Small animal in vivo imaging overview
1. Fluorescence luminescence imaging fluorescence imaging labeling object is more extensive, can be animals, cells, microorganisms, genes, but also antibodies, drugs, nanomaterials and so on. Commonly used green fluorescent protein ( GFP ), red fluorescent protein ( DsRed ) and other fluorescent reporter group, labeling method and in vitro fluorescence imaging is similar to fluorescence imaging, fluorescence imaging has the advantages of low cost and simple operation. Similar to the penetration of bioluminescence in animals, the penetration of red light is more efficient than that of blue-green light in vivo, and near-infrared fluorescence is the best choice for imaging observation.
While the fluorescence signal is much stronger than bioluminescence, the background noise generated by non-specific fluorescence makes the signal-to-noise ratio much lower than bioluminescence. Although many companies use different techniques to separate the background light, the limitations of fluorescence properties make it difficult to completely eliminate the background noise. These background noises contribute to the low sensitivity of fluorescence imaging. Although most of the high-level articles are still applying bioluminescence to study in vivo imaging of living animals. However, fluorescence imaging has the advantages of convenience, intuition, variety of labeling targets and easy acceptance by most researchers, and has been applied in some plant molecular biology studies and observation of small molecule metabolism in vivo. For different studies, the appropriate method can be selected according to the characteristics of both and the experimental requirements. For example, the use of green fluorescent protein and luciferase for double labeling of cells or animals, in vitro detection with mature fluorescence imaging technology for molecular biology and cell biology research, and then the use of bioluminescence technology for in vivo detection for in vivo studies in live animals.
Fluorescence luminescence is the generation of emitted light by excitation of fluorescent groups to a high energy state by excitation light. Considering the different emission spectrum EX (excitation spectrum) and excitation spectrum EM (emission 8pectrum) of different fluorescent substances, corresponding excitation and emission filters are selected. The excitation and emission wavelengths of commonly used fluorescent proteins and fluorescent dyes are shown in Table 1.
Table 1 Excitation and emission wavelengths of commonly used fluorescent proteins and fluorescent dyes
2. Bioluminescence Imaging
In vivo bioluminescence imaging refers to the use of the luciferase protein produced by the expression of a reporter gene - luciferase gene - with its small-molecule substrate luciferin in the presence of oxygen and Mg2+ ions in a small mammal. The oxidation reaction occurs by consuming ATP in the presence of oxygen and Mg2+ ions, which converts part of the chemical energy into visible light energy release. An image is then formed in vitro using a sensitive CCD device. The luciferase gene can be inserted into the promoters of a variety of genes to become a reporter gene for a certain gene, and monitoring of the reporter gene and thus the target gene can be realized.
Biofluorescence is essentially a form of chemical fluorescence, and firefly luciferase in the process of oxidizing its unique substrate luciferin can release visible light photons of a wide range of wavelengths, with an average wavelength of 560 nm (460-630 nm), which includes an important red component at wavelengths over 600 nm. In mammals, hemoglobin is the main component that absorbs visible light, and it can absorb most of the visible light in the middle blue-green light band; water and lipids mainly absorb infrared light, but both of them are poorly absorbing red light to near infrared light with wavelengths of 590-800 nm, so that most of the red light with wavelengths of more than 600 nm can penetrate through the mammalian tissues, although there is some scattering depletion. The mammalian tissues can be detected by the highly sensitive CCD.
The advantages of bioluminescence imaging can be non-invasive, real-time continuous dynamic monitoring of a variety of biological processes in vivo, thereby reducing the number of experimental animals, and reduce the impact of inter-individual differences; due to low background noise, so has a high sensitivity; does not require exogenous excitation light, to avoid damage to normal cells in vivo, is conducive to long-term observation; in addition to other advantages such as non-radioactive The advantages are as follows.
However, bioluminescence also has its own shortcomings: for example, wavelength-dependent tissue penetration, light will be scattered and absorbed in mammalian tissues, and photons will be refracted when encountering cell membranes and cytoplasm, and the photon absorption properties of different types of cells and tissues are different, of which hemoglobin is the main photon absorbing material; because of the detection of signals emitted from in vitro, the signal will be affected by the light emitted from in vivo. The signal is detected in vitro, which is affected by the location and depth of the light source in vivo; in addition, it requires exogenous substrates of various luciferase enzymes, and the distribution and pharmacokinetics of the substrates in vivo also affect the signal generation; because the biochemical reactions catalyzed by luciferase enzymes require the participation of oxygen, magnesium ions and ATP, which is affected by the state of the body's environment.
Advantages of molecular imagingMolecular imaging has significant advantages over traditional in vitro imaging or cell culture. First, molecular imaging can reflect the spatial and temporal distribution of cellular or gene expression to understand relevant biological processes, specific gene functions and interactions in living animals. Secondly, since the same research individual can be repeatedly tracked and imaged for a long period of time, it can not only improve the comparability of data and avoid the influence of individual differences on the test results, but also save a large amount of scientific research costs as there is no need to kill the model animal. Third, especially in drug development, molecular imaging is of epoch-making significance. According to the current statistics, most of the drugs entering clinical research are terminated because of safety issues, resulting in a large amount of money wasted in clinical research, and the introduction of molecular imaging technology provides a broad space to solve this problem, which will enable drugs to be used in preclinical research through the use of molecular imaging methods, to obtain a more detailed molecular or genetic level of data, which can not be understood by the traditional methods. understand the field, so molecular imaging will bring a revolutionary change to the mode of new drug research. Secondly, in the process of transgenic animals, animal gene targeting or pharmaceutical research, molecular imaging can track and detect animal traits, and conduct direct observation and (quantitative) analysis of phenotypes;
Small-animal in vivo imaging
1. The production of animal models can be done according to the experimental needs of the tail-vein injection, subcutaneous transplantation, in situ transplantation, and other methods of inoculation of the labeled cells or tissues. In the modeling should carefully consider the purpose of the experiment and the choice of fluorescent markers, such as labeling fluorescence wavelength is short, the penetration efficiency is not high, modeling should not be inoculated with deep organs and observation of in vivo metastasis, but can be observed in subcutaneous tumors and anatomical organs after direct imaging. Most of the deep organs and in vivo metastases are labeled with luciferase.
2. In vivo imaging
The mice are placed into the imaging dark box platform after routine anesthesia (gas anesthesia or needle anesthesia is acceptable), and the software controls the elevation of the platform to a suitable field of view, and automatically turns on the illumination (bright field) to take the first background image. In the next step, the illumination is automatically turned off, and the specific photons emitted from the mouse body are captured in the absence of external light source (dark field). The superposition of the bright-field and dark-field background images can visualize the site and intensity of the specific photons in the animal and complete the imaging operation. It is worth noting that appropriate excitation and emission filters should be selected for fluorescence imaging, while bioluminescence requires in vivo injection of substrate to stimulate luminescence before imaging.
3. In addition to providing the imaging images containing the photon intensity scale, the data processing software for small animal in vivo imaging can also calculate and analyze the luminescence area, the total number of photons, and the relevant parameters of photon intensity for the reference of the experimenters.
4. Experimental influence factors In principle, if the pre-test shot out of the picture non-specific noise more, need to reduce the exposure time; Conversely, if the signal is too weak can be appropriate to extend the exposure time. However, the extension of the exposure time, not only increases the target signal, for the background noise there is also an amplification effect. The same batch of experiments should maintain a consistent exposure time, but also to maintain the relative position of the specimen and the morphology of the same, thus reducing the experimental error.
When performing fluorescence imaging, the experimenter can choose a black paper with low background fluorescence that is not easily reflective and place it under the animal specimen to minimize the reflective interference from the metal carrier. Many substances in the animal's body will emit fluorescence after being excited by excitation light, and the non-specific fluorescence produced will affect the detection sensitivity. Background fluorescence is mainly derived from the autofluorescence of the fur and blood, melanin in the fur is the main source of autofluorescence in the fur, and the peak wavelength of its luminescent light is around 500 a 520 nm, which has the most serious impact when utilizing the green fluorescence as an imaging object, and the non-specific fluorescence generated will affect the detection sensitivity and specificity. Animal urine or other impurities that are not removed in a timely manner will also show non-specific signals in imaging.
Due to the different image analysis software of each manufacturer, the experimental data analysis methods are also different. When the in vivo imaging system is used, the experimenter may adjust the threshold value of the signal in consideration of non-specific impurity signals, as well as the aesthetics of the imaging picture, etc. Therefore, when analyzing the number of photons of the signal or the area of the signal, the effect of the change in the threshold value on the experimental results should be considered. Correct selection of the ROI area can improve the accuracy of analyzing experimental data.
Classification
Molecular imaging techniques are mainly classified into five major categories: optical imaging, nuclide imaging, magnetic **** vibration imaging and ultrasound imaging, and CT imaging.
Outlook
Small animal in vivo imaging technology has the advantages of high sensitivity, intuition, simple operation, simultaneous observation of multiple experimental specimens, compared with PET, SPECT without radiation damage, but also has its own shortcomings, such as the absorption of photons by animal tissues, lower spatial resolution and other issues, and thus still need to be constantly improved and improved. In vivo imaging of small animals belongs to functional imaging according to the nature of imaging, how to better combine with structural imaging technology (microCT, ultrasound, etc.), so that the experimental results can not only be quantitative, but also can be accurately localized, which is one of the directions of the future development of in vivo imaging technology.