Method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy

Abstract

The present invention belongs to the field of magnetic nanoparticle imaging, and specifically relates to a method, an electronic device, and a computer-readable storage medium for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy, aiming to solve the problem that the influence of cell survival is not considered and the influence of free SPIO after the death of labeled cells cannot be excluded at the signal level. The present invention includes: according to the periodicity of magnetic nanoparticle signals, by calculating the attenuation ratio of the amplitudes of odd harmonics in MPS, indicating the aggregation or dispersion state of SPIO inside and outside cells, constructing a distribution model, calculating the proportionality coefficient, and performing correction to obtain the SPIO signal inside cells of an unwashed labeled cell sample, so as to realize the extraction and separation of intracellular and extracellular SPIO signals. The present invention separates and extracts the SPIO signal inside cells, realizes the tracing of viable labeled cells through MPI, and makes MPI stem cell tracing more accurate and reliable in clinical applications.

Description

Background Art

[0002] Magnetic Particle Imaging (MPI) is an imaging technique based on superparamagnetic iron oxide nanoparticles (SPIOs). It generates images by measuring the response of magnetic nanoparticles in a specific magnetic field. The imaging principle of MPI is based on the nonlinear magnetization curve of Langevin paramagnetic law. By constructing a gradient field, a field-free point (FFP) or a field-free line (FFL) is generated. The magnetic nanoparticles (Superparamagnetic Iron Oxide Nanoparticle, SPIO) at the FFP or FFL are not magnetically saturated and can produce a nonlinear magnetization response to the excitation magnetic field. A signal voltage is detected in the receiving coil, and the voltage signal is assigned to each position for reconstruction to obtain the spatial distribution information of the magnetic particles, that is, the MPI signal. Since body tissues are diamagnetic and do not produce any interfering signals, MPI does not display anatomical structures.

[0003] Currently, the research on MPI is in its infancy. It has advantages such as high sensitivity, no radiation, and no need for external radiation sources, and has broad application potential in the field of biomedical imaging. Compared with traditional magnetic resonance imaging (MRI), MPI can provide higher temporal resolution and contrast, and is especially suitable for monitoring dynamic processes such as blood flow, cell migration, and drug distribution. In addition, MPI also shows great potential in quantitative analysis, and can accurately measure the concentration of magnetic particles in tissues, providing important information for disease diagnosis and treatment monitoring.

[0004] However, in current quantitative stem cell tracing studies based on MPI, most studies do not consider the influence of cell survival: whether the label persists after cell death, and whether the cell membrane ruptures after cell death, causing the SPIO label to transfer to other cells, resulting in signal interference. Summary of the Invention

[0005] To solve the above problems in the prior art, that is, the prior art does not consider the influence of cell survival, and cannot exclude the influence of free SPIO after the death of labeled cells at the signal level, resulting in signal interference, and further leading to the problem that the SPIO signal in the live cells of the unwashed labeled cell sample cannot be accurately obtained. In the first aspect of the present invention, a method for quantitatively distinguishing the signals of magnetic nanoparticles inside and outside cells based on magnetic particle spectroscopy is proposed, which is used to extract and separate the SPIO signals inside and outside cells, and then obtain the SPIO signal inside the cells of the unwashed labeled cell sample. The method includes the following steps:

[0006] Obtain the response voltage signal U1 of the washed labeled cell sample under excitation;

[0007] Obtain the response voltage signal U2 of the sample with ruptured cell membranes under excitation;

[0008] Obtain the response voltage signal U3 of the unwashed labeled cell sample under excitation;

[0009] Perform Fourier transform on the U1 to obtain the magnetic particle spectrum of SPIO in the cells, and then calculate the ratio of the difference in amplitudes of adjacent odd harmonics, which is recorded as K1;

[0010] Perform Fourier transform on the U2 to obtain the magnetic particle spectrum of SPIO outside the cells, and then calculate the ratio of the difference in amplitudes of adjacent odd harmonics, which is recorded as K2;

[0011] Based on the K1 and the K2, construct a distribution model of SPIO inside and outside the cells;

[0012] Perform Fourier transform on the U3 to obtain the magnetic particle spectrum of SPIO in the unwashed labeled cell sample, and then calculate the ratio of the difference in amplitudes of adjacent odd harmonics, which is recorded as K3;

[0013] Combine the distribution model and the K3 to solve the proportionality coefficient of SPIO inside and outside the cells in the unwashed labeled cell sample;

[0014] According to the proportionality coefficient, correct the U3 to obtain the SPIO signal inside the cells of the unwashed labeled cell sample.

[0015] In some preferred embodiments, the ratio of the difference in amplitudes of adjacent odd harmonics is calculated as: (A3 - A5) / (A5 - A7), where A3 is the amplitude of the third harmonic, A5 is the amplitude of the fifth harmonic, and A7 is the amplitude of the seventh harmonic.

[0016] In some preferred embodiments, the distribution model of SPIO inside and outside the cells is:

[0017] ;

[0018] In the formula, is (A5 - A7) of the washed labeled cell sample, is (A5 - A7) of the sample with ruptured cell membranes, m is the mixed sample, α is the proportion of the washed labeled cell sample in the mixed sample, is the proportion of the sample with ruptured cell membranes in the mixed sample, It is the ratio of the odd harmonic differences of the mixed sample, (A3 - A5) / (A5 - A7).

[0019] In some preferred embodiments, the calculation method of the proportionality coefficient is as follows:

[0020] Perform an equation transformation on the distribution model to solve for the proportionality coefficient α:

[0021] ;

[0022] For the unwashed labeled cell sample to be measured, substitute the ratio K of its odd harmonic differences 3 into , and solve to obtain the proportion α of the signal generated by intracellular SPIO in the total signal U3 of the unwashed labeled cell sample as:

[0023] .

[0024] In some preferred embodiments, according to the proportionality coefficient, correct U3 to obtain the intracellular SPIO signal of the unwashed labeled cell sample. The method is as follows:

[0025] Step S71, obtain the standard unit signal u1 generated by intracellular SPIO, where u1 is U1 divided by the total number of cells in the washed labeled cell sample;

[0026] Step S72, obtain the standard unit signal u2 generated by extracellular SPIO, where u2 is U2 divided by the total number of cells in the sample with ruptured cell membranes;

[0027] Step S73, combine u1 and u2 to correct the U3 signal of the unwashed labeled cell sample:

[0028] ;

[0029] In the formula, is the signal generated by intracellular SPIO in the unwashed labeled cell sample, and n is the number of cells in the unwashed labeled cell sample.

[0030] In some preferred embodiments, after obtaining the intracellular SPIO signal of the unwashed labeled cell sample, further rapid correction of the obtained SPIO signal is included. The method is as follows:

[0031] Fit the U1 harmonic curve and K1 curve of SPIO particle-incubated cells R at different incubation concentrations of magnetic nanoparticles;

[0032] According to the characteristics of the voltage response signal U2 of SPIO outside the cell and the voltage response signal U of the SPIO-diluted sample 2-pbsConstruct a relational expression based on the relationship between features;

[0033] Fit the U harmonic curves and K curves of SPIO dilution samples with different concentrations; 2-pbs harmonic curves, K 2-pbs curves, and obtain the U2 harmonic curve and K2 curve based on the relational expression;

[0034] According to the SPIO concentration of the incubated cells, find K1 and K2 on the K1 curve and the K2 curve respectively, and calculate Δ based on the corresponding harmonic amplitudes on the U1 harmonic curve and the U2 harmonic curve; 1 Δ 2 ;

[0035] Substitute the obtained K2, K1, Δ 1 Δ 2 , the cell sample signals U3 and K3 of the unknown SPIO distribution state measured, into the proportional coefficient α solution formula, and then obtain the proportional coefficient of SPIO inside and outside the cells;

[0036] Calculate the measurement deviation of the signal based on the relational expression and the proportional coefficient of SPIO inside and outside the cells;

[0037] Based on the measurement deviation of the signal, perform signal correction on the cell sample signal U3 of the unknown SPIO distribution state measured to obtain the SPIO signal inside the cells .

[0038] In some preferred embodiments, the relational expression is:

[0039] ;

[0040] The measurement deviation of the signal:

[0041] ;

[0042] The SPIO signal U inside the cells in :

[0043] .

[0044] In the second aspect of the present invention, a system for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy is used to extract and separate intracellular and extracellular SPIO signals, and then obtain the intracellular SPIO signal of an unwashed labeled cell sample. The system includes:

[0045] An MPI signal detection instrument for respectively exciting, scanning and measuring a washed labeled cell sample, a sample with ruptured cell membranes, and an unwashed labeled cell sample to obtain response voltage signals U1, U2, and U3;

[0046] The central processing unit includes a CPU and a GPU; the central processing unit is configured to perform a Fourier transform on the U1 to obtain the magnetic particle spectrum of SPIO inside the cell, and then calculate the ratio of the difference in the amplitudes of adjacent odd harmonics, which is recorded as K1;

[0047] Perform a Fourier transform on the U2 to obtain the magnetic particle spectrum of SPIO outside the cell, and then calculate the ratio of the difference in the amplitudes of adjacent odd harmonics, which is recorded as K2;

[0048] Based on the K1 and the K2, construct a distribution model of SPIO inside and outside the cell;

[0049] Perform a Fourier transform on the U3 to obtain the magnetic particle spectrum of SPIO in the unwashed labeled cell sample, and then calculate the ratio of the difference in the amplitudes of adjacent odd harmonics, which is recorded as K3;

[0050] Combined with the distribution model and the K3, solve the proportional coefficient of SPIO inside and outside the cell in the unwashed labeled cell sample;

[0051] According to the proportional coefficient, correct the U3 to obtain the SPIO signal inside the cell of the unwashed labeled cell sample.

[0052] In a third aspect of the present invention, an electronic device is proposed, including:

[0053] At least one processor; and a memory communicatively connected to at least one of the processors; wherein, the memory stores instructions executable by the processor, and the instructions are used to be executed by the processor to implement the above-mentioned method for quantitatively distinguishing magnetic nanoparticle signals inside and outside cells based on magnetic particle spectroscopy.

[0054] In a fourth aspect of the present invention, a computer-readable storage medium is proposed, and the computer-readable storage medium stores computer instructions, and the computer instructions are used to be executed by the computer to implement the above-mentioned method for quantitatively distinguishing magnetic nanoparticle signals inside and outside cells based on magnetic particle spectroscopy.

[0055] Advantages of the present invention:

[0056] 1. The established distribution model of SPIO inside and outside cells extracts the ratio of magnetic particle spectra (A3 - A5) / (A5 - A7) of standard intracellular and extracellular SPIO. Without the aid of other detections, it obtains the ratio of the response voltage signal intensities generated by intracellular SPIO and extracellular SPIO, separates and extracts the response voltage signal generated by intracellular SPIO at the signal level, calculates the proportionality coefficient and corrects it, obtains the intracellular SPIO signal of the unwashed labeled cell sample, can separate and extract the intracellular SPIO signal, realizes the precise tracing of viable labeled cells by MPI, and makes up for the problem that the influence of free SPIO cannot be excluded at the signal level in the current MPI cell tracing research;

[0057] 2. It can also promote the improvement of the detection sensitivity of MPI. By the method proposed in this study, the detection of more trace SPIO signals in units of cells is realized, which meets the development needs and directions of precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] By reading the detailed description of the non - restrictive embodiments with reference to the following drawings, other features, purposes and advantages of this application will become more obvious:

[0059] Figure 1 It is the SPIO signal spectrum diagram of viable cells inside and dead cells outside of the present invention;

[0060] Figure 2 It is the difference diagram of viable cells and dead cells outside under different incubation concentrations of the present invention;

[0061] Figure 3 It is the SPIO magnetic particle spectrum diagram outside cells of the present invention;

[0062] Figure 4 It is the SPIO magnetic particle spectrum diagram inside cells of the present invention;

[0063] Figure 5 It is the standard curve diagram of the odd - numbered harmonic amplitudes under different incubation concentrations of the present invention;

[0064] Figure 6 It is the standard curve diagram of K1 and K2 under different incubation concentrations of the present invention;

[0065] Figure 7 It is the diagram of extracting intracellular SPIO magnetic particle signals from the mixed sample signals of the present invention;

[0066] Figure 8 It is the flow chart of the method steps for quantitatively distinguishing magnetic nanoparticles signals inside and outside cells based on magnetic particle spectroscopy of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0067] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the relevant invention and are not intended to limit the invention. Additionally, it should be noted that for the convenience of description, only the parts related to the relevant invention are shown in the drawings.

[0068] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application will be described in detail below with reference to the drawings and embodiments.

[0069] To more clearly illustrate the method for quantitatively distinguishing the signals of magnetic nanoparticles inside and outside cells based on magnetic particle spectroscopy of the present invention, the following will be combined with Figures 1 to 8 to elaborate on each step in the embodiments of the present invention.

[0070] It was found in the experiment that, under the same measurement environment, the signal of SPIO existing in intact living cells is different from the signal after the cell membrane ruptures and the cell dies. Specifically, the amplitudes of the odd harmonics in the MPS are different. Among them, the attenuation of the amplitudes of the odd harmonics of the SPIO signal after the cell membrane ruptures and the cell dies is greater than that of the SPIO signal in the surviving cells. See Figure 1 , which shows the signal frequency spectrum characteristics of MSC cells incubated with Synomag50nm magnetic nanoparticle solution in the surviving state and after the cell membrane ruptures and the cell dies. See Figure 2 , which shows the difference in (A3 - A5) / (A5 - A7) outside the surviving cells and the dead cells at different incubation concentrations. Table 1 shows the difference obtained by subtracting the SPIO signal (A3 - A5) / (A5 - A7) of the surviving cells from the SPIO signal (A3 - A5) / (A5 - A7) of the dead cells in the 12 measurement results. Based on this, the first embodiment of the present invention proposes a method for quantitatively distinguishing the signals of magnetic nanoparticles inside and outside cells based on magnetic particle spectroscopy. By using the non-linear magnetization response of SPIO in an alternating magnetic field, the response voltage signal is transformed into the frequency domain, and the attenuation of the odd harmonics (A3 - A5) / (A5 - A7) in its MPS is analyzed. This ratio can indicate the aggregation or dispersion state of SPIO inside and outside the cell. By using the (A3 - A5) / (A5 - A7) characteristics of the SPIO signals inside and outside the cell, a distribution model of SPIO inside and outside the cell is constructed to achieve the distinction of the SPIO signals inside and outside the cell and the extraction of the SPIO signal inside the cell.

[0071] Table 1

[0072] Concentration 10 μg / mL 25 μg / mL 50 μg / mL 75 μg / mL 100 μg / mL 200 μg / mL -2.4369497 6.45305542 1.84719034 3.18464156 0.23368361 0.5155164 -2.0462797 0.76660302 1.86137282 0.04648958 0.80739233 0.77167248

[0073] See Figure 8 , specifically as follows:

[0074] Step S1: Obtain the response voltage signal U1 of the washed labeled cell sample under excitation;

[0075] In this embodiment, the process of obtaining the response voltage signal U1 under excitation is as follows: Place the washed labeled cell sample (i.e., the intracellular SPIO standard) into the MPI signal detection instrument and perform a scanning measurement at the center of the FOV to obtain the response voltage signal U1 under excitation. The preparation process of the washed labeled cell sample is as follows: First, prepare a culture medium dilution solution of SPIO. The standard requires an iron concentration of 50 μg / mL. Here, it should be noted that in actual preparation, the total volume is not limited and is based on the area of the culture dish or culture bottle, and the iron concentration is also not limited. Second, incubate the cells with the SPIO culture medium dilution solution standard. The incubation time is calculated according to one cell cycle of the labeled cells. Finally, after incubation, centrifuge the labeled cells, remove the SPIO-containing culture medium, wash twice with PBS to remove extracellular SPIO particles, digest the SPIO-labeled cells and add 1 mL of culture medium without SPIO to resuspend the cells, which serves as the intracellular SPIO standard.

[0076] Step S2: Obtain the response voltage signal U2 of the sample with ruptured cell membranes under excitation;

[0077] In this embodiment, the process of obtaining the response voltage signal U2 of the sample with ruptured cell membranes under excitation is as follows: Place the sample with ruptured cell membranes (i.e., the extracellular SPIO standard) into the MPI signal detection instrument and perform a scanning measurement at the center of the FOV to obtain the response voltage signal U2 under excitation. The preparation process of the sample with ruptured cell membranes is as follows: Rupture the cell membranes of the intracellular SPIO standard prepared in Step S1 to release SPIO particles into the solution, which serves as the extracellular SPIO standard.

[0078] Step S3: Obtain the response voltage signal U3 of the unwashed labeled cell sample under excitation;

[0079] In this embodiment, the process of obtaining the response voltage signal U3 of the unwashed labeled cell sample under excitation is as follows: Place the unwashed labeled cell sample into the MPI signal detection instrument and perform a scanning measurement at the center of the FOV to obtain the response voltage signal U3 under excitation. The preparation process of the unwashed labeled cell sample is as follows: Re-inoculate the intracellular SPIO standard into the culture dish, incubate for another 48 h, then aspirate the old culture medium for later use. After digesting and centrifuging the cells, resuspend the cells with the old culture medium directly as the unwashed labeled cell sample.

[0080] Step S4: Perform Fourier transform on the U1 to obtain the magnetic particle spectrum of SPIO inside the cells, and then calculate the ratio of the differences in the amplitudes of adjacent odd harmonics, which is recorded as K1;

[0081] Perform Fourier transform on the U2 to obtain the magnetic particle spectrum of SPIO outside the cells, and then calculate the ratio of the differences in the amplitudes of adjacent odd harmonics, which is recorded as K2;

[0082] In this embodiment, the calculation methods of the K1 and K2 are as follows:

[0083] Step S41: Obtain the U1, and use Fourier transform to convert the U1 from the time domain to the frequency domain to obtain the magnetic particle spectrum of SPIO inside the cells, see Figure 3 ;

[0084] Obtain the U2, and use Fourier transform to convert the U2 from the time domain to the frequency domain to obtain the magnetic particle spectrum of SPIO outside the cells, see Figure 4 ;

[0085] Step S42: Since the amplitudes of the high-order harmonics are too small, only compare the attenuation rate differences of the amplitudes of the 3rd, 5th, and 7th harmonics (i.e., A3, A5, A7), and calculate the ratio of the differences of the odd harmonics of the magnetic particle spectrum of SPIO inside the cells (A3 - A5) / (A5 - A7), which is recorded as K1;

[0086] Since the amplitudes of the high-order harmonics are too small, only compare the attenuation rate differences of the amplitudes of the 3rd, 5th, and 7th harmonics (i.e., A3, A5, A7), and calculate the ratio of the differences of the odd harmonics of the magnetic particle spectrum of SPIO outside the cells (A3 - A5) / (A5 - A7), which is recorded as K2;

[0087] Step S5: Based on the K1 and the K2, construct a distribution model of SPIO inside and outside the cells;

[0088] In this embodiment, this model is used to describe the changes in the ratio (A3 - A5) / (A5 - A7) of the sample caused by the processes of SPIO being phagocytosed into the cells and SPIO being released back outside the cells due to cell death and other reasons. The distribution model is:

[0089] ;

[0090] In the formula, is (A5 - A7) of the labeled cell sample after washing, is (A5 - A7) of the sample with ruptured cell membranes, m is the mixed sample, α is the proportion of the labeled cell sample after washing in the mixed sample, is the proportion of the sample with ruptured cell membranes in the mixed sample, It is the ratio (A3 - A5) / (A5 - A7) of the odd - order harmonic differences of the mixed sample.

[0091] Step S6: Perform Fourier transform on the U3 to obtain the magnetic particle spectrum of the unwashed labeled cell sample SPIO, and then calculate the ratio of the differences in the amplitudes of adjacent odd - order harmonics, which is recorded as K3.

[0092] In this embodiment, the process of calculating K3 is as follows: Obtain the U3, and use Fourier transform to convert the U3 from the time domain to the frequency domain to obtain the magnetic particle spectrum of the unwashed labeled cell sample. See Figure 4 ; Since the amplitudes of the higher - order harmonics are too small, only compare the attenuation rate differences of the amplitudes of the 3rd, 5th, and 7th harmonics (i.e., A3, A5, A7), calculate the ratio of the odd - order harmonic differences (A3 - A5) / (A5 - A7) of the magnetic particle spectrum of the unwashed labeled cell sample SPIO, and record it as K3.

[0093] Combined with the distribution model and the K3, solve the proportionality coefficient of the intracellular SPIO and extracellular SPIO in the unwashed labeled cell sample. The method for calculating the proportionality coefficient is as follows:

[0094] Step S61: Perform equation transformation on the distribution model to solve for the proportionality coefficient α:

[0095] ;

[0096] Step S62, for the unwashed labeled cell sample to be measured, substitute its odd - order harmonic difference ratio K 3 into , and solve to obtain the proportion α of the signal generated by intracellular SPIO in the total signal U3 of the unwashed labeled cell sample as:

[0097] ;

[0098] Step S7: According to the proportionality coefficient, correct the U3 to obtain the intracellular SPIO signal of the unwashed labeled cell sample.

[0099] In this embodiment, according to the proportionality coefficient, the specific process of correcting the U3 to obtain the intracellular SPIO signal of the unwashed labeled cell sample is as follows:

[0100] Step S71, obtain the standard unit signal u1 generated by intracellular SPIO. The u1 is U1 divided by the total number of cells in the washed labeled cell sample, where the total number of cells in the labeled cell sample is obtained by cell counting during sample preparation.

[0101] Step S72: Obtain the standard unit signal u2 generated by extracellular SPIO, where u2 is U2 divided by the total number of cells in the sample with ruptured cell membranes. The total number of cells in the sample with ruptured cell membranes is obtained by cell counting during sample preparation.

[0102] Step S73: Combine u1 and u2 to correct the U3 signal of the unwashed labeled cell sample:

[0103] ;

[0104] In the formula, is the signal generated by intracellular SPIO in the unwashed labeled cell sample, and n is the number of cells in the unwashed labeled cell sample.

[0105] In addition, to further quickly correct the signal generated by intracellular SPIO, a rapid correction model for intracellular SPI signal is constructed. The main difference from the previous method is that the former requires preparing multiple labeled cell samples and performing cell membrane rupture operations on the stem cell samples to be injected to obtain U2 and K2, while rapid correction only needs to pre-construct solutions with different concentrations of labeled particles (diluted with PBS) to approximately replace the extracellular signal. Thus, the signal measurement deviation (caused by particles released due to cell death) can be calculated by retrieving the K2 curve and U2 curve corresponding to the labeled concentration, and the intracellular signal can be obtained by excluding the deviation interference. The method is as follows:

[0106] Step S81: Fit the U1 harmonic curve and K1 curve of SPIO particle-incubated cells R at different incubation concentrations of magnetic nanoparticles;

[0107] Step S82: Based on the (approximate) relationship between the characteristics of the voltage response signal U2 of SPIO extracellularly and the voltage response signal U 2-pbs characteristics of the SPIO-diluted sample, construct a relational expression; ;

[0108] Step S83: Fit the U2-pbs harmonic curve and K2-pbs curve of SPIO-diluted samples at different concentrations, and obtain the U2 harmonic curve and K2 curve based on the relational expression; among them, the U1 harmonic curve, K1 curve, U2 harmonic curve, and K2 curve are specifically obtained by the method of steps S1 - S4 to get the point values at each concentration, and then fit the A3, A5, and A7 harmonic curves of SPIO particle-incubated cells R and SPIO-diluted samples, that is, SPIO in two states: intracellular and extracellular (see Figure 5 ), as well as the K 1 and K 2 curves (see Figure 6 );

[0109] Step S84: According to the SPIO concentration of the incubated cells, find K1 and K2 on the K1 curve and the K2 curve respectively, and calculate Δ based on the corresponding harmonic amplitudes on the U1 harmonic curve and the U2 harmonic curve. 1 and Δ 2 ;

[0110] Step S85: Substitute the obtained K2, K1, Δ 1 and Δ 2 , the signal U3 and K3 of the cell sample with unknown SPIO distribution state measured (specifically obtained by the method of the above steps S1 - S4) into the proportionality coefficient α solution formula, and then obtain the proportionality coefficient of SPIO inside and outside the cell.

[0111] ;

[0112] Step S86: Calculate the measurement deviation of the signal based on the relational expression and the proportionality coefficient, the measurement deviation of the signal;

[0113] ;

[0114] Step S87: Based on the measurement deviation of the signal, perform signal correction on the signal of the cell sample with unknown SPIO distribution state to obtain the SPIO signal inside the cell :

[0115] .

[0116] This signal correction method uses the signal measurement deviation to quickly correct the sample signal, and can optimize the image reconstruction model of imaging systems such as MPI, so as to make the signal extraction inside the cell more accurate and improve the sensitivity of cell tracing. See Figure 7 , the intraSPIO curve is the spectrum of the response voltage signal generated only by the SPIO inside the cell after model screening and excluding the SPIO signal that is free outside the cell due to reasons such as cell death.

[0117] Although the above embodiments describe the various steps in the above - mentioned sequential order, those skilled in the art can understand that in order to achieve the effects of this embodiment, different steps do not have to be executed in such an order. They can be executed simultaneously (in parallel) or in a reversed order, and these simple changes are all within the protection scope of the present invention.

[0118] The second embodiment of the present invention, a system for quantitatively distinguishing the signals of magnetic nanoparticles inside and outside cells based on the magnetic particle spectrum, is used to extract and separate the SPIO signals inside and outside the cells, and then obtain the SPIO signal inside the cells of the unwashed labeled cell sample. The system includes:

[0119] An MPI signal detection instrument is used to respectively excite and scan and measure a washed labeled cell sample, a sample with ruptured cell membranes, and an unwashed labeled cell sample to obtain response voltage signals U1, U2, and U3;

[0120] The central processing unit includes a CPU and a GPU; the central processing unit is used to perform Fourier transform on the U1 to obtain the magnetic particle spectrum of SPIO in the cells, and then calculate the ratio of the difference in the amplitudes of adjacent odd harmonics, which is recorded as K1;

[0121] Perform Fourier transform on the U2 to obtain the magnetic particle spectrum of SPIO outside the cells, and then calculate the ratio of the difference in the amplitudes of adjacent odd harmonics, which is recorded as K2;

[0122] Based on the K1 and the K2, construct a distribution model of SPIO inside and outside the cells;

[0123] Perform Fourier transform on the U3 to obtain the magnetic particle spectrum of SPIO in the unwashed labeled cell sample, and then calculate the ratio of the difference in the amplitudes of adjacent odd harmonics, which is recorded as K3;

[0124] Combined with the distribution model and the K3, solve the proportionality coefficient of SPIO inside and outside the cells in the unwashed labeled cell sample;

[0125] According to the proportionality coefficient, correct the U3 to obtain the SPIO signal inside the cells of the unwashed labeled cell sample.

[0126] It should be noted that the above-mentioned quantitative discrimination system for magnetic nanoparticles signals inside and outside cells based on magnetic particle spectroscopy provided by the above embodiment only takes the division of the above-mentioned functional modules as an example. In actual applications, the above functions can be allocated to different functional modules according to needs, that is, the modules or steps in the embodiments of the present invention can be further decomposed or combined. For example, the modules in the above embodiment can be combined into one module, or further split into multiple sub-modules to complete all or part of the functions described above. For the names of the modules and steps involved in the embodiments of the present invention, they are only used to distinguish each module or step, and are not regarded as an improper limitation of the present invention.

[0127] An electronic device according to the third embodiment of the present invention includes: at least one processor; and a memory communicatively connected to at least one of the processors; wherein, the memory stores instructions executable by the processor, and the instructions are used to be executed by the processor to implement the above-mentioned method for quantitatively discriminating magnetic nanoparticle signals inside and outside cells based on magnetic particle spectroscopy.

[0128] A computer-readable storage medium according to a fourth embodiment of the present invention, wherein the computer-readable storage medium stores computer instructions for being executed by a computer to implement the above-mentioned method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy.

[0129] Those skilled in the art can clearly understand that for the convenience and brevity of description, the specific working processes and related descriptions of the above-described electronic devices and computer-readable storage media can refer to the corresponding processes in the foregoing method embodiments and will not be repeated here.

[0130] Those skilled in the art should be able to realize that the modules and method steps of each example described in combination with the embodiments disclosed herein can be implemented by electronic hardware, computer software, or a combination of the two. The programs corresponding to the software modules and method steps can be placed in a random access memory (RAM), memory, read-only memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, register, hard disk, removable disk, CD-ROM, or any other form of storage medium known in the art. To clearly illustrate the interchangeability of electronic hardware and software, the components and steps of each example have been generally described according to their functions in the above description. Whether these functions are executed in the form of electronic hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered to exceed the scope of the present invention.

[0131] The terms "first", "second", etc. are used to distinguish similar objects, rather than to describe or represent a specific order or sequence.

[0132] The term "comprising" or any other similar term is intended to cover non-exclusive inclusion, so that a process, method, article, or device / apparatus comprising a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to these processes, methods, articles, or devices / apparatus.

[0133] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy, which is used to extract and separate intracellular and extracellular SPIO signals, and then obtain the intracellular SPIO signals in unwashed labeled cell samples, characterized in that: The method comprises the following steps: Obtaining a response voltage signal U1 of the washed labeled cell sample under stimulation; Obtaining a response voltage signal U2 of the sample with ruptured cell membrane under excitation; Acquire a response voltage signal U3 of the unwashed labeled cell sample under stimulation; Perform Fourier transformation on U1 to obtain the magnetic particle spectrum of SPIO in the cell, and then calculate the ratio of the difference between the amplitudes of adjacent odd-order harmonics, which is recorded as K1; Performing Fourier transformation on U2 to obtain the magnetic particle spectrum of extracellular SPIO, and then calculating the ratio of the difference between the amplitudes of adjacent odd-order harmonics, which is recorded as K2; Based on the K1 and the K2, constructing a distribution model of SPIO inside and outside the cell; Performing Fourier transformation on the U3 to obtain the magnetic particle spectrum of the unwashed labeled cell sample SPIO, and then calculating the ratio of the difference between the amplitudes of adjacent odd-order harmonics, which is recorded as K3; In combination with the distribution model and K3, a proportional coefficient between intracellular SPIO and extracellular SPIO in the unwashed labeled cell sample is solved; The U3 is corrected according to the proportional coefficient to obtain the SPIO signal in the cells of the unwashed labeled cell sample.

2. The method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy according to claim 1, characterized in that: The ratio of the difference between the amplitudes of adjacent odd-order harmonics is calculated as: (A3-A5) / (A5-A7), where A3 is the amplitude of the third harmonic, A5 is the amplitude of the fifth harmonic, and A7 is the amplitude of the seventh harmonic.

3. The method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy according to claim 2, characterized in that: The distribution model of SPIO inside and outside the cell is: ； In the formula, For the labeled cell samples after washing (A5-A7), is the sample with ruptured cell membrane (A5-A7), m is the mixed sample, α is the proportion of the labeled cell sample after washing in the mixed sample, is the proportion of samples with ruptured cell membranes in the mixed samples, It is the ratio of the difference in amplitude of odd harmonics of the mixed samples (A3-A5) / (A5-A7).

4. The method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy according to claim 3, characterized in that: The proportionality coefficient is calculated as follows: Transform the distribution model equation and solve for the proportionality coefficient α: ； For the unwashed labeled cell sample to be tested, the ratio K3 of the difference between the amplitudes of adjacent odd harmonics is substituted into , the ratio α of the intracellular SPIO signal in the unwashed labeled cell sample to the total signal U3 is obtained as: 。 5. The method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy according to claim 1, characterized in that: According to the proportional coefficient, the U3 is corrected to obtain the SPIO signal in the cells of the unwashed labeled cell sample, and the method is: Step S71, obtaining a standard unit signal u1 generated by SPIO in the cell, wherein u1 is U1 divided by the total number of cells in the labeled cell sample after washing; Step S72, obtaining a standard unit signal u2 generated by extracellular SPIO, wherein u2 is U2 divided by the total number of cells in the sample with ruptured cell membranes; Step S73, combining the u1 and the u2, calibrating the U3 signal of the unwashed labeled cell sample: ； In the formula, is the signal generated by intracellular SPIO in the unwashed labeled cell sample, n is the number of cells in the unwashed labeled cell sample, and α is the proportionality coefficient.

6. The method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy according to claim 5, characterized in that: After obtaining the SPIO signal in the cells of the unwashed labeled cell sample, the method further includes quickly correcting the obtained SPIO signal, the method being: Fitting the U1 harmonic curve and K1 curve of SPIO particle incubated cells R at different magnetic nanoparticle incubation concentrations; According to the voltage response signal U2 of SPIO outside the cell and the voltage response signal U 2-pbs The relationship between features, constructing a relational expression; Fitting the U of SPIO diluted samples with different concentrations 2-pbs Harmonic curve, K 2-pbs Curve, and obtain U2 harmonic curve and K2 curve based on the relationship; According to the SPIO concentration of the incubated cells, K1 and K2 are respectively found on the K1 curve and the K2 curve, and Δ1 and Δ2 are calculated according to the corresponding harmonic amplitudes on the U1 harmonic curve and the U2 harmonic curve, where Δ1 is (A5-A7) of the labeled cell sample after washing, and Δ2 is (A5-A7) of the sample with ruptured cell membrane; Substitute the K2, K1, Δ1, Δ2 obtained by the search, the measured response voltage signal U3 of the unwashed labeled cell sample under stimulation, and the ratio K3 of the difference between the amplitudes of adjacent odd-order harmonics into the proportionality coefficient α solution formula, and then obtain the proportionality coefficient of SPIO inside and outside the cell; Calculating the signal measurement deviation based on the relationship and the proportionality coefficient of SPIO inside and outside the cell; Based on the measured deviation of the signal, the measured cell sample signal U3 of the unknown SPIO distribution state is corrected to obtain the SPIO signal in the cell .

7. A system for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectrum, based on the method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectrum according to any one of claims 1 to 6, for extracting and separating intracellular and extracellular SPIO signals, and then obtaining intracellular SPIO signals of unwashed labeled cell samples, characterized in that: The system comprises: MPI signal detection instrument, used to stimulate and scan the washed labeled cell samples, the samples with ruptured cell membranes, and the unwashed labeled cell samples, respectively, to obtain response voltage signals U1, U2, and U3; The central processing unit includes a CPU and a GPU; the central processing unit is used to perform Fourier transform on U1 to obtain the magnetic particle spectrum of SPIO in the cell, and then calculate the ratio of the difference between the amplitudes of adjacent odd harmonics, which is recorded as K1; Performing Fourier transformation on U2 to obtain the magnetic particle spectrum of extracellular SPIO, and then calculating the ratio of the difference between the amplitudes of adjacent odd-order harmonics, which is recorded as K2; Based on the K1 and the K2, constructing a distribution model of SPIO inside and outside the cell; Performing Fourier transformation on the U3 to obtain the magnetic particle spectrum of the unwashed labeled cell sample SPIO, and then calculating the ratio of the difference between the amplitudes of adjacent odd-order harmonics, which is recorded as K3; In combination with the distribution model and K3, a proportional coefficient between intracellular SPIO and extracellular SPIO in the unwashed labeled cell sample is solved; The U3 is corrected according to the proportional coefficient to obtain the SPIO signal in the cells of the unwashed labeled cell sample.

8. An electronic device, characterized in that: include: at least one processor; as well as a memory communicatively connected to at least one of the processors; wherein, The memory stores instructions executable by the processor, and the instructions are used to be executed by the processor to implement the method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy as described in any one of claims 1-6.

9. A computer-readable storage medium storing computer instructions, wherein the computer instructions are used to be executed by the computer to implement the method for quantitatively distinguishing intracellular and extracellular magnetic nanoparticle signals based on magnetic particle spectroscopy as described in any one of claims 1-6.

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