In vivo imaging technique refers to the technology of using imaging methods and a set of very sensitive optical detection instruments to conduct qualitative and quantitative research on biological processes at the cellular and molecular levels in living bodies without causing harm to experimental animals. Experimenters can use this technology to non-invasively and intuitively observe biological processes such as tumor growth, metastasis, disease development, and gene expression changes in living animals, and track and observe various biological behaviors of the same experimental subject at different time points. Due to its extremely simple operation, intuitive results, high sensitivity, and low experimental cost, it has been widely used in life sciences, medical research, and drug development.
1. Experimental principle
1.1 Optical principle
When light propagates in mammalian tissues, it will be scattered and absorbed. When photons encounter cell membranes and cytoplasm, refraction will occur, and different types of cells and tissues have different characteristics of absorbing photons. In the reddish light region, a large amount of light can pass through tissues and skin and be detected. At the same depth, the detected luminous intensity and the number of cells have a very good linear relationship. The basic principle of visible light in vivo imaging technology is that light can penetrate the tissues of experimental animals and the light intensity can be quantified by the instrument, which also reflects the number of cells.
1.2 Labeling principle
Currently, in vivo imaging technology mainly uses two technologies: bioluminescence and fluorescence.
Bioluminescence technology is to label cells or DNA with luciferase genes in mammals, that is, to integrate luciferase genes into cell chromosome DNA to express luciferase. When the substrate luciferin is given exogenously (intraperitoneally or intravenously), luminescence can be generated within a few minutes. This enzyme can only emit light by catalyzing the oxidation reaction of luciferin in the presence of ATP and oxygen. Therefore, luminescence can only be generated in living cells, and the intensity of light is linearly related to the number of labeled cells.
Genes, cells and living animals can be labeled with luciferase genes. The method of labeling cells is basically to insert the luciferase gene into the chromosome of the expected cell through molecular biological cloning technology, and to cultivate cell lines that can stably express luciferase through screening of monoclonal cell technology. After the labeled cells are injected into the mouse, the substrate of luciferase, luciferin, needs to be injected before observation. Luciferin is very lipid-soluble and can easily pass through the blood-brain barrier. One injection of luciferin can keep the luciferase-labeled cells in the mouse glowing for 30-45 minutes. Each luciferase-catalyzed reaction only produces one photon. These photons can be observed and recorded using a bio-optical molecular imaging system, a highly sensitive cooled CCD camera, a specially designed imaging darkroom, and imaging software.
Fluorescence technology is a method of labeling cells or proteins with fluorescent proteins (such as green fluorescent protein GFP, red fluorescent protein DsRed and other fluorescent reporter groups), stimulating the fluorescent group to a high energy state through excitation light, and generating fluorescence after absorbing the excitation light to form a biological light source in the body, and detecting the emitted light through a highly sensitive instrument.
2. Experimental steps
2.1 Cell labeling
2.1.1 Amplification and purification of plasmids
Prepare eukaryotic expression plasmids with luciferase reporter genes or genes encoding fluorescent proteins and amplify and purify them.
2.1.2 Cell transfection
Take the target cells in the logarithmic growth period, inoculate the cells in a 6-well plate, and wait until the cell fusion reaches 80% to 90% in the well culture plate, and then TM can be transfected. When transfecting, the target cells, liposome transfection reagent and sufficient plasmid vector suspension are co-cultured for 6 hours, and then fresh culture medium is added.
2.1.3 Screening of monoclonal cells
48 hours after transfection, cells were digested with trypsin and inoculated into 6-well plates at a ratio of 1:6. Antibiotic G418 was added at the same time. The culture medium was then replaced every 2 days and G418 screening was maintained until a single cell resistant clone appeared. Single resistant clones were selected and transferred to 96-well plates respectively. After they gradually proliferated, they were transferred to 24-well plates for continued subculture.
2.1.4 Identification of positive clones by luciferase activity and screening of stable and highly expressed cell lines
Luciferase activity was detected by Luciferase As-say Aystem when the single resistant clone was subcultured to the fifth generation. During the test, each clone was inoculated into a 24-well plate at 1×105 cells/well. After 24 hours, the cells were lysed with cell lysate and centrifuged at 12000r and 4℃ for 10 minutes. The lysate was collected and 10μl of the supernatant was added to a 96-well white plate. 50μl of luciferase was added to each well. The substrate was continuously read by 96microplateluminometer. After 2s, the fluorescence value (RLU) of 10s was taken. Three replicate wells were set for each clone. Cell clones with high RLU values were retained for continued subculture. After another 5 generations, luciferase activity was detected. The clones with high RLU values were retained until the 30th generation. The clones with the highest luciferase activity, MCF-7-luc, were positive clones. Cell lines with high expression and a positive rate close to 100% were screened for culture.
2.2 Construction of animal model
Choose tail vein injection, subcutaneous transplantation, orthotopic transplantation and other methods to inoculate labeled cells according to the purpose of the experiment.
2.3 In vivo imaging
2.3.1 After the mouse is anesthetized by the anesthesia system, it is placed on the imaging dark box platform. The software controls the platform to rise and fall to a suitable field of view, and automatically turns on the lighting to take the first background image.
2.3.2 Automatically turn off the lighting. In the absence of external light sources, the light emitted by the mouse is photographed, which is bioluminescence imaging. After superimposing with the first background image, the position of the light source in the animal body can be clearly displayed to complete the imaging operation. Fluorescence imaging should select appropriate excitation and emission filters, and bioluminescence requires the injection of substrates in vivo before imaging to excite luminescence.
2.3.3 Use software to complete the image analysis process. Users can easily select the area of interest for measurement, data processing and storage. After selecting the area to be measured, the software can calculate the number of photons emitted by this area and obtain experimental data.
3. Example of experimental results
Bioluminescence imaging was used to non-invasively measure bioluminescence in the brain and spinal cord at different time points in experimental autoimmune encephalomyelitis (EAE) model mice, and the mice were killed at different time points to evaluate clinical and pathological changes. Pearson correlation analysis was used to statistically analyze the correlation between bioluminescence and clinical pathological EAE.
4. References
[1] Luo, J. , Ho, P. , Steinman, L. , & Wyss-Coray, T. . (2008). Bioluminescence in vivo imaging of autoimmune encephalomyelitis predicts disease. Journal of Neuroinflammation,5,1(2008-02-01), 5(1), 6.
[2] Syed, A. J. , & Anderson, J. C. . (2021). Applications of bioluminescence in biotechnology and beyond. Chemical Society Reviews, 50,5668.
[3] Pogue, B. W. , Zhang, R. , Cao, X. , Jia, J. M. , & Vinogradov, S. A. . (2021). Review of in vivo optical molecular imaging and sensing from x-ray excitation. Journal of Biomedical Optics, 26(1).