How do you feel when you see an experimental animal locked in an animal husbandry hospital?

Overview of small animal in vivo imaging

1. Fluorescent imaging Fluorescent imaging has a wide range of labeling objects, which can be animals, cells, microorganisms, genes, antibodies, drugs, nanomaterials, etc. Green fluorescent protein (GFP), red fluorescent protein (DsRed) and other fluorescent reporter groups are commonly used. The labeling method is similar to in vitro fluorescence imaging, and fluorescence imaging has the advantages of low cost and simple operation. Similar to the penetration of bioluminescence in animals, the penetration efficiency of red light in vivo is higher than that of blue-green light, and near infrared fluorescence is the best choice for imaging observation.

Although the fluorescence signal is far stronger than bioluminescence, the background noise caused by non-specific fluorescence makes its signal-to-noise ratio far lower than bioluminescence. Although many companies use different technologies to separate background light, it is difficult to completely eliminate background noise due to the limitation of fluorescence characteristics. These background noises cause low sensitivity of fluorescence imaging. Although at present, most high-level articles still use bioluminescence to study in vivo imaging of living animals. However, fluorescence imaging has the advantages of convenience, intuition, diverse labeling targets and easy acceptance by most researchers, and has also been applied in some plant molecular biology research and observation of metabolism of small molecules in vivo. For different research, we can choose the appropriate method according to their characteristics and experimental requirements. For example, using green fluorescent protein and luciferase to double label cells or animals, using mature fluorescence imaging technology for in vitro detection, molecular biology and cell biology research, and then using bioluminescence technology for in vivo detection of animals and in vivo research of living animals.

Fluorescent luminescence is to excite fluorescent groups to reach a high-energy state through excitation light, and then generate emission light. Considering the difference of emission spectrum EX(excitation spectrum) and excitation spectrum EM (emission 8 spectrum) of different fluorescent substances, the corresponding excitation and emission filters should be selected. The excitation and emission wavelengths of common fluorescent proteins and dyes are shown in Table 1.

table 1 excitation and emission wavelengths of common fluorescent proteins and dyes

2. bioluminescence imaging

In vivo bioluminescence imaging technology refers to the oxidation reaction of luciferase protein produced by the expression of reporter gene-luciferase gene and its small molecular substrate fluorescein in the presence of oxygen and Mg2+ ions, which converts part of chemical energy into visible light energy and releases it. Then an image is formed in vitro using a sensitive CCD device. Luciferase gene can be inserted into promoters of various genes to become a reporter gene of a certain gene, and the target gene can be monitored by monitoring the reporter gene.

Biofluorescence is essentially a kind of chemical fluorescence. Firefly luciferase can release visible light photons with a wide range of wavelengths in the process of oxidizing its unique substrate fluorescein, with an average wavelength of 56 nm (46-63 nm), including important red light components with wavelengths exceeding 6 nm. In mammals, hemoglobin is the main component of absorbing visible light, which can absorb most of the visible light in the blue-green band; Water and lipids mainly absorb infrared rays, but both of them have poor absorption ability for red light to near infrared rays with wavelength of 59—8 nm. Therefore, although some red light with wavelength over 6 nm is scattered and consumed, most of it can penetrate mammalian tissues and be detected by highly sensitive CCD.

bioluminescence imaging has the advantages of non-invasive, real-time and continuous dynamic monitoring of various biological processes in vivo, thus reducing the number of experimental animals and the influence of individual differences; Because of the low background noise, it has high sensitivity; No external excitation light is needed, which avoids damage to normal cells in vivo and is beneficial to long-term observation; In addition, there are other advantages such as no radioactivity.

However, bioluminescence also has its own shortcomings: for example, wavelength-dependent tissue penetration, light will be scattered and absorbed when it propagates in mammalian tissues, and photons will be refracted when it meets cell membrane and cytoplasm, and different types of cells and tissues have different characteristics of absorbing photons, among which hemoglobin is the main substance to absorb photons; Because the signal emitted from the body is detected in vitro, it is affected by the position and depth of the light source in the body; In addition, it is necessary to provide substrates for various luciferases, and the distribution and pharmacokinetics of substrates in vivo will also affect the signal generation; Because the biochemical reaction catalyzed by luciferase needs the participation of oxygen, magnesium ions and ATP, it is affected by the environmental state in the body.

advantages of molecular imaging Compared with traditional in vitro imaging or cell culture, molecular imaging has obvious advantages. First of all, molecular imaging can reflect the spatial and temporal distribution of cell or gene expression, so as to understand the related biological processes, specific gene functions and interactions in living animals. Secondly, because the same individual can be tracked and imaged repeatedly for a long time, the comparability of data can be improved, and the influence of individual differences on the test results can be avoided, and model animal does not need to be killed, thus saving a lot of scientific research expenses. Thirdly, especially in drug development, molecular imaging is of epoch-making significance. According to the current statistical results, most drugs entering clinical research are terminated due to safety problems, which leads to a lot of money waste in clinical research. The advent of molecular imaging technology provides a broad space for solving this problem, which will enable drugs to obtain more detailed molecular or gene description data in preclinical research by using molecular imaging, which is an area that cannot be understood by traditional methods, so molecular imaging will bring revolutionary changes to the model 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 directly observe and (quantitatively) analyze phenotypes;

in vivo imaging of small animals

1. To make animal models, labeled cells or tissues can be inoculated by tail vein injection, subcutaneous transplantation, in situ transplantation and other methods according to experimental needs. When modeling, we should carefully consider the experimental purpose and choose fluorescent markers. If the fluorescence wave length is marked, the penetration efficiency is not high, and it is not suitable to inoculate deep organs and observe in vivo metastasis, but we can observe subcutaneous tumors and dissected organs for direct imaging. The observation of deep organs and metastasis in vivo is mostly marked by luciferase.

2. In vivo imaging

Mice were put into the imaging camera platform after conventional anesthesia (both gas anesthesia and acupuncture anesthesia), and the software controlled the platform to rise and fall to a suitable field of vision, and automatically turned on the lighting (bright field) to take the first background image. Next, turn off the lighting automatically, and shoot the special photons emitted by the mouse without external light source (dark field). After the background images of bright field and dark field are superimposed, the position and intensity of specific photons in animals can be displayed intuitively, and the imaging operation can be completed. It is worth noting that proper excitation and emission filters should be selected for fluorescence imaging, while bioluminescence requires in vivo injection of substrates to stimulate luminescence before imaging.

3. Data processing The image processing software for small animal in vivo imaging can not only provide imaging pictures with photon intensity scale, but also calculate and analyze the relevant parameters of luminous area, total photon number and photon intensity for the reference of experimenters.

4. experimental influencing factors: in principle, if there are many nonspecific impurities in the pictures taken in the pre-experiment, the exposure time should be reduced; Conversely, if the signal is too weak, the exposure time can be appropriately extended. But the extension of exposure time not only increases the target signal, but also has an amplification effect on the background noise. The same batch of experiments should keep consistent exposure time, and at the same time, the relative position and morphology of specimens should be consistent, so as to reduce experimental errors.

When performing fluorescence imaging, the experimenter can choose black paper with low background fluorescence and not easy to reflect light under the animal specimen to reduce the reflection interference of the metal stage. Many substances in animals will emit fluorescence after being excited by excitation light, and the non-specific fluorescence will affect the detection sensitivity. Background fluorescence mainly comes from the autofluorescence of fur and blood. Melanin in fur is the main autofluorescence source in fur, and the peak wavelength of its luminescent light is about 5-52 nm. When green fluorescence is used as the imaging object, the influence is the most serious, and the generated nonspecific fluorescence will affect the detection sensitivity and specificity. If animal urine or other impurities are not removed in time, nonspecific signals will also appear in imaging.

Because the image analysis software of different manufacturers is different, the experimental data analysis methods are also different. When the in-vivo imaging system is used, the experimenter may adjust the threshold of the signal in consideration of the nonspecific clutter and the beautiful image, so the influence of the change of the threshold on the experimental results should be considered when analyzing the signal photon number or signal area. Correct selection of ROI region can improve the accuracy of analyzing experimental data.

classification

molecular imaging techniques are mainly divided into five categories: optical imaging, radionuclide imaging, magnetic resonance imaging, ultrasonic imaging and CT imaging.

Prospect

Small animal in vivo imaging technology has the advantages of high sensitivity, intuition, simple operation, and no radiation damage compared with PET and SPECT, but it also has its own shortcomings, such as the absorption of photons by animal tissues and low spatial resolution, so it still needs to be continuously improved and improved. In-vivo imaging of small animals belongs to functional imaging according to its imaging nature. How to better combine with structural imaging technology (such as microCT and ultrasound) so that the experimental results can not only be quantified, but also accurately located is one of the future development directions of in-vivo imaging technology.