Application of Quantitative Imaging Flow Cytometry in Cardiovascular Research
"One in every five adults in China has one cardiovascular disease, and one in 10 seconds died of cardiovascular disease." Cardiovascular disease ranks first among the deaths of urban and rural residents in China. Therefore, it can be seen that the prevention and treatment of cardiovascular diseases is the research focus of future clinical and scientific research. The concept of translational medicine has promoted clinical practice to propose new propositions to basic research. Basic research proposes possible solutions for clinical verification and mutual transformation. At present, the research of translational medicine in China focuses on cardiovascular diseases and tumors, focusing on the pathogenesis of diseases, early non-invasive diagnosis of diseases, standardized treatment of diseases and development of new drugs and new technologies. It is an important direction for cardiovascular disease to establish an in vitro disease model at the cellular level to study related pathogenesis and treatment. Flow cytometry and microscopy are two traditional methods of cell-level research. Using flow cytometry, researchers can analyze tens of thousands of cells, obtain the relative size, granularity, and fluorescence signal values ​​of each cell, thereby obtaining various statistical data of the cell population and screening out rare cell subpopulations. However, the traditional flow cytometry technology has certain limitations, and high-throughput is obtained while ignoring the rich information carried by the cells. The researchers only got a point on the scatter plot, not the real cell image, lacking information about cell morphology, subcellular organ structure, and spatial distribution of fluorescent signals. In order to obtain data based on cell images, researchers must use a variety of microscopic imaging equipment to observe, but the number of cells that can be observed by the microscope is very limited, it is easy to miss rare events, and manual analysis of data takes a lot of manpower and time, and Influenced by the subjective factors of the experimental personnel, the stability of the experimental results is very poor, and it is difficult to provide accurate cell population quantification and statistical data. Therefore, the emergence of quantitative imaging flow cytometry ( ImageStream ) combines the high content of flow detection with the high content of fluorescence microscopy, while providing cell image and population statistics, which has brought significant changes to traditional cell analysis. In addition to the traditional techniques of cell analysis and sorting, the development direction of “imaging flow†next-generation expert flow cell technology has been pioneered.
Cardiovascular and cerebrovascular studies often involve the separation and analysis of stem cells, precursor cells, etc. in in situ tissues. It is difficult to accurately identify cells using only conventional flow analysis methods. Fluorescence microscopy is limited by flux and it is difficult to capture rare phenomena. The advantages of quantitative imaging flow cytometry are that high-power lasers and 785nm SSC-specific lasers are suitable for analyzing low-level cells, combining the high-throughput features of quantitative imaging high content and traditional flow analysis to accurately display the morphological characteristics of rare cells. The biological function of the cell population can be further confirmed by binding to the cell surface and internal markers. Very small embryonic stem cells (VSELs) are a very small and very rare pluripotent stem cell found in human blood and bone marrow. They are considered to have the potential to replace embryonic stem cells, and they are highly valued in the scientific community. However, does this cell really exist? A cardiovascular disease research team from the University of Kentucky used ImageStream to find evidence of migration of VSELs to peripheral blood in patients with myocardial ischemia (Figure 1). The proportion of cells in the bone marrow is only 0.01%, expressing CXCR4+, SSEA+, Oct4+, and Nanog+. . The researchers accurately learned that the diameter of VSELs is about 3.6 microns, while the diameter of HSCs for hematopoietic stem cells is larger, about 6.5 microns. For the nuclear/mass ratio analysis, the nuclear/mass ratio of VSELs was significantly higher than that of HSCs, and the cytoplasmic region was significantly smaller than HSCs. When tissue is damaged (such as the myocardial infarction confirmed by the study), VSELs may be released from the bone marrow into the blood circulation to participate in the damage repair process. The release of damaged tissue inflammatory factors or chemokines also affects the homing of VSELs to damaged areas or other organs. Similarly, ImageStream can be applied to study myocardial tissue stem cell research, combined with high-throughput and morphological identification to study activation of signaling pathways.
Figure 1: Minimal embryonic stem cell VSELs in patients with myocardial atrophy migrate to peripheral blood. Lin/CD45 is a marker for hematopoietic stem cell HSCs, Oct4 and CD34 are markers of minimal embryonic stem cell VSELs, and 7-AAD labeled nuclei. This study used the ImageStream system to detect VSELs, showing brightfield images and biomarker fluorescence imaging. It was found that Oct4 can colocalize with the nucleus . Source: Evidence of Mobilization of Pluripotent Stem Cells into Peripheral Blood of Patients with Myocardial Ischemia . Exp Hematol. 2010 Dec; 38(12): 1131–1142.e1.
Autophagy is a highly conserved cell degradation process that utilizes lysosomes to decompose their organelles and recycle the resulting macromolecular substances. Hunger, ischemia, oxidative stress, etc. can induce it. Regulation of autophagy is also associated with cardiovascular disease, including cardiac hypertrophy, ischemic heart disease, heart failure, and ischemia-reperfusion injury. Normal autophagy has a protective effect on cardiomyocytes, and insufficient autophagy or autophagy can promote disease or aggravate lesions . Quantitative imaging analysis techniques can (1) track changes in autophagy and autophagosomes with a variety of cell surfaces and internal markers, (2) colocalization of autophagy and lysosomes, and (3) study of autophagy Protein-receptor interaction, and can be combined with a variety of application modules for quantitative analysis (4) the relationship between autophagy and apoptosis, and the upstream and downstream signaling molecules involved. The research results of ImageStream technology in autophagy have been published in high-level professional journals such as Science, Oncogene, Autophage and Journal of Immunology.
Based on traditional flow cytometry, ImageStream technology combines fluorescence microscopy imaging with 12 detection channels to image every cell in the flow and achieve various morphologies of cell images. Quantitative analysis of parameters to obtain new cell morphology statistics. ImageStream technology is very similar to traditional flow cytometry. Its system platform is also composed of three parts: liquid flow system, optical system and detection system. The flow system injects the sample cell suspension and the system sheath into the flow chamber through a syringe pump, and the cells are focused on the center of the flow under the constraint of the sheath flow, flowing through the detection window one by one. The light source in the optical system illuminates the cells passing through the detection window to produce an optical signal. There are two types of light sources, one for producing brightfield cell images and the other for generating fluorescent cell images. A unique, custom all-solid-state laser that is high-powered and adjustable to facilitate simultaneous detection of multiple fluorescent or weak signals. The system has a unique 785 nm laser for detecting lateral angle (SSC) parameters, greatly improving the detection sensitivity of this parameter. The light signal generated by the light source illuminating the cells is collected by an objective lens of large numerical aperture and then transmitted through an optical path system to a filter stack composed of dichroic mirrors. Here, the optical signal is split into different bands to be projected onto the corresponding detection channels of the CCD, producing a bright field cell image, a dark field cell image, and a cell image of a plurality of fluorescent channels, that is, each cell can acquire 12 different images. The detection system of ImageStream technology is very unique. It adopts the PMT detection method which is not the traditional flow cytometer, but the CCD (TDI (time delay integration) CCD) acquisition based on time delay integration technology, which ensures the system's high-speed movement of fluid cells. Ability to capture high quality images.
Figure 2: Systemic lupus erythematosus SLE patients have an increased degree of autophagy in immune cells compared with the normal population. Green fluorescently labeled LC3 autophagy, in the case of High high autophagy, LC3 aggregates. CD19, CD4, and CD14 mark B cells, T cells, and monocytes, respectively. Source: Autophagy is activated in systemic lupuserythematosus and required for plasmablast development. Ann Rheum Dis. 2015 May; 74(5): 912–920.
The ImageStream system is equipped with the powerful data analysis software IDEAS®, which enables over 100 quantitative parameter analysis of each cell's graphics. These parameters include not only the scattered light and fluorescence signal intensity of the cell as a whole, but also the analysis of cell morphology, cell structure and subcellular signal distribution. Click on the point on the scatter plot to visually see the image of the cell represented by this point. It has been widely used in typical applications such as cell signal transduction, cell colocalization, cell morphology change, intercellular interaction, and autophagy, thus improving the ease of use of the software. In addition, users can customize the parameters according to the special needs of their own research, and conduct more in-depth analysis. Therefore, scientists can apply it to basic life science research, such as immunology, biochemistry, transcriptomics, etc., and can be used to understand the pathogenesis of certain diseases. Since 2005, more than 500 peer-reviewed articles have been published using quantitative imaging flow cytometry. Many of the articles have been published in top journals such as Science, Nature, and Immunity, indicating that the technology has been obtained by the research community. Widely affirmed.
With the continuous application of ImageStream technology, scientists have expanded a large number of flexible and novel applications. For example, quantitative imaging flow technology can identify 20nm diameter particles, which is very suitable for analyzing circulating microparticles and microvesicles involved in cardiovascular diseases; detecting platelet aggregation by multicolor markers, assessing thrombosis; and quantifying the effectiveness of nanotargeted drugs Wait. It is believed that the next generation of expert-level flow will definitely contribute to the research of cardiovascular disease refinement.
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