresponds to the relationship between the one before the convolution is performed and the one after the convolution is performed. That is, the present inventors assumed that the relationship between the sandwich immune complex (which is actually detected by the microscope) and the cytokine captured by the capture antibody (which cannot be detected by the microscope) corresponds to the relationship between the one before the deconvolution is performed and the one after the deconvolution is performed. Furthermore, the present inventors have found through intensive research that the relationship between the sandwich immune complex (which is actually detected by the microscope) and the cytokine captured by the capture antibody (which cannot be detected by the microscope) corresponds to the relationship between the one before the deconvolution is performed and the one after the deconvolution is performed. That is, the inventors have found that by performing deconvolution on the captured image of the sandwich immune complex obtained by a microscope, it is possible to accurately estimate the image of the cytokine captured by the capture antibody, which cannot be obtained by some microscopes. That is, an object of the present invention is to provide a substance estimation device, a total internal reflection fluorescence microscope, a substance estimation method, and a program that can accurately estimate an image of a substance (for example, an image of a substance at a stage before becoming a complex such as a sandwich immune complex), such as a cytokine, captured by a capture body such as a capture antibody, which could not be visualized by conventional imaging devices (for example, a total internal reflection fluorescence microscope). In other words, an object of the present invention is to provide a substance estimation device, a total internal reflection fluorescence microscope, a substance estimation method, and a program that can accurately estimate an image of a substance captured by a capture body (that is, an image of a substance at a stage before becoming a complex).

[0006] One aspect of the present invention is a substance estimation device including: an acquisition unit that acquires a captured image of a complex; and a calculation unit that calculates an estimated image of a substance, which is any one of a mediator, a messenger substance, a carrier, and an antigen captured by a capture body, based on the captured image of the complex acquired by the acquisition unit. The calculation unit calculates the estimated image of the substance captured by the capture body by performing deconvolution on the captured image of the complex. The substance captured by the capture body is a substance released from a substance release source in a solution containing a phosphor and captured by a capture body disposed at the bottom of the solution, and the complex is formed by binding of the substance captured by the capture body and the phosphor in the solution. According to the substance estimation device of the present invention, by performing deconvolution on the captured image of the complex, it is possible to accurately estimate the image of the substance captured by the capture body.

[0007] In intensive research, the inventors of the present invention have found that as the sampling interval of the captured image of the complex used for estimating the image of the substance is increased, the estimation accuracy of the image of the substance captured by the capturer increases (that is, the signal-to-noise ratio of the estimated image of the substance captured by the capturer increases). Therefore, a substance estimation apparatus according to an aspect of the present invention includes a sampling interval setting unit that sets a sampling interval of the captured image of the complex acquired by the acquisition unit, and when the signal-to-noise ratio of the estimated image of the substance captured by the capturer calculated by the calculation unit is lower than a first threshold value, the sampling interval setting unit may increase the sampling interval of the captured image of the complex. When configured in this way, the estimation accuracy of the image of the substance captured by the capturer can be maintained at a high value (that is, the signal-to-noise ratio of the estimated image of the substance captured by the capturer can be maintained at or higher than the first threshold value).

[0008] The value obtained by performing deconvolution on the captured image of the complex corresponds to the image signal intensity of the estimated image of the substance captured by the capturer, and cannot be a negative value. That is, when the value obtained by performing deconvolution on the captured image of the complex becomes a negative value, it can be considered that the noise included in the estimated image of the substance obtained by performing deconvolution on the captured image of the complex is large. Therefore, in a substance estimation apparatus according to an aspect of the present invention, when the value obtained by performing deconvolution on the captured image of the complex is less than zero, the calculation unit may correct the value obtained by performing deconvolution on the captured image of the complex to zero. When configured in this way, by correcting to zero the value obtained by performing deconvolution on the captured image of the complex (the image signal intensity of the estimated image of the substance captured by the capturer estimated by the calculation unit), the noise included in the estimated image of the substance captured by the capturer can be reduced.

[0009] When calculating the estimated image of the substance captured by the capture body at a certain time (the first time), it is considered that the imaging image of the complex imaged at a time (the second time) with a large time difference from that time (the first time) has almost no influence on the estimated image of the substance captured by the capture body at a certain time (the first time). However, if the calculation unit performs an operation in which the imaging image of the complex imaged at the second time affects the estimated image of the substance captured by the capture body at the first time, the calculation unit will substantially add noise to the estimated image of the substance captured by the capture body. In view of the above points, in the substance estimation device according to one aspect of the present invention, when calculating the estimated image of the substance captured by the capture body at the first time, the calculation unit does not calculate the estimated image of the substance captured by the capture body at the first time based on the imaging image of the complex imaged at the second time whose time difference from the first time is greater than the second threshold value, and may calculate the estimated image of the substance captured by the capture body at the first time. When configured in this way, it is possible to suppress the risk that the calculation unit substantially adds noise to the estimated image of the substance captured by the capture body, and it is possible to reduce the noise included in the estimated image of the substance captured by the capture body.

[0010] One aspect of the present invention is a total reflection illumination microscope including the substance estimation device. According to the total reflection illumination microscope of the present invention, it is possible to obtain an image of the substance captured by the capture body, which cannot be obtained by a conventional total reflection illumination microscope.

[0011] One aspect of the present invention includes an acquisition step of acquiring a captured image of a complex, and a calculation step of calculating an estimated image of a substance that is any one of a transfer factor, a transfer substance, a transfer carrier, and an antigen captured by a capture body based on the captured image of the complex acquired in the acquisition step. In the calculation step, an estimated image of the substance captured by the capture body is calculated by performing deconvolution on the captured image of the complex. The substance captured by the capture body is a substance released from a substance release source in a solution containing a phosphor and captured by a capture body disposed at the bottom portion of the solution. The complex is formed by binding the substance captured by the capture body and the phosphor in the solution. This is a method for estimating a substance. According to the method for estimating a substance of the present invention, by performing deconvolution on the captured image of the complex, it is possible to accurately estimate an image of a substance captured by a capture body that cannot be visualized by some imaging devices.

[0012] One aspect of the present invention is a program for causing a computer to execute an acquisition step of acquiring a captured image of a complex, and a calculation step of calculating an estimated image of a substance that is any one of a transfer factor, a transfer substance, a transfer carrier, and an antigen captured by a capture body based on the captured image of the complex acquired in the acquisition step. In the calculation step, an estimated image of the substance captured by the capture body is calculated by performing deconvolution on the captured image of the complex. The substance captured by the capture body is a substance released from a substance release source in a solution containing a phosphor and captured by a capture body disposed at the bottom portion of the solution. The complex is formed by binding the substance captured by the capture body and the phosphor in the solution. According to the program of the present invention, by performing deconvolution on the captured image of the complex, it is possible to accurately estimate an image of a substance captured by a capture body that cannot be visualized by some imaging devices.

Advantages of the Invention

[0013] According to the present invention, it is possible to provide a substance estimation device, a total internal reflection illumination microscope, a substance estimation method, and a program that can accurately estimate an image of a substance captured by a capture body.

Brief Description of Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0015] Hereinafter, embodiments of the substance estimation device, total internal reflection illumination microscope, substance estimation method, and program of the present invention will be described.

[0016] <First Embodiment> FIG. 1 is a diagram showing an example of the functional block of the substance estimation device 1 of the first embodiment. In the example shown in FIG. 1, the substance estimation device 1 visualizes an image of a substance that is any one of a transfer factor, a transmitter substance, a transfer carrier, and an antigen captured by a capture body, which could not be visualized by a conventional imaging device. The substance estimation device 1 includes an acquisition unit 1A, a calculation unit 1B, and a sampling interval setting unit 1C. The acquisition unit 1A acquires an imaging image MY of a complex (for example, a sandwich immune complex, etc.) imaged by a microscope. Based on the imaging image MY of the complex acquired by the acquisition unit 1A, the calculation unit 1B calculates an estimated image MA of a substance (for example, a cytokine, etc.) captured by a capture body (for example, a capture antibody, etc.). Specifically, the calculation unit 1B calculates the estimated image MA of the substance captured by the capture body by performing deconvolution on the imaging image MY of the complex. The substance captured by the capture body to which the substance estimation device 1 of the first embodiment is applied is a substance released from a substance release source (for example, a cell, etc.) in a solution (for example, a culture solution, etc.) containing a phosphor (for example, a fluorescent detection antibody, etc.) and captured by a capture body arranged at the bottom of the solution. Moreover, the complex to which the substance estimation device 1 of the first embodiment is applied is formed by the binding of the substance captured by the capture body and the phosphor in the solution.

[0017] In the example shown in FIG. 1, the sampling interval setting unit 1C sets the sampling interval Δt of the imaging image MY of the complex acquired by the acquisition unit 1A. Depending on the sampling interval Δt of the imaging image MY of the complex, the S / N ratio of the estimated image MA of the substance captured by the capture body calculated by the calculation unit 1B may become low. Therefore, in the example shown in FIG. 1, when the S / N ratio of the estimated image MA of the substance captured by the capture body calculated by the calculation unit 1B is lower than the first threshold value, the sampling interval setting unit 1C increases the sampling interval Δt of the imaging image MY of the complex.

[0018] In some cases, the value obtained by performing deconvolution on the captured image MY of the complex (the image signal intensity of the estimated image MA of the substance) may be less than zero. Therefore, in the example shown in FIG. 1, when the value obtained by performing deconvolution on the captured image MY of the complex is less than zero, the calculation unit 1B corrects the value obtained by performing deconvolution on the captured image MY of the complex to zero.

[0019] When calculating the estimated image MA of the substance captured by the capture object at a certain time (the first time), it is considered that the captured image MY of the complex captured at a time (the second time) with a large time difference from that time (the first time) has almost no influence on the estimated image MA of the substance captured by the capture object at a certain time (the first time). Therefore, in the example shown in FIG. 1, when calculating the estimated image MA of the substance captured by the capture object at the first time, the calculation unit 1B does not calculate the estimated image MA of the substance captured by the capture object at the first time based on the captured image MY of the complex captured at the second time whose time difference from the first time is greater than the second threshold (that is, without using the captured image MY of the complex captured at the second time).

[0020] <Application Example> FIG. 2 is a diagram showing an example of a functional block of a total internal reflection illumination microscope A to which the substance estimation device 1 of the first embodiment is applied. In the example shown in FIG. 2, the total internal reflection illumination microscope A includes the substance estimation device 1 shown in FIG. 1 and a total internal reflection illumination microscope main body A1. That is, in the example shown in FIG. 2, the captured image MY of the complex captured by the total internal reflection illumination microscope main body A1 is acquired by the acquisition unit A1 of the substance estimation device 1. Further, in the calculation unit 1B of the substance estimation device 1, the estimated image MA of the substance captured by the capture object is calculated based on the captured image MY of the complex acquired by the acquisition unit A1.

[0021] <Second Embodiment> Hereinafter, a second embodiment of the substance estimation apparatus, total internal reflection illumination microscope, substance estimation method, and program of the present invention will be described. The substance estimation apparatus 1 of the second embodiment is configured in the same manner as the substance estimation apparatus 1 of the first embodiment described above, except for the points described later. Therefore, according to the substance estimation apparatus 1 of the second embodiment, the same effects as those of the substance estimation apparatus 1 of the first embodiment described above can be obtained, except for the points described later. In the substance estimation apparatus 1 of the second embodiment, an estimated image of a substance (that is, a substance other than a cytokine) released from something other than a cell and captured by a capture body is calculated.

[0022] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples and can be implemented with appropriate modifications without departing from the gist thereof.

[0023] [Examples] The present inventors applied the substance estimation apparatus, total internal reflection illumination microscope, substance estimation method, and program of the present invention to immunoassay. FIG. 3 is a diagram for explaining a mathematical simulation of deconvolution for immunoassay. Specifically, in FIG. 3(A), the middle graph shows the convolution of the spike increase of the antigen (upper graph) and the staining dynamics of the detection antibody, and such an immunoassay signal is actually obtained in the LCI-S experiment. The lower graph shows the estimated antigen amount obtained by moving deconvolution every 5 frames from the immunoassay signal. FIG. 3(B) shows the influence of the noise reduction processing sampling interval on deconvolution. Deconvolution was performed by sampling a convolution immunoassay signal containing white noise at intervals of 1, 3, 5, and 10. Figure 3(C) shows the correlation between the sampling interval and the S / N ratio. The mathematical simulation of signal deconvolution was repeated 100 times for each interval. The S / N ratio was calculated using the height of the peak of the deconvolved signal and the standard deviation of the background data. As the sampling interval increased, the temporal reproduction accuracy deteriorated, but the S / N ratio improved, and the relationship between the sampling interval and the S / N ratio was a positive proportional relationship (R2>0.999). Figure 3(D) shows an example of the simulation of a random walk of about 500 steps of cells secreting antigens. The amount of antigen secreted at each step was randomly given along a lognormal distribution. The thin broken line indicates the trajectory of the center of gravity of the cell, and the round marks indicate the maximum points of the reproduced signal increments. The gray-scale filling of the round marks represents the passage of time. Figure 3(E) shows the calibration curves of antibody binding at four different sampling intervals. Figure 3(F) shows the ensemble average of the displacement from the cell centroid position at a certain time point to the maximum point of the captured antigen increment distribution reproduced at each relative time point (-12 to +12 hours, horizontal axis). The simulation was repeated 100 times for each interval time.

[0024] Figure 4 is an image showing the spatial position of the increment of the secreted substance at each time point after subtracting the antibody binding kinetics by moving deconvolution. From the cumulative secretion signal image (middle), moving deconvolution was performed to subtract the antibody binding kinetics of the immunoassay, and an increment distribution image of the captured antigen at a specific time point (bottom: secretion activity image) was calculated. The center of the secretion activity was detected from the secretion activity image by a spot detection algorithm on the NIS-elements image software. The cell position at each time point was defined as the center of gravity of the cell body obtained by image analysis from the bright-field image (top). The contour (white line) of the cell body obtained from the bright-field image was overlaid on each corresponding secretion activity image. The size of the nanoliter well is a cube with a side length of 80 μm.

[0025] Figure 5 is a diagram showing the tracking of cell movement and secretion activity. Specifically, in the graph on the left side of Fig. 5, the thin broken line indicates the trajectory of the cell's center of gravity, and the solid circles indicate the centers of the reproduced signal increments. The gray-scale filling of the solid circles represents the passage of time. The graph in the upper right of Fig. 5 shows the change in the intensity of the secretion activity. The graph in the lower right of Fig. 5 shows the displacement from the cell to the center of the secretion activity at each time point. In particular, during the time period when the secretion activity intensity is high, it is considered that the displacement from the cell is stably suppressed to a low level because the detection accuracy of the center is high. The displacement distance was substantially 10 μm or less, with an average of about 5 μm.

[0026] Fig. 6 shows the evaluation of the detection simultaneity of the cell center of gravity and the secretion activity center. The graph on the left side of Fig. 6 shows the ensemble average of the displacement from the maximum point of the captured antigen increment distribution reproduced by deconvolution operation at a certain time point to the cell center of gravity position at each relative time point (-15 to +15 minutes, horizontal axis). The displacement is minimized at -2 minutes, indicating that the secretion activity was detected 2 minutes after the time point when the cells actually existed and exhibited secretion activity. The graph on the right side of Fig. 6 shows the frequency distribution of the time difference that minimizes the displacement from the cell to the maximum point of the secretion activity. It shows that the detection of the secretion activity at the same point where the cells existed often had a delay of 2 minutes.

[0027] Fig. 7 is a diagram showing the antibody-binding standard curve measured by microinjection of the recombinant protein. Under the same conditions as the cell secretion measurement in the presence of the fluorescence-detecting antibody at concentrations of 10 nM, 30 nM, and 90 nM, the recombinant protein was microinjected as the antigen standard protein to obtain the binding standard curve of the fluorescence-detecting antibody.

[0028] Assume that the number of antigen molecules captured on the glass bottom surface increases with the passage of time and simply accumulates without dissociation. When antigen molecules are captured at a certain point on the bottom surface at t = 0, the detection antibody binds to the antigen according to the law of mass action. In this case, the increment △X of the antibody-antigen complex in a very short time △t is expressed as in the following formula (1).

[0029]

Number

[0030] In equation (1), X, Ag, and Ab represent the concentrations of the antibody-antigen complex, antigen molecule, and antibody, respectively, and k on , k off are the association rate constant and dissociation rate constant, respectively. Under the LCI-S condition, since the detection antibody solution in the nanoliter well is continuous with the large pool above it, the fluctuation of the unbound antibody concentration can be ignored.

[0031]

Number

[0032] Substituting (2) into (1) and solving the equation gives equations (3) to (5).

[0033]

Number

[0034]

Number

[0035]

Number

[0036] The increment of the captured antigen at Ag(t) at each time is independently stained with the detection antibody at each time. Therefore, the complex concentration Y(τ) at that time is given by the convolution of equation (6) using Ag(t) (where t ≤ τ).

[0037]

Number

[0038] When measured n times at intervals of △t, the complex concentration Y for each measurement n is given by equations (7) and (8).

[0039]

Number

[0040]

Number

[0041] On the other hand, the increment of the captured antigen Ag n can be described as in equation (9).

[0042]

Number

[0043] The above equation (9) shows that the increment of the captured antigen at a certain point in time is the difference between the complex concentration Y n+1 at the next time point and the sum of the complex concentrations Ag i Z n+1-i derived at all previous time points immediately before that, minus the initial staining amount Z 1 and is given as the ratio to Z

[0044] (Mathematical Simulation of the Separation of Antigen Binding Kinetics and Detection Antibody Binding Kinetics) Figure 3(A) described above shows the convolution result of the antibody staining kinetics when Ag 20 = 1 is given. When performing moving deconvolution with a sampling interval of 5, as shown in Figure 3(A), the increment of the captured antigen Ag n is reproduced. In actual measurement, the detected signal contains noise. Ag nTo evaluate the effect of noise on the reproduction by simulation, white noise was added during the generation of the convolution signal (Fig. 3(B)). As a result of performing moving deconvolution with sampling intervals of 1, 3, 5, and 10, it was shown that the difference in sampling intervals gives a difference in the effect on both the time resolution and the improvement of the signal / noise ratio in the reproduction of the captured antigen increment (Fig. 3(B), Fig. 3(C)). In order to obtain a reproduced signal that is sufficiently detectable and has as high a time resolution as possible, moving deconvolution was performed for all of the two time points with a sampling interval of 5. Next, a secretion dynamics simulation was performed assuming that the cells that secrete while migrating move in a two-dimensional random walk. The secretion activity was given as a random event that varies according to a logarithmic distribution. Fig. 3(D) shows the position of the cells at each time point and the position having the maximum value of the spatial distribution of the reproduced captured antigen increment. However, it is assumed that the secreted molecules are immediately captured by the capture antibody on the glass surface without diffusion, and the antigen given at time point n is captured in place to give the captured antigen Ag n . The maximum value points of the reproduced secretion signal were observed to deviate from the points where the cells were present. This displacement is due to the occurrence of tailing by deconvolution that depends on the sampling time interval. This phenomenon can be explained by the following example. The averaged displacement between two points of the cell presence location at the same time or at relative times (-12 to +12, 25 points) before and after the location of the maximum value point of the secretion signal reproduced at each time point at sampling intervals of 1, 3, 5, and 10 was calculated for 100 trials of a 500-step random walk (Fig. 3(E), Fig. 3(F)). As a result, it was found that the displacement is minimized in the comparison with the location of the cells at the same time as the maximum secretion point, and the width of the valley of the minimum displacement widens as the sampling time increases. These results indicate that spatio-temporal correlation analysis of the deconvolved secretion activity and other dynamics of individual cells at that time point is possible.

[0045] (Example with data using cells that actually secrete) Type 2 innate lymphoid cells (ILC2s) obtained from Fat associated lymphoid cluster, a lymphocyte accumulation present in the mesentery of mice, were grown in IL-2 supplemented culture and used in the experiment. ILC2s were stimulated with IL-2 / IL-33 to induce IL-5 secretion activity, and visualized with LCI-S every minute. The concentration of the detection antibody was set at a relatively high concentration (90 nM) to obtain a signal intensity sufficient to distinguish from background noise. The position of the cells was defined as the area centroid of the contour extracted from the bright-field image. The incremental distribution of the captured antigen was reproduced based on the antibody-binding standard curve (Figure 7) empirically obtained from an injection experiment using microinjection with recombinant IL-5 protein under the same experimental conditions. Image calculations for reproduction were performed using NIS-elements software. Briefly, first, the time-dependent spatial drift due to the instability during image acquisition was corrected, and then sporadic noise was removed by smoothing using a 5-frame moving median filter along the time axis. Next, the image was subjected to 5×5 pixel binning processing, and the moving deconvolution was performed at a sampling interval of 5 as described above. As shown in Figure 4, we succeeded in reproducing the distribution of the increase in captured antigen at each time point from the cumulative secretion signal amount. The position of the maximum point of the increase in captured antigen was determined by the particle detection algorithm on NIS-elements, and the center of the distribution of the increase in captured antigen was determined. When multiple centers were detected, the one with the highest intensity was adopted (left in Figure 5). Comparing the centroid of the cells with the center of the increase in captured antigen, it was found that at each time point, the center of the distribution of the increase in captured antigen was within approximately 5 μm from the cell position. However, although the temporal synchronization was ensured in the simulation (Figure 3), it was found that the appearance timing of the signal increment was delayed by about 2 minutes (Figure 6). Several possibilities can be considered for the cause of this phenomenon. First, a certain delay is assumed due to the time required for the molecule secreted from the cell to move by diffusion until it is captured by the capture antibody on the bottom surface. In fact, the signal increment is observed as a distribution with a diameter of up to several tens of μm, but the diffusion rate of the molecule with a molecular weight of about 30 kDa in the medium is at least 100 μm 2Considering that it is in seconds, taking into account that it only takes a few seconds for most of the secreted molecules to be captured, it suggests that the delay due to diffusion was not the main cause. As another possibility, since the cells during secretion themselves cover the immobilized bottom surface of the capture antibody, it is conceivable that it significantly inhibits the access of the detection antibody to the captured molecules.

[0046] As described above, the embodiments for carrying out the present invention have been described using embodiments. However, the present invention is not limited to such embodiments at all, and various modifications and substitutions can be made without departing from the gist of the present invention. The configurations described in the above-described respective embodiments and respective examples may be combined.

[0047] Note that the entire function or a part of each part provided in the substance estimation device 1 and the total reflection illumination microscope A in the above-described embodiments may be realized by recording a program for realizing these functions on a computer-readable recording medium, reading the program recorded on this recording medium into a computer system, and executing it. Here, the "computer system" is assumed to include hardware such as an OS and peripheral devices. In addition, the "computer-readable recording medium" refers to a portable medium such as a flexible disk, a magneto-optical disk, a ROM, a CD-ROM, etc., and a storage unit such as a hard disk built in a computer system. Further, the "computer-readable recording medium" also includes, like a communication line when transmitting a program via a network such as the Internet or a communication line such as a telephone line, a medium that dynamically holds a program for a short time, and in that case, a volatile memory inside a computer system serving as a server or a client, which holds a program for a certain period of time. Also, the above program may be for realizing a part of the above-described functions, and further, it may be realized in combination with a program already recorded in the computer system for the above-described functions.

Explanation of Reference Numerals

[0048] 1... Substance estimation device, 1A... Acquisition unit, 1B... Calculation unit, 1C... Sampling interval setting unit, A... Total internal reflection fluorescence microscope, A1... Total internal reflection fluorescence microscope main body

Claims

1. An acquisition unit that acquires an imaging image of a complex at a predetermined sampling time interval; An arithmetic unit that calculates an estimated image of a substance that is any one of a transfer factor, a transfer substance, a transfer carrier, and an antigen captured by a capture body based on the imaging image of the complex acquired by the acquisition unit; The arithmetic unit performs deconvolution in the time axis direction for each predetermined frame using the relationship shown in Equation (A) on the fluorescence signal indicated by the convolution of the increase in the substance captured by the capture body and the time change in the staining state of the phosphor obtained from the imaging image of the complex, thereby calculating an estimated image of the substance captured by the capture body. The substance captured by the capture body is a substance released from a substance release source in a solution containing a phosphor and captured by a capture body disposed at the bottom portion of the solution. The complex is formed by binding the substance captured by the capture body and the phosphor in the solution. A substance estimation device. However, in Equation (A), X is the concentration of the complex, Ag is the concentration of the substance, Ab is the concentration of the capture body, kon is the binding rate constant, and koff is the dissociation rate constant. 【Number 1】

2. A sampling interval setting unit that sets the sampling time interval of the imaging image of the complex acquired by the acquisition unit. When the S / N ratio of the estimated image of the substance captured by the capture body calculated by the arithmetic unit is lower than a first threshold value, The sampling interval setting unit increases the sampling time interval of the imaging image of the complex. The substance estimation device according to Claim 1.

3. When the value obtained by performing the deconvolution on the imaging image of the complex becomes less than zero, the arithmetic unit corrects the value obtained by performing the deconvolution on the imaging image of the complex to zero. The arithmetic unit is as follows: The substance estimation device according to Claim 1.

4. When calculating the estimated image of the substance captured by the capture body at a first time, the arithmetic unit calculates the estimated image of the substance captured by the capture body at the first time without using the imaging image of the complex imaged at a second time whose time difference from the first time is greater than a second threshold value. The arithmetic unit is as follows: The substance estimation device according to Claim 1.

5. A total internal reflection illumination microscope including the substance estimation device according to Claim 1.

6. The total internal reflection illumination microscope according to Claim 5, further including a fluorescence signal detection unit that detects a fluorescence signal from the complex.

7. The total internal reflection illumination microscope according to Claim 6, further including a control unit that controls the acquisition unit, the arithmetic unit, and the fluorescence signal detection unit.

6. An acquisition step of acquiring a captured image of a complex at a predetermined sampling time interval; Based on the captured image of the complex acquired in the acquisition step, a calculation step of calculating an estimated image of a substance that is any one of a transfer factor, a transfer substance, a transfer carrier, and an antigen captured by a capture body; and In the calculation step, for the fluorescence signal indicated by the convolution of the increase in the substance captured by the capture body and the temporal change in the staining state of the phosphor obtained from the captured image of the complex, a deconvolution in the time axis direction for each predetermined frame using the relationship shown in formula (A) is performed, whereby an estimated image of the substance captured by the capture body is calculated. The substance captured by the capture body is a substance released from a substance release source in a solution containing a phosphor and captured by a capture body disposed at the bottom portion of the solution. The complex is formed by binding the substance captured by the capture body and the phosphor in the solution. Substance estimation method. However, in formula (A), X is the concentration of the complex, Ag is the concentration of the substance, Ab is the concentration of the capture body, kon is the binding rate constant, and koff is the dissociation rate constant. 【Number 2】

7. A program for causing a computer to perform an acquisition step of acquiring a captured image of a complex at a predetermined sampling time interval; and a calculation step of calculating an estimated image of a substance that is any one of a transfer factor, a transfer substance, a transfer carrier, and an antigen captured by a capture body based on the captured image of the complex acquired in the acquisition step, wherein in the calculation step, for the fluorescence signal indicated by the convolution of the increase in the substance captured by the capture body and the temporal change in the staining state of the phosphor obtained from the captured image of the complex, a deconvolution in the time axis direction for each predetermined frame using the relationship shown in formula (A) is performed, whereby an estimated image of the substance captured by the capture body is calculated. The substance captured by the capture body is a substance released from a substance release source in a solution containing a phosphor and captured by a capture body disposed at the bottom portion of the solution. The complex is formed by binding the substance captured by the capture body and the phosphor in the solution. Program. However, in formula (A), X is the concentration of the complex, Ag is the concentration of the substance, Ab is the concentration of the capture agent, kon is the association rate constant, and koff is the dissociation rate constant. 【Number 3】

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