CN115452929A - Imaging mass spectrum flow signal calibration method - Google Patents
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Abstract
The invention provides a signal calibration method of imaging mass spectrometry flow type, which carries out positive and negative twice linear regression on a sample signal through a standard signal: firstly, selecting a sample as a standard sample, establishing a first standard curve and a first regression model according to a standard sample on a slide, then establishing a second standard curve and a second regression model according to the standard sample on the slide of the sample to be calibrated, scanning the sample to be calibrated with a first resolution, performing log processing on an obtained original signal value, inputting the log processing into the second regression model to obtain the actual metal content of each pixel of the sample to be calibrated, and finally converting the actual metal content of each pixel into a calibration signal value through the first regression model to finish the calibration of data, thereby eliminating the influence caused by the fluctuation of the sensitivity of an instrument.
Description
Technical Field
The invention relates to a multichannel imaging technology, in particular to a signal calibration method of imaging mass spectrum streaming.
Background
Imaging Mass spectrometry (Imaging Mass Cytometry) is a tissue multichannel Imaging technology platform. The method comprises the steps of marking a tissue sample by using a metal tag antibody to form a tissue section, then scanning and sampling point by point through laser, and sending the tissue section into an inductively coupled plasma mass spectrometry (ICP-mass) host machine for element analysis, so that distribution information of metal tags in a detected area is obtained, and images of dozens of channels in the same visual field are reconstructed. Compared with other fluorescence-based tissue imaging technologies, the imaging mass spectrometry flow has the advantages of multiple channels, no cross color, no interference of tissue background fluorescence and the like, and therefore, plays an important role in the research of tissue microenvironments of tumors, type I diabetes and some infectious diseases.
However, due to the comprehensive influence of factors such as oxidation of the environment and parts related to the sampling cone, the sensitivity of the imaging mass spectrum flow fluctuates, the intensity of the detected signal is directly influenced, and further certain influence is generated on the consistency of data and subsequent confidence data analysis. In order to obtain more accurate data analysis results, how to eliminate the influence caused by the fluctuation of the sensitivity of the instrument is very important.
Disclosure of Invention
In order to eliminate the influence of instrument sensitivity fluctuation on a sample signal, the invention provides a signal calibration method of imaging mass spectrometry, which calibrates the sample signal through a standard signal, and the signal calibration method comprises the following steps:
selecting a sample as a standard sample, and establishing a first standard curve according to the standard sample;
establishing a first regression model according to the first standard curve, wherein the first regression model is used for converting the actual metal content into a signal value;
establishing a second standard curve according to a standard substance on a sample to be calibrated; establishing a second regression model according to the second standard curve, wherein the second regression model is used for converting the original signal value subjected to log processing into actual metal content;
scanning a sample to be calibrated, and calculating according to the second regression model to obtain the actual metal content of each pixel of the sample to be calibrated;
and converting the calculated actual metal content of each pixel into a calibration signal value through a first regression model, and completing the calibration of data.
Further, the establishing of the first standard curve and/or the second standard curve comprises:
defining a scanning area for a standard on a sample slide; and
scanning the region of interest for the standards to create a standard curve, wherein at least three standards are included on the sample slide.
Further, the area of interest of the standard is scanned with a resolution lower than the resolution with which the sample to be calibrated is scanned.
Further, the method further comprises eliminating sensitivity differences within the sample region and between the sample region and the standard region based on the change in signal ratio of argon dimer, or xenon, or iodine elements of the sample region and the standard region.
Further, the standard includes one or more halides and/or soluble salts comprising lanthanide metals, and the atomic weight of the standard covers the lanthanide element range 139 to 176.
Further, the standard substance comprises cerium chloride, samarium nitrate, holmium chloride, and lutetium chloride.
Further, the standard is disposed on a slide of a specimen by:
forming a plurality of standard dilutions of different concentrations;
mixing the plurality of standard substance diluents with trypan blue with specified concentration respectively to obtain a plurality of working solutions;
heating a local part of the slide; and
after the specified time, the specified amounts of the various working solutions are respectively spotted on the heating positions of the glass slides.
Further, the forming of the standard dilution comprises:
the metal salt is diluted with dilute hydrochloric acid of the indicated concentration.
Further, the concentration of the dilute hydrochloric acid is 0.01M, and the concentration of the standard substance diluent is 10 -4 M to 10 -8 M is greater than or equal to the total weight of the composition.
Further, the standard dilution is three, and the three standards areThe concentrations of the dilutions were: 10 - 6 M、10 -7 M and 10 -8 M。
Further, the concentration of trypan blue is 0.5%, which is mixed with the standard dilution 1.
Further, heating a part of the slide includes:
and heating the part of the slide, which needs to be provided with the standard product, by the heating module.
Further, the heating range of the heating module is 40-70 ℃.
The invention provides a flow-type signal calibration method for imaging mass spectrometry, which takes a standard solution containing a series of lanthanide metals as a standard substance at one side of a sample. Before the instrument detects the sample, the standard area is scanned, and then the sample is scanned. Because the metal content in the standard substance area is fixed, and the signal intensity of the standard substance area can change along with the sensitivity of the instrument, the signal of the sample can be calibrated by using the signal of the standard substance, and the influence caused by the sensitivity fluctuation of the instrument can be eliminated. The signal calibration method further comprises the step of performing positive and negative linear regression on a sample to be calibrated through a standard sample, and further eliminating the influence caused by the fluctuation of the sensitivity of the instrument.
Drawings
To further clarify the above and other advantages and features of various embodiments of the present invention, a more particular description of various embodiments of the invention will be rendered by reference to the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. In the drawings, the same or corresponding parts will be denoted by the same or similar reference numerals for clarity.
FIG. 1 is a schematic flow chart of a method of imaging mass spectrometry signal calibration according to an embodiment of the present invention;
FIG. 2 shows a schematic view of locally heating a slide according to one embodiment of the present invention;
FIGS. 3a and 3b are schematic diagrams of images scanned at different sensitivities by an imaging mass spectrometer;
FIGS. 3c and 3d are schematic diagrams of the imaging mass spectrometer signal calibration method according to an embodiment of the present invention after signal calibration of FIGS. 3a and 3 b;
FIG. 3e shows a statistical data diagram of FIGS. 3a to 3 d;
FIGS. 4a and 4b are schematic diagrams of an imaging mass spectrometry signal calibration method according to an embodiment of the present invention before and after calibration of a tissue region image;
FIGS. 5a and 5b are schematic diagrams of an imaging mass spectrometry flow signal calibration method according to an embodiment of the present invention before and after calibration of a further tissue region image;
FIGS. 5c and 5d are schematic diagrams of the tsne dimension reduction analysis corresponding to FIGS. 5a and 5b, respectively;
FIG. 6a shows a sample region argon dimer signal variation in one embodiment of the present invention;
FIG. 6b shows the calculated adj.factor for each row based on the argon dimer signal change shown in FIG. 6 a; and
fig. 6c and 6d respectively show the signal distribution of HistoneH3 before and after calibration by the argon dimer signal calibration method according to an embodiment of the present invention.
Detailed Description
In the following description, the present invention is described with reference to examples. One skilled in the relevant art will recognize, however, that the embodiments may be practiced without one or more of the specific details, or with other alternative and/or additional methods, materials, or components. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention. Similarly, for purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the embodiments of the invention. However, the invention is not limited to these specific details. Further, it should be understood that the embodiments shown in the figures are illustrative representations and are not necessarily drawn to scale.
Reference in the specification to "one embodiment" or "the embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.
It should be noted that the method steps are described in a specific order according to the embodiments of the present invention, which is only for illustrating the specific embodiments and not for limiting the sequence of the steps. On the contrary, in different embodiments of the present invention, the sequence of the steps can be adjusted according to the adjustment of actual requirements.
Aiming at the problem that the existing imaging mass spectrometry flow type tissue multichannel imaging is easily affected by instrument sensitivity change, the invention provides a signal calibration method of the imaging mass spectrometry flow type, which calibrates a signal of a sample by using a signal of a standard substance and eliminates the influence caused by instrument sensitivity fluctuation.
In embodiments of the invention, the standard refers to a standard solution containing a series of lanthanide metals spotted on one side of the sample, and the instrument scans the area of the standard with a lower resolution, e.g., 5 to 10um, and then scans the sample with a normal higher resolution, e.g., 1um, before detecting the sample. Because the content of the metal contained in the standard substance area is fixed and the signal intensity of the standard substance area can change along with the sensitivity of the instrument, the signal of the sample can be calibrated by using the signal of the standard substance, and the influence caused by the sensitivity fluctuation of the instrument is eliminated. In one embodiment of the invention, the template of the standard sample is set as: laser energy was set to 2, x-step and y-step were set to 10, and a larger number of lanthanide metal channels were selected from 139 to 176, and a scan area was required to be delineated for each standard area before scanning to ensure that the entire standard area was delineated, in one embodiment of the invention, scanning the ROI of a single standard took approximately 5 minutes.
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 similar in chemical properties, or halides, nitrates, acetates, or other soluble salt forms of elements including lanthanum, praseodymium, neodymium, promethium, europium, gadolinium, terbium, dysprosium, erbium, thulium, ytterbium, and the like. In one embodiment of the invention, the atomic weight of the metal salt should cover the 139 to 176 lanthanide range, and table 1 gives the isotopic abundance of the relevant elements, which can be selected according to table 1.
| 1 3 9 | 1 4 0 | 1 4 1 | 1 4 2 | 1 4 3 | 1 4 4 | 1 4 5 | 1 4 6 | 1 4 7 | 1 4 8 | 1 4 9 | 1 5 0 | 1 5 1 | 1 5 2 | 1 5 3 | 1 5 4 | 1 5 5 | 1 5 6 | 1 5 7 | 1 5 8 | 1 5 9 | 1 6 0 | 1 6 1 | 1 6 2 | 1 6 3 | 1 6 4 | 1 6 5 | 1 6 6 | 1 6 7 | 1 6 8 | 1 6 9 | 1 7 0 | 1 7 1 | 1 7 2 | 1 7 3 | 1 7 4 | 1 7 5 | 176 | |
| C e | 8 8 | 1 1 | ||||||||||||||||||||||||||||||||||||
| |
3 . 1 | 1 5 | 1 1 | 1 4 | 7 . 4 | 2 7 | 2 3 | |||||||||||||||||||||||||||||||
| H o | 1 0 0 | |||||||||||||||||||||||||||||||||||||
| L u | 9 7 | 2.6 |
TABLE 1
In one embodiment of the invention, cerium chloride, samarium nitrate, holmium chloride and lutetium chloride are selected as standard substances, after all metal salts are accurately weighed, the metal salts are diluted into a plurality of standard substance diluents with different concentrations by using 0.01M dilute hydrochloric acid and stored for later use. In one embodiment of the invention, the standard dilution is at a concentration of 10 -4 M to 10 -8 And M is between the two. In one embodiment of the present invention, three standards were used, and the concentration of each standard dilution was 10 -6 M、10 -7 M and 10 -8 M。
The standard substance diluent is further mixed with trypan blue with specified concentration according to a preset proportion to obtain the required standard solution. In one embodiment of the present invention, a standard solution is prepared by mixing 0.5% trypan blue with standard dilutions of different concentrations 1:1, respectively.
In one embodiment of the present invention, when spotting the standard solution to one side of the sample, it is first necessary to preheat a slide of the sample slice. FIG. 2 shows a schematic view of locally heating a slide, in accordance with one embodiment of the present invention. As shown in fig. 2, in an embodiment of the present invention, a portion of a slide where a standard is to be disposed is heated by a heating module, specifically, the heating module is first preheated to a temperature in a range from 40 degrees celsius to 70 degrees celsius, preferably to 60 degrees celsius, and then a support with the same height is disposed on one side of the heating module, so that one end of a sample slice, that is, a portion where a point standard is to be disposed, is lapped on the heating module to achieve local heating, the other end of the sample slice is lapped on the support to maintain a horizontal state, and after heating for a specified time, standard solutions with different concentrations are sequentially sucked and respectively spotted on the slide, where in an embodiment of the present invention, the specified time is 1 minute. In yet another embodiment of the invention, 0.3ul of each concentration of standard is spotted on the slide and the standard solution can typically be air dried within 20 seconds, forming a circular area of about 1.3mm in diameter. In order to eliminate the sensitivity difference between the sample region and the standard region, the influence of signal drift (signal drift) can be eliminated according to the signal ratio change of argon dimer, xenon or iodine of the sample region and the standard region.
On the basis of setting the standard, the method further adopts forward and backward two-time linear regression to realize signal calibration, that is, firstly, converting a signal obtained by scanning into metal content through one regression Model, and then converting the metal content into a calibration signal through the other regression Model.
The scheme of the invention is further described by combining the embodiment drawings.
Fig. 1 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 fig. 1, a signal calibration method of imaging mass spectrometry flow comprises:
first, in step 101, a first regression model is established. In one embodiment of the present invention, the first regression Model R is built by standard samples. Specifically, the establishing of the first regression Model R includes:
firstly, selecting a sample as a standard sample, carrying out antibody and Ir staining on the standard sample, and air-drying a slice;
next, the method as described above is adopted, and a plurality of standard samples are arranged on one side of the standard sample; and
finally, establishing a first standard curve according to the standard product, and establishing a first regression Model R converted from actual metal content to a signal value (count);
next, at step 102, a second regression model is established. In one embodiment of the invention, the second regression Model F is built from the samples to be calibrated. Specifically, the establishing of the second regression Model F includes:
firstly, carrying out antibody and Ir staining on the sample to be calibrated, and air-drying the section;
next, the method as described above is sampled, and a plurality of standards are arranged on one side of the sample to be calibrated; and
finally, establishing a second standard curve according to the standard product, and establishing a second regression Model F for converting the original signal value count after log processing, namely logarithm operation, into the actual metal content;
next, in step 103, raw signal values are acquired. Scanning a sample to be calibrated to obtain an original signal value;
next, at step 104, the actual metal content is calculated. Performing log processing on the original signal value, inputting the log processed original signal value to the second regression model, and further calculating to obtain the actual metal content of each pixel of the sample to be calibrated; in one embodiment of the present invention, before the calculation, the sensitivity difference inside the sample region and between the sample region and the standard region is also eliminated, specifically, the ratio of the median of the signal of the argon dimer, or xenon, or iodine element to the median of the signal of the argon dimer, or xenon, or iodine element in the standard region is first calculated for each line of the sample, and then the signal value of each channel is divided by the value of the signal of each line by the value of the plus factor to eliminate the influence caused by signal drift (signal drift). Fig. 6a shows a sample region argon dimer signal variation in an embodiment of the present invention, and fig. 6b shows adj. Factors of respective rows calculated according to the argon dimer signal variation shown in fig. 6 a; and FIGS. 6c and 6d are schematic diagrams showing the signal distribution of HistoneH3 before and after calibration by the argon dimer signal calibration method according to an embodiment of the present invention, wherein before calibration, at the position of the shear of the argon dimer signal, i.e. the arrow, a significant signal intensity change can be seen, and the image signal distribution after calibration becomes uniform, wherein HistoneH3 is a nucleosome protein distributed in the nucleus; and
finally, in step 105, a calibration signal value is calculated. And converting the calculated actual metal content of each pixel into a calibration signal value through a first regression model, wherein the calibration signal value is equivalent to a value detected under the condition of completely same sensitivity as the standard sample, and thereby completing the data standardization.
In order to verify the effect of the signal calibration method provided by the invention, different images are calibrated by adopting the signal calibration method.
Fig. 3a and 3b respectively show images obtained by scanning at different sensitivities by using an imaging mass spectrometry flow. Where fig. 3a is the image scanned at high sensitivity and fig. 3b is the image scanned at low sensitivity, it can be seen that there is a significant difference in signal between the images scanned at different sensitivities. Fig. 3c and 3d respectively show schematic diagrams of the calibration of fig. 3a and 3b by using an imaging mass spectrometry streaming signal calibration method according to an embodiment of the present invention, and it can be seen that, after signal calibration, monitoring data at two sensitivities are substantially the same. To visually illustrate the difference between the two, fig. 3e further shows the statistical data diagram of fig. 3a to 3d, wherein the darker colored bars show the statistical data of fig. 3a and 3b, and the lighter colored bars show the statistical data of fig. 3c and 3 d. Specifically, in fig. 3e, the left, middle and right regions are respectively shown to obtain the content values of Ce, sm and Lu according to the image statistics, in each region, the two columns on the left side are shown in fig. 3a and 3c, which are the metal content values corresponding to the images acquired under the high sensitivity, and the two columns on the right side are shown in fig. 3b and 3d, which are the metal content values corresponding to the images acquired under the low sensitivity.
Fig. 4a and 4b respectively show schematic diagrams before and after calibration of an image of a certain tissue region (tonsil) by using an imaging mass-flow signal calibration method according to an embodiment of the present invention, where the upper part of fig. 4a is scanned at a high sensitivity, and the lower part is scanned at a low sensitivity, and it can be seen that the lower part is significantly weaker, and after signal calibration, as shown in fig. 4b, the upper part and the lower part are not significantly distinguished, which significantly improves the comparability of tissue images.
Fig. 5a and 5b respectively show schematic diagrams before and after a further tissue region is calibrated by using an imaging mass spectrometry signal calibration method according to an embodiment of the present invention, and fig. 5c and 5d respectively show schematic diagrams of tsne dimension reduction analysis corresponding to fig. 5a and 5 b. The upper part of fig. 5a is scanned under low sensitivity, and the lower part is scanned under high sensitivity, it can be seen that there is a significant difference in signals of the two regions, which is evident from the ctsne dimension reduction analysis chart of fig. 5 that the cells in the upper low sensitivity region are intensively distributed at one place, indicating that there is a significant phenotypic difference with the cells in the other regions. After signal calibration, as shown in fig. 5b and 5d, the cell distribution in the region is fused into a large population, and the influence of sensitivity is substantially eliminated, thereby significantly reducing the influence of sensitivity difference on the result of the signal analysis.
While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various combinations, modifications, and changes can be made thereto without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
Claims (13)
1. A method of imaging mass spectrometry streaming signal calibration, comprising:
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 of the sample, wherein the first regression model is configured to convert actual metal content into a signal value;
establishing a second standard curve and a second regression model according to a standard substance on a sample slide to be calibrated, wherein the second regression model is configured to convert the original signal value after the logarithm operation into the actual metal content;
scanning the sample to be calibrated by using a first resolution, carrying out log processing on an obtained original signal value, and inputting the obtained original signal value into the second regression model to obtain the actual metal content of each pixel of the sample to be calibrated;
the actual metal content of each pixel is converted to a calibration signal value by a first regression model.
2. The signal calibration method of claim 1, wherein the establishing of the first standard curve and/or the second standard curve comprises:
demarcating a scanning area for a standard on a sample slide; and
scanning the region of interest of the standard with a second resolution to create a standard curve, wherein at least three standards are included on the sample slide.
3. The signal calibration method of claim 2, wherein the first resolution is higher than the second resolution.
4. The signal calibration method of claim 1, further comprising:
and eliminating sensitivity differences inside the sample region and between the sample region and the standard region according to the signal ratio change of the argon dimer, or xenon, or iodine element of the sample region and the standard region.
5. The method for signal calibration according to claim 1, wherein the standard comprises one or more halides and/or soluble salts comprising lanthanide metals, and wherein the atomic weight of the standard covers the lanthanide range 139 to 176.
6. The signal calibration method of claim 1, wherein the standards comprise cerium chloride, samarium nitrate, holmium chloride, and lutetium chloride.
7. The signal calibration method of claim 1, wherein the standard is disposed on a slide of a sample by:
forming a plurality of standard dilutions of different concentrations;
respectively mixing the plurality of standard substance diluents with trypan blue with specified concentration to obtain a plurality of working solutions;
heating a local part of the slide; and
after the specified time, the specified amounts of the various working solutions are respectively spotted on the heating positions of the glass slides.
8. The signal calibration method of claim 7, wherein the forming of the standard dilution comprises:
the metal salt is diluted with dilute hydrochloric acid of a specified concentration.
9. The signal calibration method of claim 8, wherein the dilute hydrochloric acid has a concentration of 0.01M and the standard dilution has a concentration of 10M -4 M to 10 -8 M is greater than or equal to the total weight of the composition.
10. The method of signal calibration according to claim 7, wherein there are three standard dilutions, and wherein there are three standard dilutionsThe concentrations of the standard dilution are respectively as follows: 10 -6 M、10 -7 M and 10 -8 M。
11. The method for signal calibration according to claim 7, wherein the concentration of trypan blue is 0.5% mixed with the standard dilution 1.
12. The signal calibration method of claim 7, wherein heating the portion of the slide comprises:
and heating the part of the slide where the standard product needs to be arranged by the heating module.
13. The signal calibration method of claim 12, wherein the heating range of the heating module is 40 degrees celsius to 70 degrees celsius.
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| PCT/CN2023/112512 WO2024066762A1 (en) | 2022-09-30 | 2023-08-11 | Signal calibration method for imaging mass cytometry |
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| WO2024066762A1 (en) * | 2022-09-30 | 2024-04-04 | 上海立迪生物技术股份有限公司 | Signal calibration method for imaging mass cytometry |
Also Published As
| Publication number | Publication date |
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| CN115452929B (en) | 2023-04-21 |
| WO2024066773A1 (en) | 2024-04-04 |
| WO2024066762A1 (en) | 2024-04-04 |
| CN116429870A (en) | 2023-07-14 |
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