WO2024066773A1

WO2024066773A1 - Method for eliminating sensitivity difference in imaging mass cytometry

Info

Publication number: WO2024066773A1
Application number: PCT/CN2023/113007
Authority: WO
Inventors: 陈新蕾; 闻丹忆
Original assignee: 上海立迪生物技术股份有限公司
Priority date: 2022-09-30
Filing date: 2023-08-15
Publication date: 2024-04-04
Also published as: CN115452929A; CN116429870A; CN115452929B; WO2024066762A1

Abstract

Provided in the present invention is a signal calibration method for imaging mass cytometry, which method performs forward and reverse linear regressions on a sample signal by means of a standard substance signal. The method comprises: first selecting a sample as a standard sample, and establishing a first standard curve and a first regression model according to a standard substance on a slide thereof; then establishing a second standard curve and a second regression model according to a standard substance on a slide of a sample to be subjected to calibration; then scanning, by using a first resolution, the sample to be subjected to calibration, performing log processing on an obtained original signal value, and inputting, into the second regression model, the original signal value that has been subjected to the log processing, so as to obtain the actual metal content of each pixel of the sample to be subjected to calibration; and finally, converting the actual metal content of each pixel into a calibrated signal value by means of the first regression model, so as to complete data calibration, thereby eliminating the influence caused by sensitivity fluctuations of an instrument.

Description

A method to eliminate sensitivity differences in imaging mass spectrometry

Technical Field

The invention relates to a multi-channel imaging technology, and in particular to a signal calibration method for imaging mass spectrometry flow.

Background technique

Imaging Mass Cytometry is a tissue multi-channel imaging technology platform. It uses metal-labeled antibodies to label tissue samples to form tissue slices, and then scans the samples point by point through laser scanning, and then sends them to the inductively coupled plasma mass spectrometry (ICP-mass) host for elemental analysis, thereby obtaining the distribution information of the metal labels in the detected area and reconstructing images of dozens of channels in the same field of view. Compared with other fluorescence-based tissue imaging technologies, imaging mass cytometry has the advantages of multiple channels, no cross-color, and no interference from tissue background fluorescence. Therefore, it has played an important role in the study of tissue microenvironment of tumors, type I diabetes and some infectious diseases.

However, due to the combined influence of factors such as the environment and oxidation of sampling cone-related components, the sensitivity of imaging mass spectrometry will fluctuate, directly affecting the intensity of the detected signal, which in turn will have a certain impact on the consistency of the data and subsequent bioinformatics data analysis. In order to obtain more accurate data analysis results, it is crucial to eliminate the impact of instrument sensitivity fluctuations.

Summary of the invention

In order to eliminate the influence of instrument sensitivity fluctuation on sample signals, the present invention provides a signal calibration method for imaging mass spectrometry, wherein the sample signal is calibrated by a standard signal, and the signal calibration method comprises:

Selecting a sample as a standard sample and establishing a first standard curve based on the standard sample;

Establishing a first regression model according to the first standard curve, wherein the first regression model is used to convert the actual metal content into a signal value;

Establishing a second standard curve based on the standard substance on the sample to be calibrated; establishing a second regression model based on the second standard curve, wherein the second regression model is used to convert the original signal value after log processing into the actual metal content;

Scanning the sample to be calibrated, and calculating the actual metal content of each pixel of the sample to be calibrated according to the second regression model;

The calculated actual metal content of each pixel is converted into a calibration signal value through the first regression model to complete the data calibration.

Further, the establishment of the first standard curve and/or the second standard curve includes:

Define the scanning area for the standards on the sample slide; and

The standard region of interest is scanned to establish a standard curve, wherein at least three standards are included on the sample slide.

Furthermore, the resolution used for scanning the region of interest of the standard is lower than the resolution used for scanning the sample to be calibrated.

Furthermore, the method also includes eliminating the sensitivity difference within the sample area and between the sample area and the standard area according to the change in the signal ratio of argon dimer, or xenon, or iodine element in the sample area and the standard area.

Furthermore, the standard comprises one or more halides and/or soluble salts containing lanthanide metals, and the atomic weight of the standard covers the range of 139 to 176 lanthanide elements.

Furthermore, the standard substances include cerium chloride, samarium nitrate, holmium chloride, and lutetium chloride.

Furthermore, the standard is placed on the glass slide of the sample by the following steps:

Form multiple dilutions of the standard with different concentrations;

mixing the plurality of standard dilution solutions with trypan blue of a specified concentration respectively to obtain a plurality of working solutions;

applying heat to a local area of the slide; and

After a specified time, a specified amount of various working solutions are applied to the heated area of the slide.

Furthermore, the formation of the standard dilution solution includes:

The metal salt is diluted with dilute hydrochloric acid of the specified concentration.

Furthermore, the concentration of the dilute hydrochloric acid is 0.01M, and the concentration of the standard dilution is between 10 ^-4 M and 10 ^-8 M.

Furthermore, there are three standard dilution solutions, and the concentrations of the three standard dilution solutions are 10 ^-6 M, 10 ^-7 M and 10 ^-8 M, respectively.

Furthermore, the concentration of the trypan blue is 0.5%, and it is mixed with the standard diluent at a ratio of 1:1.

Further, heating the local part of the glass slide includes:

The portion of the slide where the standard sample is to be set is heated by the heating module.

Furthermore, the heating range of the heating module is 40 degrees Celsius to 70 degrees Celsius.

The present invention provides a signal calibration method for imaging mass spectrometry flow, in which a standard solution containing a series of lanthanide metals is applied to one side of a sample as a standard. Before the instrument detects the sample, it first scans the standard area and then scans the sample. Since the metal content contained in the standard area is fixed, its signal intensity will vary with the sensitivity of the instrument. Therefore, the signal of the standard can be used to calibrate the signal of the sample, thereby eliminating the influence of the fluctuation of the instrument sensitivity. The signal calibration method also performs two positive and negative linear regressions on the sample to be calibrated through the standard sample, thereby further eliminating the influence of the fluctuation of the instrument sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

To further illustrate the above and other advantages and features of the various embodiments of the present invention, a more detailed description of the various embodiments of the present invention will be presented with reference to the accompanying drawings. It is to be understood that these drawings only depict the present invention. The present invention is only a typical embodiment of the present invention, and therefore will not be considered to limit its scope. In the accompanying drawings, for the sake of clarity, the same or corresponding parts will be represented by the same or similar marks.

FIG1 is a flowchart showing a signal calibration method for imaging mass spectrometry according to an embodiment of the present invention;

FIG2 is a schematic diagram showing local heating of a glass slide according to an embodiment of the present invention;

Figures 3a and 3b are schematic diagrams of images obtained by scanning at different sensitivities using imaging mass spectrometry;

3c and 3d are schematic diagrams showing the signal calibration of FIGS. 3a and 3b respectively using a signal calibration method for imaging mass spectrometry according to an embodiment of the present invention;

Fig. 3e is a schematic diagram showing the statistical data of Figs. 3a to 3d;

4a and 4b are schematic diagrams showing respectively before and after calibration of a tissue region image using a signal calibration method for imaging mass spectrometry according to an embodiment of the present invention;

5a and 5b are schematic diagrams showing before and after calibration of another tissue region image using a signal calibration method for imaging mass spectrometry according to an embodiment of the present invention, respectively;

Figures 5c and 5d are schematic diagrams showing the tsne dimensionality reduction analysis corresponding to Figures 5a and 5b, respectively;

FIG6 a shows the change of argon dimer signal in the sample area in one embodiment of the present invention;

FIG. 6 b shows the argon calibration coefficient (adj. factor) of each row calculated according to the change of the argon dimer signal shown in FIG. 6 a ; and

6c and 6d are schematic diagrams showing the signal distribution of histone H3 before and after calibration using the argon dimer signal calibration method according to an embodiment of the present invention.

Detailed ways

In the following description, the invention is described with reference to various embodiments. However, one skilled in the art will recognize that the invention may be implemented without one or more of the specific details or with other alternatives and/or Additional methods, materials or components are used to implement various embodiments. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid blurring the inventive point of the present invention. Similarly, for the purpose of explanation, specific quantities, materials and configurations are described to provide a comprehensive understanding of the embodiments of the present invention. However, the present invention is not limited to these specific details. In addition, it should be understood that the various embodiments shown in the drawings are illustrative representations and are not necessarily drawn in correct proportions.

In this specification, reference to "one embodiment" or "the embodiment" means that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. The phrase "in one embodiment" appearing in various places in this specification does not necessarily all refer to the same embodiment.

It should be noted that the embodiments of the present invention describe the method steps in a specific order, but this is only for the purpose of illustrating the specific embodiment, rather than limiting the order of the steps. On the contrary, in different embodiments of the present invention, the order of the steps can be adjusted according to actual needs.

In view of the problem that the existing imaging mass spectrometry flow cytometry for multi-channel tissue imaging is easily affected by changes in instrument sensitivity, the present invention provides a signal calibration method for imaging mass spectrometry flow cytometry, which uses the signal of a standard to calibrate the signal of a sample to eliminate the impact of fluctuations in instrument sensitivity.

In an embodiment of the present invention, the standard refers to a standard solution containing a series of lanthanide metals, which are dotted on one side of the sample. Before detecting the sample, the instrument first scans the standard area at a lower resolution, such as 5 to 10 μm, and then scans the sample at a normal higher resolution, such as 1 μm. Since the metal content contained in the standard area is fixed, and its signal intensity varies with the sensitivity of the instrument, the signal of the standard can be used to calibrate the signal of the sample, thereby eliminating the impact of fluctuations in instrument sensitivity. In one embodiment of the present invention, the template for standard sampling is set as follows: laser energy is set to 2, x-step and y-step are both set to 10, and a lanthanide metal channel between 139 and 176 is selected. Before scanning, A scanning area is defined for each standard area to ensure that the entire standard area is within the range. In one embodiment of the present invention, it takes about 5 minutes to scan the ROI area of a single standard.

In one embodiment of the present invention, the standard refers to a diluted metal salt, which includes, for example, lanthanide metals, such as cerium Ce, samarium Sm, holmium Ho, lutetium Lu and other lanthanide metals with similar chemical properties, or halides, nitrates, acetates or other soluble salt forms of elements such as lanthanum, praseodymium, neodymium, promethium, europium, gadolinium, terbium, dysprosium, erbium, thulium, ytterbium, etc. In one embodiment of the present invention, the atomic weight of the metal salt should cover the range of 139 to 176 lanthanide elements. Table 1 gives the isotopic abundance of the relevant elements, and the metal salt can be selected according to Table 1.

Table 1

In one embodiment of the present invention, cerium chloride, samarium nitrate, holmium chloride, and lutetium chloride are selected as standard substances. After all metal salts are accurately weighed, they are diluted with 0.01M dilute hydrochloric acid into multiple portions of standard dilutions of different concentrations, which are stored for later use. In one embodiment of the present invention, the concentration of the standard dilution is between 10 ^-4 M and 10 ^-8 M. In one embodiment of the present invention, three standards are used, and the concentrations of the standard dilutions are 10 ^-6 M, 10 ^-7 M, and 10 ^-8 M, respectively.

The standard diluent needs to be further mixed with trypan blue of a specified concentration in a preset ratio to obtain the required standard solution. In one embodiment of the present invention, 0.5% trypan blue and standard diluents of different concentrations are mixed in a ratio of 1:1 to obtain the standard solution.

In one embodiment of the present invention, when the standard solution is applied to one side of the sample, the glass slide of the sample slice needs to be preheated first. FIG2 is a schematic diagram of local heating of a glass slide according to one embodiment of the present invention. As shown in FIG2, in one embodiment of the present invention, the heating module is used to heat the glass slide. The part of the glass slide where the standard is to be placed is heated. Specifically, the heating module is first preheated to a range of 40 degrees Celsius to 70 degrees Celsius, preferably to 60 degrees Celsius, and then a support of equal height is set on one side of the heating module so that one end of the sample slice, i.e., the part where the standard is to be placed, is placed on the heating module to achieve local heating, and the other end of the sample slice is placed on the support to maintain horizontality. After heating for a specified time, standard solutions of different concentrations are successively drawn and placed on the glass slide. In one embodiment of the present invention, the specified time is 1 minute. In another embodiment of the present invention, 0.3 μl of each concentration of standard is placed on the glass slide, and the standard solution can usually be air-dried within 20 seconds to form a circular area with a diameter of about 1.3 mm. In order to eliminate the sensitivity difference within the sample area and between the sample area and the standard area, the influence of signal drift can also be eliminated according to the change in the signal ratio of argon dimer, xenon, or iodine element in the sample area and the standard area.

On the basis of setting the standard, the present invention further adopts two positive and negative linear regressions to realize signal calibration, that is, firstly, the scanned signal is converted into metal content through a regression model, and then the metal content is converted into a calibration signal through another regression model. In one embodiment of the present invention, the two regression models are respectively recorded as the first regression model Model R and the second regression model Model F, wherein the first regression model Model R is used to convert the metal content into a signal value, and the second regression model Model F is used to convert the signal value into a metal content.

The scheme of the present invention is further described below in conjunction with the accompanying drawings of the embodiments.

FIG1 is a schematic flow chart of a signal calibration method for imaging mass spectrometry according to an embodiment of the present invention. As shown in FIG1 , a signal calibration method for imaging mass spectrometry comprises:

First, in step 101, a first regression model is established. In one embodiment of the present invention, the first regression model Model R is established by using a standard sample. Specifically, the establishment of the first regression model Model R includes:

First, a sample is selected as a standard sample, and the standard sample is stained with antibodies and Ir, and air-dried and sliced;

Next, the method described above is used to set a plurality of standards on one side of the standard sample; and

Finally, a first standard curve is established based on the standard product, and a first regression model Model R is established to convert the actual metal content into a signal value (count);

Next, in step 102, a second regression model is established. In one embodiment of the present invention, the second regression model Model F is established by the sample to be calibrated. Specifically, the establishment of the second regression model Model F includes:

First, the sample to be calibrated is stained with antibodies and Ir, and the slices are air-dried;

Next, the method described above is adopted to set a plurality of standards on one side of the sample to be calibrated; and

Finally, a second standard curve is established based on the standard product, and a second regression model Model F is established to convert the original signal value (count) after logarithmic processing into the actual metal content;

Next, in step 103, the original signal value is obtained. The sample to be calibrated is scanned to obtain the original signal value;

Next, in step 104, the actual metal content is calculated. The original signal value is log processed and input into the second regression model, and then the actual metal content of each pixel of the sample to be calibrated is calculated; in one embodiment of the present invention, before the calculation, the sensitivity difference within the sample area and between the sample area and the standard area is eliminated. Specifically, the argon calibration coefficient of each row of the sample is first calculated, that is, the ratio of the median signal of the argon dimer, or xenon, or iodine element in the row to the median signal of the argon dimer, or xenon, or iodine element in the standard area, and then The signal value of each channel is divided row by row by the argon calibration coefficient to eliminate the influence of signal drift. Figure 6a shows the change of argon dimer signal in the sample area in one embodiment of the present invention, and Figure 6b shows the argon calibration coefficient of each row calculated according to the change of argon dimer signal shown in Figure 6a; and Figures 6c and 6d respectively show the signal distribution schematic diagrams of Hi stoneH3 before and after calibration using the argon dimer signal calibration method of one embodiment of the present invention. As shown in the figure, before calibration, at the position where the argon dimer signal is sheared, that is, where the arrow points, an obvious signal intensity change can be seen, and after calibration, the image signal distribution becomes uniform, wherein the HistoneH3 is a nucleosomal protein distributed in the cell nucleus; and

Finally, in step 105, the calibration signal value is calculated. The calculated actual metal content of each pixel is converted into a calibration signal value through the first regression model. The calibration signal value is equivalent to the value detected at the same sensitivity as the standard sample, thereby completing data standardization.

In order to verify the effect of the signal calibration method provided by the present invention, different images were calibrated using the signal calibration method.

Figures 3a and 3b are schematic diagrams of images obtained by scanning at different sensitivities using imaging mass spectrometry. Among them, Figure 3a is an image obtained by scanning at high sensitivity, and Figure 3b is an image obtained by scanning at low sensitivity. It can be seen that there are obvious signal differences in the images obtained at different sensitivities. Figures 3c and 3d are schematic diagrams of Figures 3a and 3b after calibration using a signal calibration method for imaging mass spectrometry according to an embodiment of the present invention. It can be seen that after signal calibration, the monitoring data at the two sensitivities are basically the same. In order to intuitively show the difference between the two, Figure 3e further shows a schematic diagram of statistical data of Figures 3a to 3d, wherein the darker colored columns show the statistical data of Figures 3a and 3b, and the lighter colored columns show the statistical data of Figures 3c and 3d. Specifically, Figure 3e is divided into three areas, left, middle and right, which respectively show the content values of Ce, Sm and Lu obtained according to image statistics. In each area, the two columns on the left show Figures 3a and 3c, i.e., the images obtained at high sensitivity. The metal content value corresponding to the image and the two columns on the right are shown in Figures 3b and 3d, which are the metal content values corresponding to the images obtained under low sensitivity.

Figures 4a and 4b respectively show schematic diagrams of the image of a certain tissue area (human tonsil) before and after calibration using a signal calibration method for imaging mass spectrometry according to an embodiment of the present invention, wherein the upper part of Figure 4a is obtained by scanning under high sensitivity, and the lower part is obtained by scanning under low sensitivity. It can be seen that the lower part is obviously weaker, and after the signal calibration, as shown in Figure 4b, there is no obvious distinction between the upper and lower parts, which significantly improves the comparability of tissue images.

Figures 5a and 5b respectively show schematic diagrams of another tissue region before and after calibration using a signal calibration method of an imaging mass spectrometry flow according to an embodiment of the present invention, and Figures 5c and 5d respectively show schematic diagrams of tSNE dimensionality reduction analysis corresponding to Figures 5a and 5b. The upper part of Figure 5a is obtained by scanning at low sensitivity, and the lower part is obtained by scanning at high sensitivity. It can be seen that there are significant differences in the signals of the two regions. This can be clearly seen from the tSNE dimensionality reduction analysis diagram of Figure 5. The cells in the low-sensitivity region above are concentrated in one place, indicating that there are obvious phenotypic differences between them and cells in other regions. After signal calibration, as shown in Figures 5b and 5d, the cell distribution in this region is integrated into a large group, and the influence of sensitivity is basically eliminated, which significantly reduces the influence of sensitivity differences on the results of bioinformatics analysis.

Although various embodiments of the present invention are described above, it should be understood that they are presented as examples only and not as limitations. It is obvious to those skilled in the relevant art that various combinations, modifications and changes can be made thereto without departing from the spirit and scope of the present invention. Therefore, the breadth and scope of the present invention disclosed herein should not be limited by the exemplary embodiments disclosed above, but should only be defined according to the attached claims and their equivalents.

Claims

A method for eliminating sensitivity differences in imaging mass spectrometry flow, characterized by comprising the steps of:

Calculating the signal median of the argon dimer, xenon, or iodine element of the sample row by row to obtain a first value;

Calculating the median of the signal of argon dimer or xenon or iodine element in the standard area on the sample row by row to obtain a second value;

Calculating the ratio of the first value to the second value row by row; and

The signal value of each channel is divided by the ratio row by row to eliminate the sensitivity difference of the signal value of each channel.
The method according to claim 1, further comprising:

Scan the sample to be measured to obtain the original signal value;

Performing log processing on the signal value after eliminating the sensitivity difference, and inputting it into a second regression model to obtain the actual metal content of each pixel of the sample to be measured; and

The actual metal content of each pixel is converted into a measurement signal value through a first regression model.
The method according to claim 2, characterized in that the first regression model is established according to the following steps:

Select one sample as a standard sample, perform antibody and I r staining on the standard sample, and air-dry and slice it;

Disposing a plurality of standards on one side of the standard sample; and

A first standard curve is established based on the standard product, and a first regression model is established to convert the actual metal content into a signal value.
The method according to claim 2, characterized in that the second regression model is established according to the following steps:

Perform antibody and Ir staining on the sample to be measured, and air-dry the slices;

Disposing a plurality of standards on one side of the sample to be measured; and

A second standard curve is established based on the standard product, and a second regression model is established to convert the original signal value after logarithmic operation into the actual metal content.
The method according to claim 3 or 4, characterized in that the standard comprises one or more halides and/or soluble salts containing lanthanide metals, and the atomic weight of the standard covers the range of 139 to 176 lanthanide elements.
The method according to claim 3 or 4, characterized in that the standard is set on the slide of the sample by the following steps:

Dilute the metal salt with dilute hydrochloric acid of a specified concentration to form a plurality of standard dilution solutions of different concentrations;

mixing the plurality of standard dilution solutions with trypan blue of a specified concentration respectively to obtain a plurality of working solutions;

applying heat to a local area of the slide; and

After a specified time, a specified amount of various working solutions are applied to the heated area of the slide.
The method according to claim 6, characterized in that the concentration of the dilute hydrochloric acid is 0.01 M, and the concentration of the standard diluent is between 10 -4 M and 10 -8 M.
The method according to claim 6, characterized in that the concentration of trypan blue is 0.5%, and it is mixed with the standard diluent in a 1:1 ratio.
The method according to claim 6, wherein heating the local portion of the glass slide comprises:

The portion of the slide where the standard sample is to be set is heated by the heating module.
The method according to claim 9, characterized in that the heating range of the heating module is 40 degrees Celsius to 70 degrees Celsius.