Synchronous imaging method for multi-cell structure in multi-cell type

Abstract

The invention discloses a synchronous imaging method for a multicellular structure in a multicellular type. The synchronous imaging method comprises the following steps: S1, marking the cellular structure in the multicellular type by using fluorescent protein; s2, forming a spectrum-spatial feature joint coding set according to the spectrum and spatial features; s3, performing spectral unmixing on the spectral image to obtain an abundance image of each fluorescent component; s4, performing cell segmentation and cell structure positioning, and extracting spatial features; and S5, carrying out joint decoding on the spectral features and the spatial features to distinguish different cell types, and carrying out synchronous imaging on the multi-cell structure. The method has the beneficial effects that different cell types are marked by using corresponding fluorescent proteins, and a spectrum-space coupling coding strategy is formed in combination with spatial position characteristics, so that simultaneous observation and dynamic analysis of different cell types and a plurality of internal structures in the same imaging view field are realized; and the limitation that traditional spectral imaging can only work in a single cell type is broken through.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical microscopic imaging and spectral analysis, in particular to a method for synchronous imaging of multiple cell structures in multiple cell types. BACKGROUND

[0002] Cells have very complex internal structures, and detecting the internal structures of cells is of great significance in basic scientific research, drug development and clinical medical detection. At present, specific observation of the complex internal structures of cells mainly depends on fluorescence spectral imaging. However, the existing spectral imaging method is mainly limited to the imaging of multiple targets in the internal structure of a single cell type, and cannot distinguish multiple different types of cells and their internal structures in the same imaging field, which limits the systematic study of researchers on the structural heterogeneity, dynamic behavior difference and potential interaction mechanism between different cell types. Therefore, there is an urgent need for a method that can realize synchronous imaging of multiple cell structures in multiple cell types in the same imaging process. SUMMARY

[0003] The purpose of the present application is to overcome the shortcomings of the prior art and provide a method for synchronous imaging of multiple cell structures in multiple cell types.

[0004] The purpose of the present application is achieved by the following technical solution: a method for synchronous imaging of multiple cell structures in multiple cell types, comprising the following steps:

[0005] S1: using fluorescent proteins to label the cell structures within the cell types;

[0006] S2: forming a spectral-spatial feature joint coding set according to the spectral and spatial features;

[0007] S3: performing spectral unmixing on the spectral image to obtain the abundance map of each fluorescent component;

[0008] S4: performing cell segmentation and cell structure positioning to extract spatial features;

[0009] S5: jointly decoding the spectral features and spatial features to distinguish different cell types, and synchronously imaging the multiple cell structures.

[0010] Preferably, in step S1, three fluorescent proteins are assigned to each cell type.

[0011] Preferably, step S2 further comprises the following steps:

[0012] S21: selecting three fluorescent proteins in the excitation wavelength range of 480nm-560nm and collecting their fluorescence excitation spectra;

[0013] S22: Calculate the mean area and standard deviation of mitochondria, lysosomes, and endoplasmic reticulum, and use them as spatial distribution characteristics for identifying organelle types.

[0014] ;

[0015] ;

[0016] in, For average area, The standard deviation of the area. The number of a certain organelle within a cell. The area of ​​each organelle;

[0017] S23: Combine the fluorescence excitation spectra of the three fluorescent proteins with the spatial distribution characteristics of the three organelles in pairs to form a spectral-spatial feature joint coding set.

[0018] Preferably, step S3 further includes the following step:

[0019] S31: Label cells according to the coding combination in step S23, mix and culture different types of cells, and simultaneously acquire multi-band spectral images of the mixed samples under the same field of view;

[0020] S32: At the excitation wavelength , , , The mixed fluorescence images acquired under irradiation are as follows: , , , The individual images after spectral decomposition of the three fluorescent proteins are as follows: , and ;

[0021] S33: In mixed samples, due to the combined contributions of different fluorescent proteins, the relationships are as follows:

[0022] ;

[0023] in, For the first Fluorescence excitation spectra of several fluorescent proteins;

[0024] S34: Individual decomposition images of each fluorescent protein were solved using least-squares fitting.

[0025] ;

[0026] ;

[0027] ;

[0028] wherein, is the transpose matrix of.

[0029] The present application has the following advantages: the present application labels different cell types by using corresponding fluorescent proteins, and combines spatial position characteristics to form a spectral-spatial coupling coding strategy, so that different cell types and internal multiple structures in the same imaging field of view are simultaneously observed and dynamically analyzed, thereby breaking through the limitation of traditional spectral imaging which can only work in a single cell type. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a schematic diagram of the principle of synchronous imaging of multiple cell structures in multiple cell types;

[0031] Figure 2 is a schematic diagram of the synchronous imaging result of multiple cell structures in multiple cell types. DETAILED DESCRIPTION

[0032] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. The components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations.

[0033] Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0034] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0035] It should be noted that: similar reference numerals and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0036] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship commonly understood by those skilled in the art, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation of the present application. In addition, the terms "first", "second" and the like are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0037] In the description of the present application, it should be noted that, unless otherwise explicitly specified and limited, the terms "arrangement", "installation", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be connected inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0038] In the present embodiment, as shown in Figure 1 and Figure 2 A method for synchronously imaging multi-cell structures in multiple cell types, comprising the following steps:

[0039] S1: Labeling the cell structure within the cell type using fluorescent proteins; preferably, three fluorescent proteins are assigned to each cell type. Specifically, guided by operability, decoding uniqueness and biocompatibility, each cell type is assigned 3 labels (cell structure type and fluorescent protein combination), ensuring that the labels within the cell do not interfere with each other, and the encoding across cell types also has differences.

[0040] S2: Forming a spectral-spatial feature joint encoding set according to the spectral and spatial features;

[0041] S3: Spectral unmixing of the spectral image to obtain the abundance map of each fluorescent component; specifically, the spectral unmixing uses a linear decomposition algorithm or a non-negative matrix factorization algorithm.

[0042] S4: cell segmentation and cell structure localization, extract spatial features; Specifically, since each single decomposition image of fluorescent protein contains multiple different cells, it is necessary to first segment the decomposition image, identify the boundary of each cell, and then calculate the spatial distribution features (average area and area standard deviation) of the organelles labeled by the three fluorescent proteins, i.e. mitochondria, lysosomes and endoplasmic reticulum, in each cell, and further establish the corresponding relationship between the fluorescence spectrum and the spatial features in each cell. Here, since the endoplasmic reticulum covers the entire range of the cell, in each segmented cell, the range of the endoplasmic reticulum is directly used as the standard for segmenting the range of each cell, thereby achieving simple and fast cell segmentation; In each cell range, the spatial distribution features of the organelles corresponding to the three fluorescent proteins, i.e. the average area and the area standard deviation, are calculated, and the corresponding relationship between the fluorescence spectrum and the spatial features of each fluorescent protein in each cell is established, wherein the spatial features include the relative distance distribution, local density and spatial arrangement pattern of the organelles to the nucleus or the cell membrane.

[0043] S5: Joint decoding of spectral features and spatial features to distinguish different cell types, and synchronous imaging of multiple cell structures. Specifically, the established corresponding relationship between the fluorescence spectrum and the spatial features of each fluorescent protein after image decomposition is compared with the spectral-spatial feature code combination assigned to each cell type, so as to identify the type of the cell, and finally obtain the synchronous imaging and dynamic behavior analysis results of multiple cell structures in multiple cell types. The present application uses corresponding fluorescent proteins to mark different cell types, and combines spatial position features to form a spectral-spatial coupling coding strategy, so as to realize the simultaneous observation and dynamic analysis of different cell types and multiple structures inside the same imaging field of view, thereby breaking through the limitation of traditional spectral imaging which can only work in a single cell type. The present application is suitable for spectral microscopes, confocal microscopes or super-resolution microscopic imaging systems, and can also be used for live cell imaging, fixed sample detection and drug action kinetics analysis.

[0044] Further, in step S2, the following steps are further included:

[0045] S21: Select three fluorescent proteins in the excitation wavelength range of 480nm-560nm, and collect their fluorescence excitation spectra;

[0046] S22: Calculate the average area and area standard deviation of mitochondria, lysosomes and endoplasmic reticulum, and use them as spatial distribution features to identify organelle types,

[0047] ;

[0048] ;

[0049] wherein, is the average area, is the area standard deviation, is the number of a certain organelle in a cell, is the area of each organelle;

[0050] S23: The fluorescence excitation spectrum of the three fluorescent proteins and the spatial distribution characteristics of the three organelles are combined in pairs to form a spectrum-spatial feature joint encoding set. Specifically, based on the spectrum database of fluorescent proteins, fluorescent proteins with significantly distinguishable spectra are screened, i.e. three fluorescent proteins with excitation peaks around 495 nm, 525 nm and 555 nm are screened in the range of 480 nm to 560 nm, which are mGod, PhiYFP and tdTomato respectively, and their fluorescence excitation spectra are collected in the experiment; in step S23, the fluorescence excitation spectra of the three fluorescent proteins and the spatial distribution characteristics of the three organelles are combined in pairs to form a spectrum-spatial feature joint encoding set, and each cell type is assigned a spectrum-spatial feature encoding combination, such as: cell 1: mGod-lysosome, PhiYFP-mitochondria, tdTomato-endoplasmic reticulum; cell 2: PhiYFP-lysosome, mGod-mitochondria, tdTomato-endoplasmic reticulum; cell 3: tdTomato-lysosome, PhiYFP-mitochondria, mGod-endoplasmic reticulum.

[0051] Further, step S3 further includes the following steps:

[0052] S31: Label the cells according to the encoding combination in step S23, mix different types of cells for culture, and simultaneously collect multi-band spectral images of the mixed sample under the same field of view;

[0053] S32: Collect mixed fluorescence images under the irradiation of excitation wavelengths , , , The single images of the three fluorescent proteins after spectral decomposition are , , , , respectively; preferably, , and ; preferably, herein is 8.

[0054] S33: In the mixed sample, due to the joint contribution of different fluorescent proteins, the relationship is as follows:

[0055] ;

[0056] wherein, is the fluorescence excitation spectrum of the nth fluorescent protein; is the fluorescence excitation spectrum of the nth fluorescent protein;

[0057] S34: a single decomposition image of each fluorescent protein is solved by least square fitting,

[0058] ;

[0059] ;

[0060] ;

[0061] wherein, is the transpose matrix of , here can be expressed as .

[0062] Although the present application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can be modified, or some of the technical features can be replaced by equivalent features, any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for simultaneous imaging of multicellular structures in multiple cell types, characterized in that: Includes the following steps: S1: Use fluorescent proteins to label cellular structures within a cell type; S2: Form a joint coding set of spectral and spatial features based on spectral and spatial features; S3: Perform spectral demixing on the spectral image to obtain the abundance map of each fluorescent component; S4: Perform cell segmentation and cell structure localization, and extract spatial features; S5: Combines spectral and spatial features for decoding to distinguish different cell types and enables simultaneous imaging of multicellular structures.

2. The method for simultaneous imaging of multicellular structures in multiple cell types according to claim 1, characterized in that: In step S1, three fluorescent proteins are assigned to each cell type.

3. The method for simultaneous imaging of multicellular structures in multiple cell types according to claim 2, characterized in that: Step S2 further includes the following steps: S21: Three fluorescent proteins were screened in the excitation wavelength range of 480nm-560nm, and their fluorescence excitation spectra were collected; S22: Calculate the mean area and standard deviation of mitochondria, lysosomes, and endoplasmic reticulum, and use them as spatial distribution characteristics for identifying organelle types. ； ； in, For average area, The standard deviation of the area. The number of a certain organelle within a cell. The area of ​​each organelle; S23: Combine the fluorescence excitation spectra of the three fluorescent proteins with the spatial distribution characteristics of the three organelles in pairs to form a spectral-spatial feature joint coding set.

4. The method for simultaneous imaging of multicellular structures in multiple cell types according to claim 3, characterized in that: Step S3 further includes the following steps: S31: Label cells according to the coding combination in step S23, mix and culture different types of cells, and simultaneously acquire multi-band spectral images of the mixed samples under the same field of view; S32: At the excitation wavelength , , , The mixed fluorescence images acquired under irradiation are as follows: , , , The individual images after spectral decomposition of the three fluorescent proteins are as follows: , and ; S33: In mixed samples, due to the combined contributions of different fluorescent proteins, the relationships are as follows: ； in, For the first Fluorescence excitation spectra of several fluorescent proteins; S34: Individual decomposition images of each fluorescent protein were solved using least-squares fitting. ； ； ； in, for The transpose of .

Images