Introduction to basic principles and applications of bioluminescence and fluorescence imaging in living animals

Bioluminescence and fluorescence imaging in living animals
Introduction to basic principles and applications
Article directory:
First, living bioluminescence imaging technology
Second, living animal fluorescence imaging technology
Third, the comparison of bioluminescence imaging and fluorescence imaging
Fourth, living animal visible light imaging instrument principle and operation process

In vivo imaging technology of living animals refers to the technique of applying qualitative and quantitative research on the biological, biological, and biological levels of biological processes in a living state. Animal in vivo imaging technique is mainly divided into visible light imaging (optical imaging), nuclear imaging (radio-nuclear imaging), MRI (magnetic resonance imaging, MRI) and ultrasound imaging (Ultrasound) imaging, computed tomography (computed tomography, CT There are five major categories of imaging, where visible light imaging and radionuclide imaging are particularly suitable for studying molecular, metabolic, and physiological events, often referred to as functional imaging; ultrasound imaging and CT are suitable for anatomical imaging, commonly referred to as structural imaging. Compared with structural imaging, functional imaging can reflect the spatial and temporal distribution of cell or gene expression to understand related biological processes, specific gene functions and interactions in living animals. Therefore, in vivo functional imaging techniques in living animals can be used to observe and track the expression of target cells and genes, simultaneously detect multiple molecular events, optimize drug and gene therapy protocols, and observe drug efficacy at the molecular and cellular levels, from the overall animal level. Assess disease progression and track the effects of time, environment, development and treatment on the same animal. Due to the many advantages of functional imaging, this technology is widely used in life science, medical research and drug development. This paper focuses on the visible imaging technology of living animals.
In vivo imaging of visible light (optical in vivo imaging) technologies include bioluminescence (Bioluminescence) and fluorescence (Fluorescence) forming two techniques. Bioluminescence imaging is luciferase (Luciferase) cell marker gene or DNA, either to generate optical probe signal generated in vivo by proteases and their corresponding substrate biochemical reaction occurs; the fluorescence imaging is the use of a fluorescent reporter gene (such as GFP Fluorescent dyes such as RFP) or Cyt and dyes are labeled, and fluorescence generated by fluorescent proteins or dyes can be used to form fluorescent light sources in the body. The former is self-luminous in the animal body, does not need to excite the light source, can directly capture the light signal through a highly sensitive CCD, while the latter requires the excitation of the external excitation light source to capture the illuminating signal. Traditional animal experiment methods require slaughtering experimental animals at different time points to obtain data, and experimental results at multiple time points are obtained. In contrast, in vivo visible light imaging technology records the movement and changes of the same observation target (labeled cells and genes) by recording the same group of subjects at different time points, and the obtained data is more authentic. In addition, this technology has been widely used in life sciences, medical research, and drug development in the few years since it was developed because it does not involve radioactive materials and has simple operation, intuitive results, and high sensitivity.
(1) Technical principle
Marking principle
In mammalian bioluminescence, the Firefly luciferase gene (constituted by 554 amino acids, about 50KD), that is, the luciferase gene, is integrated into the chromosomal DNA of the cell to be observed to express luciferase, and the luciferase can be stably expressed. In cell lines, luciferase is also stably expressed when cells divide, metastasize, and differentiate. Genes, cells, and living animals can all be labeled with a luciferase gene. After the labeled cells are inoculated into the experimental animal, luminescence can be produced in a few minutes when exogenously (either intraperitoneally or intravenously) is administered to the substrate luciferin. This enzyme catalyzes the oxidation reaction of fluorescein in the presence of ATP and oxygen, so that luminescence is only produced in living cells, and the intensity of luminescence is linearly related to the number of labeled cells.
In addition to Firefly Luciferase, Renilla Luciferase is sometimes used. Both substrates are not the same, the former substrate is luciferin (D-luciferin), which substrate is coelentarizine. The wavelengths of the two are different. The former emits light at a wavelength of 540-600 nm, and the latter emits at a wavelength of 460-540 nm. The light emitted by the former is easier to pass through the tissue. The latter is faster in metabolism than the former, and the specificity is not as good as the former. Therefore, most living experiments use Firefly Luciferase as a reporter gene. If double labeling is required, the latter can also be used. As an alternative.
The luminescence of luciferase is bioluminescence and does not require excitation light, but requires the substrate fluorescein. Luciferin and luciferase occurs under oxygen, the presence of ATP in the reaction to produce oxidized luciferin (oxyluciferin), and generates a light emission phenomenon.
For bacterial labeling, the luciferase gene operon luxABCDE or luxCDABE is typically utilized, which consists of a controlled gene encoding a luciferase and a gene encoding a luciferase substrate synthetase. Bacteria that are labeled by this method will continue to emit light and do not require an exogenous substrate. However, general bacterial markers require the help of a transposon to insert a foreign gene into the bacterial chromosome for stable expression.
2. Characteristics of the substrate fluorescein
Fluorescein is favored by many researchers because of its many advantages. The main features are as follows:
(1) Fluorescein does not affect the normal physiological function of animals.
(2) Fluorescein is a small molecule of 280 Dalton, which is very water-soluble and fat-soluble, and easily penetrates the cell membrane and the blood-brain barrier.
(3) Fluorescein diffuses rapidly in the body and can be injected into the animal by intraperitoneal injection or tail vein injection. Intraperitoneal injection spreads slowly and continues to emit light. After about 1 minute of intraperitoneal injection of fluorescein, the cells expressing luciferase began to emit light. After 10 minutes, the intensity reached the highest point of stability. After the highest point lasted for about 20~30 min, the attenuation began. After about 3 h, the fluorescein was eliminated and the luminescence disappeared. The optimal detection time is between 15 and 35 minutes after injection; if fluorescein is injected intravenously, the diffusion is fast, but the duration of illumination is short. According to a large number of experiments, the researchers concluded that the appropriate amount of fluorescein is 150 mg/kg, that is, mice weighing 20 g require 3 mg of fluorescein.
(4) There is no minimum limit on the interval of observation time, as long as the observed conditions are consistent. Although the substrate has a certain metabolic process in the animal, the residual curve of the previous substrate can be known to control the effect on the next observation.
3. Optical principle
Light is scattered and absorbed as it travels through mammalian tissues. Photons collide when they encounter cell membranes and cytoplasm, and the characteristics of different types of cells and tissues absorb photons are not the same. Hemoglobin is a major factor in the absorption of visible light in the body, which absorbs most of the blue-green light in visible light. However, in the red light band with visible light greater than 600 nm, the absorption of hemoglobin is small. Therefore, in the reddish light region, a large amount of light can be detected through the tissue and the skin. At least a few hundred cells under the skin can be detected using live animal bioluminescence imaging technology. Of course, the minimum number of cells that can be seen due to the difference in depth of the illuminating source in the mouse is different. It is generally believed that the intensity of luminescence is attenuated by 10 times per centimeter of depth, and the tissues or organs rich in blood (such as the heart, liver, and lungs) are attenuated, and the tissue or organs adjacent to the bones are reduced. At the same depth, the detected luminescence intensity and the number of cells have a very good linear relationship, and the intensity of the detected light can be quantified by the instrument, reflecting the number of cells.
(2) Application field of living bioluminescence imaging technology
In vivo bioluminescence imaging technology is an irreplaceable technology in some fields, such as tumor metastasis research, drug development, gene therapy, stem cell tracing and so on.
1. Oncology
In vivo bioluminescence imaging enables researchers to directly and quickly measure tumor growth, metastasis, and drug response in various cancer models. It is characterized by extremely high sensitivity, so that tiny tumor lesions (as few as a few hundred cells) can be detected, which is much more sensitive than traditional methods; it is very suitable for quantitative analysis of tumor growth in vivo; avoiding slaughtering mice The resulting differences between groups; saving animal costs. Due to the above characteristics, oncology research based on metastasis model, in situ model, and spontaneous tumor model has been developed. Establish a tumor metastasis model to observe tumor metastasis and further explore the mechanism of tumor metastasis; in situ vaccination, observation of in situ and in situ metastasis models, to make oncology research closer to the microscopic environment of clinical oncology; through the establishment of spontaneous tumors The model can observe the mechanism of tumorigenesis. (Figure 11-1).
Figure 11-1 Long-term detection of tumors, the left image is 7 days, 14 days, 30 days of imaging. From the Chinese Academy of Military Medical Sciences
2. Drug research
In terms of pharmacodynamic evaluation, the luciferase cancer model can be used for cancer in vivo for long-term efficacy tracking at the overall animal level. The detection of cancer cell growth using atraumatic in vivo imaging provides real-time observation and assessment of changes in cancer cells before and during cancer treatment. This approach provides a good pre-diagnostic approach to cancer cell response and recurrence assessment. The method of in vivo imaging has higher sensitivity than the conventional technique, and when the tumor block cannot be detected by the conventional method, a strong signal can be detected by the technique. Since this technique only detects living cells, it cannot detect cells that have already undergone apoptosis. However, traditional methods cannot distinguish between normal cells and apoptotic cells, so this technique can detect the efficacy of drugs earlier and more sensitively than conventional techniques.
Using the characteristics of high sensitivity and convenient observation of in vivo imaging technology, in the preclinical study of antitumor drugs, the optimal administration of antitumor drugs can be observed by administering different doses of mice inoculated with tumors, different administration time and different administration routes. Route, dose, and time of administration to establish the appropriate dosage form and time of administration.
In terms of drug metabolism, genes related to drug metabolism, such as CYP3A4, are examined to study the effects of different drugs on the expression of the gene, so that the metabolism of related drugs in the body can be indirectly known.
In pharmacy research, the distribution of the drug carrier to the internal organs and the body can be observed by directly loading the plasmid of the luciferase reporter gene into the drug carrier (Fig. 11-2). In pharmacology, it is also possible to observe the pathway of drug action through the application of transgenic mice, label a certain gene of interest with a luciferase gene, and observe the pathway of drug action.
Figure 11-2 Screening of anti-inflammatory drugs using IL-1Î’ transgenic mice from Shanghai Southern Model Biological Research Center
3. Gene therapy
Gene therapy is to treat a normal gene or a therapeutic gene into a target cell in a certain way to correct the defect of the gene or to exert a therapeutic effect, thereby achieving the purpose of treating the disease. At present, gene therapy mainly uses a virus as a vector, and a luciferase gene can be used as a reporter gene to add a vector to observe whether the target gene reaches a specific tissue in the animal body and whether it is continuously and efficiently expressed. This non-invasive method has low toxicity and immune response. Slightly superior and can be directly observed in real time to understand the location and time domain information of the virus or vector infection; luciferase gene can also be inserted into the liposome-encapsulated DNA molecule to observe the DNA transport of the liposome as a vector. Gene therapy; the naked DNA of the plasmid expressing the luciferase gene can be directly injected into the animal, and the effect of different vectors, different injection sites and different injection amounts on the expression of luciferase gene can be analyzed by bioluminescence imaging. At the same time, the distribution, level and duration of gene expression can be quantified in time and space. This visual method intuitively evaluates the transfection efficiency and expression efficiency of DNA and plays an important guiding role in gene therapy research.
4. Stem cells and immunology
There are several methods for labeling stem cells with luciferase: one is to label the constitutively expressed genes, and the transgenic animals are made, the stem cells are labeled, and several cells are transplanted into another animal, which can be traced by living bioluminescence imaging technology. The process of proliferation, differentiation and migration of stem cells in vivo; another method is to directly label stem cells with lentivirus, transplant them into the body to observe their proliferation, differentiation and migration process, and study the effects of repairing, treating damage or defective parts, further Explore its mechanism.
By labeling immune cells, observing the recognition and killing function of immune cells on tumor cells, etc., evaluating the immune specificity, proliferation, migration and function of immune cells; by marking allogeneic cells, observing the effects of allogeneic cells on organ transplantation; Conduct some research on immune factors and so on.
5. Gene expression pattern and gene function research
Studying gene expression can be studied from different levels affecting gene expression, such as the use of fusion proteins (p27-luc fusion protein to study its expression in the Cdk cell division cycle), luciferase controlled by the promoter of interest genes (Catenin) Signaling mechanisms in tumor metastasis), siRNA methods and methods such as transgenic animals.
In order to study when and under what stimulation the target gene is expressed, the luciferase gene is inserted downstream of the promoter of the target gene, and stably integrated into the chromosome of the experimental animal to form a transgenic animal model. Through this method, the parallel expression of luciferase and the target gene can be directly observed, thereby directly observing the expression pattern of the target gene, including the quantity, time, location and factors affecting its expression and function; and can also be used to study the specificity of the animal development process. Spatiotemporal expression of genes, observation of drug-induced expression of specific genes; and expression or closure of corresponding genes caused by other biological events.
6. Protein interaction
In vivo bioluminescence imaging techniques can be used to study the interaction of proteins and proteins in living animals. The principle is to connect the C-terminus and the N-terminus of the luciferase, which are not separately illuminated when separated, to two different proteins. If there is an interaction between the two proteins, the C-terminus and the N-terminus of the luciferase will Linked together, activating luciferase transcriptional expression, bioluminescence occurs in the presence of a substrate. By studying the effects of drugs on protein interactions under in vivo conditions, the effects of living environments that cannot be simulated in in vitro experiments on protein interactions can be observed.
7. Apoptosis
Direct observation of apoptosis in living animals using live animal bioluminescence imaging techniques. The specific principle is to use a molecular biological method to bind a protein (such as estrogen) that inhibits its luminescence at both ends of a luciferase, but at the junction thereof, a caspase (an enzyme specifically expressed during apoptosis) is cleaved. point. When the cells undergo apoptosis, the caspase is expressed, and the protein that inhibits the luminescence of the luciferase is cleaved, so that the luciferase starts to emit light.
8. Disease mechanism
A gene closely related to a disease can be labeled to make a transgenic mouse, and the pathogenesis of the disease and the effect of the drug on the treatment of the disease can be inferred by a specific drug action or a change in the expression of the gene under other conditions.
9. other
Such as RNAi, protein nuclear transport and so on. At the end of the luciferase gene, the gene of the protein to be studied is connected, and the other end of the gene that is surely expressed in the nucleus, when the extra-nuclear protein is transported into the nucleus, the N-terminal and C-terminal of the luciferase are caused. Close to, restore the glow.
(1) Technical principle
1. Marking principle
There are three main methods of labeling in vivo fluorescence imaging.
(1) fluorescent protein labeling: fluorescent protein is suitable for labeling cells, viruses, genes, etc., usually using GFP, EGFP, RFP (DsRed), etc.;
(2) fluorescent dye labeling: fluorescent dye labeling and in vitro labeling methods are the same, commonly used Cy3, Cy5, Cy5.5 and Cy7, can label antibodies, peptides, small molecule drugs, etc.;
(3) the quantum dot marks: quantum dot semiconductor nanocrystal (quantum dot) capable of emitting a fluorescent, are clusters of hundreds to tens of thousands of atoms, 100nm or less in size, like the appearance of a tiny Point. As a new type of fluorescent labeling material, quantum dots have unique application advantages in long-term life activity monitoring and live tracing. Compared with the traditional organic fluorescent reagent, the quantum dot fluorescence is 20 times stronger than the emission intensity of the organic fluorescent dye, and the stability is more than 100 times. The fluorescence spectrum is narrow, the quantum yield is high, the bleaching is difficult, and the excitation spectrum is wide. The color is adjustable, and the photochemical stability is high, and it is not easy to be decomposed and the like. It is mainly used for real-time dynamic fluorescence observation and imaging of living cells, which can perform cell differentiation and lineage observation in a few days, as well as in-situ real-time dynamic tracing of interactions between cells, cells and organelles. Not only that, quantum dots can also be labeled on other substances that need to be studied, such as drugs, specific biomolecules, etc., to trace their activities and effects.
2. Optical principle
Fluorescence luminescence is the excitation of light to excite a fluorophore to a high energy state, which then produces emitted light. Similar to the penetration of bioluminescence in animals, the penetration of red light is much better than that of blue-green light in small animals. As the depth of the luminescent signal increases in the body, the wavelength is closer to 900 nm. The stronger the penetrating ability, the more the background noise can be reduced, and the near-infrared fluorescence is the best choice for observing physiological indicators. Fluorescent proteins or dyes with a longer emission wavelength should be selected as much as possible under the conditions of the experimental conditions.
(2) Application field of fluorescent imaging technology for living animals
1. Oncology
In vivo fluorescence imaging technology enables non-invasive quantitative detection of subcutaneous tumor models in mice. Compared with bioluminescence imaging technology, in vivo fluorescence imaging technology has a faster detection time, which takes less than 1 s, and does not require injection of a substrate, thereby saving the detection cost. However, it is necessary to select near-infrared fluorescence to detect deep tissues. At present, the types of fluorescent proteins in this band are limited, and accurate quantification is difficult.
(1) GFP-labeled lung tumor model (H-460-GFP)
H-460-GFP is a green fluorescent protein-expressing cell line derived from H-460 lung small cell lung cancer, stably transfected with the green fluorescent protein gene, and expressed by the SV-40 promoter.
An experimental model of mouse lung cancer was established by the H-460-GFP subcutaneous tumor model, which can be used to screen for anticancer drugs (Fig. 11-3). It can be used to measure the growth of subcutaneous tumors and monitor the response to potential chemotherapeutic drugs.
Figure 11-3 on the left is 1w fluorescence imaging after inoculation, and the right picture is 3w fluorescence imaging (for the Shanghai Cancer Institute)
(2) Quantum dot labeled cell line
Tumor cells can be labeled by quantum dots, and MDA-MB-231 breast cancer cells are labeled with quantum dot Qtracker® 705, and their growth and changes are observed dynamically after subcutaneous inoculation (Fig. 11-4). The excitation light has a wavelength of 625 nm and the scattered light has a wavelength of 680 nm.
Figure 11-4 Quantum dot-labeled tumor cells at different time for fluorescence imaging
2. antibody
One end of the molecular probe is linked to a molecular structure (such as a peptide, an enzyme substrate, a ligand, etc.) capable of binding to a specific target in the living body, and the other end is a fluorescent dye. The distribution of liver, kidney, etc. was observed by in vivo metabolism experiments of Cy5.5-labeled antibodies (Fig. 11-5).
Figure 11-5 In vivo metabolism of Cy5.5-labeled antibodies ( photo courtesy of Shanghai Cancer Institute)
3. Pharmaceutical research
Fluorescence imaging has great advantages in pharmaceutical preparation research, especially drug targeting research, and drug carrier research. Experts are designing to label small molecule drugs with appropriate fluorescent dyes to observe the specific distribution and metabolism of drugs in animals, especially in traditional Chinese medicine research.
Using a transilluminator to excite the light source from the bottom of the sample can increase the sensitivity and depth of detection of live fluorescence imaging. Figure 11-6 shows the therapeutic effect of a drug that treats Alzheimer's disease using NIR fluorescent dye-labeled amylase. The exposure time is only 200 ms, the excitation wavelength is 680 nm, and the scattered light wavelength is 720 nm.
Figure 11-6 NIR-labeled amylase imaging
4. Tissue Engineering
The construction of tissue engineering was evaluated atraumatically by developing cell lines expressing EGFP gene. EGFP-labeled tissue engineered cells were transplanted into a special scaffold in mice to observe the growth and changes of the cells, thereby judging the success or failure of tissue engineering.
(1) Advantages of bioluminescence imaging technology
Compared with fluorescence imaging technology, the main advantages of bioluminescence imaging technology are:
1. Strong specificity, no autofluorescence
The bioluminescence method using luciferase as a source of in vivo reporting is based on the specific action of an enzyme and a substrate, and is highly specific. The animal itself does not have any self-illumination, so that the bioluminescence has a very low background and a very high signal to noise ratio. However, when using the fluorescence method, when excited by the excitation light, the skin, hair, various tissues and foods in the living body will produce fluorescence, especially if the labeled target is deep inside the tissue, and high-energy excitation light is required. When it comes, it also produces a strong background noise. Although the intensity of the fluorescent signal far exceeds that of bioluminescence, the extremely low level of self-luminescence makes the signal-to-noise ratio of bioluminescence much higher than that of fluorescence.
2. High sensitivity
Many substances in the organism will also fluoresce after being excited by the excitation light, and the non-specific fluorescence generated will affect the detection sensitivity. Especially when the luminescent cells are deeply hidden inside the tissue, a higher energy excitation light source is required, and a strong background noise is also generated. Fluorescence imaging is the most sensitive and can only detect about 10 5 cells in animals. The sensitivity of cells with 10 2 orders of magnitude is much different from that of bioluminescence in animals.
3. Depth of detection
Since bioluminescence is more sensitive than fluorescence imaging, bioluminescence imaging is the best choice for studies that require deep imaging (detection depths of 3 to 4 cm), such as stem cells, in situ tumors and metastases, and spontaneous tumors.
4. Accurate quantification
The bioluminescence signal can be used for accurate quantification because the luciferase gene is stably expressed in the inserted cell chromosome, and the amount of luminescence per unit cell is very stable. Even if the labeled cells have complex localization in the animal, the relative amount of luminescent cells can be directly derived from the signal level of the animal's body surface. For fluorescence, the excitation light needs to pass through the tissue to reach the target, and the emitted light needs to come out of the body with a longer path. The signal level depends on the intensity of the excitation light, the number of luminescent cells, the depth of the target, the absorption and scattering of the light passing through the tissue, and the fluorescence intensity is more difficult to quantify. Fluorescence imaging quantification requires the instrument's excitation light to be stable for long periods of time and evenly illuminate the animal's surface. The NightOWL IILB 983 imaging system realizes the energy control and adjustment of the excitation light through the special design of the fluorescent light path, selects the appropriate excitation device according to the size and depth of the light source, and uses the narrow band filter to improve the living body fluorescence imaging. Stability and sensitivity, and the system is simple to operate, inexpensive, and does not involve radioactivity.
(B) the advantages of fluorescent imaging technology
In the visible imaging technology of living animals, the advantages of fluorescence imaging technology are mainly reflected in the bioluminescence imaging technology:
1. Strong fluorescent dye and protein labeling ability
A wide variety of fluorescent labels, including fluorescent proteins, fluorescent molecules, quantum dots, etc., can be labeled with biomolecules such as genes, polypeptides, and antibodies, and can be used as a molecular probe in a wide range. At the same time, different fluorescent proteins or dyes can be multi-labeled and imaged simultaneously. Detected wavelengths range from 300 to 1100 nm, and some instrument companies offer full-spectrum filters for in vivo imaging of virtually all fluorescent labels.
2. Strong signal strength
Since fluorescence is an energy transfer phenomenon generated by excitation of an external light source, its photon intensity is stronger than other optical signals, and its duration is long. The amount of sample information reflected by the signal is more abundant, and the requirements for the signal receiving instrument are relatively low. It is necessary to have a low temperature cold CCD (such as absolute temperature <-80 o C) to save on experimental costs and acquisition costs.
3. Low cost of experiment
Compared to in vivo bioluminescence imaging, fluorescence imaging is inexpensive and requires no injection of the substrate fluorescein. The fluorescent luminescent group can emit a certain wavelength of the emitted light signal as long as it is excited by the excitation light of a suitable intensity, and the entire reaction does not need to inject any expensive reaction components into the animal, and as long as the fluorophore is stabilized, the excitation can be performed at any time. The effect of light.
4. Live animals, animal carcasses, and organs can all be imaged.
Since fluorescence is based on the principle of physical energy transfer, the physiological state of the experimental sample is relatively low, and optical imaging of living, cadaver, and autopsy tissue and organ samples can be achieved. For bioluminescence, luminescence is only produced in living cells.
In short, how bioluminescence and fluorescence technology complement each other, complement each other and meet different research fields, and the future development direction is equal to both technologies. For different studies, the appropriate method can be selected according to the characteristics of the two and the experimental requirements (Table 11-1).
Table 11-1 Comparison of bioluminescence and fluorescence characteristics
        
         excellent      point
lack     point
Bioluminescence
Strong specificity, no autofluorescence
High sensitivity, hundreds of cells can be detected in the body
Detected depth is 3-4 cm
Precise quantification
The signal is weak, the detection time is long, and a sensitive CCD lens is required, and the precision of the instrument is high;
Need to inject fluorescein, the experiment cost is high;
Cells or genes require transgenic markers;
Some substances cannot be labeled with bioluminescence, such as antibodies, peptides, etc.
Hard to use in the human body.
Fluorescence
Fluorescent dyes, protein labeling ability, a variety of proteins and dyes can be used for multiple markers;
High signal intensity and fast imaging speed;
Low experimental cost;
Live animals, animal carcasses, and organs can all be imaged;
Can be linked to in vivo and in vitro experiments to maintain consistency in the study;
The future may be used in the human body.
Non-specific fluorescence limits sensitivity and detects at least about 10 5 cells in vivo;
The depth of detection is limited;
It is more difficult to accurately quantify in vivo.
(1) Instrument principle
Take the NightOWL IILB 983 as an example to illustrate the instrument design principle of the visible imaging system of living animals. The entire instrument consists of a CCD with a very good containment, fluorescent fittings, anesthesia system and software. The CCD lens is located at the upper left of the black box, the fluorescent light source and the light path are located at the upper right, and the animal platform (heatable to maintain the temperature of the experimental animal) is located below the black box, and the anesthesia system is connected to the black box through a pipe.
Choosing the right CCD lens is very important for visible light imaging in the body. The CCD lens used must have very high sensitivity and quantum efficiency for light with a wavelength of 450-700 nm, and the noise signal should be as small as possible because the light source to be detected is a few centimeters below the skin. Researchers have discovered that the back-illuminated thinned cold CCD is the only suitable choice. The temperature of the CCD chip can reach <-80 0 C. At this temperature, the dark current and reading noise of the chip drop to an almost negligible level, and the dark box with very good tightness makes the system detect bioluminescence and Fluorescence has unparalleled sensitivity. The CCD is controlled by software to raise and lower, auto focus, and a continuous field of view from 3.5 cm to 25 cm.
The imaging dark box shields the cosmic rays and all the light sources, so that the inside of the dark box can be completely dark. The light detected by the CCD is completely emitted from the animal to be inspected to avoid light pollution from the external environment.
In the design of fluorescent accessories, the light source uses a 75W tungsten halogen lamp. The system first realizes that the fluorescence excitation light source energy can be adjusted from 0% to 100%, and the excitation energy is controlled by the optical fiber feedback to maintain stability during the detection time. Quantitative accuracy. In addition, by using a uniform illumination excitation device, a narrow-band filter, etc., it is ensured that the fluorescence imaging can better remove the background noise, obtain clear detection results, and accurately quantify the data.
In order to perform visible light imaging on experimental animals, the experimental animals are anesthetized to obtain the desired viewing angle and stable data. For bioluminescence imaging, gas anesthesia is generally recommended due to the long time of detection. The gas anesthesia system consists of a gas evaporator, an induction anesthesia box, a flow regulating valve, a five-channel mouse anesthesia chamber with separate control switches, and an exhaust gas absorption device. Before imaging, the experimental animals were placed in an induction anesthesia box and anesthetized, and then placed in an imaging dark box for observation.
The software system is responsible for instrument control and image analysis. The software controls the focal length of the lens, the lifting and lowering of the CCD, the exposure time, the replacement of the filter and the opening of the light, etc., with a friendly user interface and easy operation.
(two) experimental operation process
Cell marker or animal marker
For bioluminescence experiments, first, tumor cells, stem cells, viruses, drug carriers or animals are labeled with a luciferase gene, or the bacteria are labeled with a Lux operon, depending on the experimental content. Labeling with a luciferase gene can be carried out by a method such as a plasmid, a lentivirus or a retrovirus.
If a fluorescent experiment is performed, tumor cells, stem cells, viruses, or animals are labeled with GFP, EGFP, or RFP, and the latter uses fluorescent dyes (including quantum dots) to label substances to be detected, such as antibodies, drug carriers, and the like.
2. In vitro pre-experimental testing
After labeling cells, viruses, bacteria, etc., which need to be detected, before the in vivo imaging, the label can be pre-tested for the success of the label, the expression intensity of luciferase or fluorescent protein, and the positive clones and marker markers can be screened accordingly. Luminous gradient curve, etc. In vitro pre-experiment is an integral part of a small animal in vivo imaging solution. For example, an in vitro pre-experiment is used to initially detect the luminescence value of tumor cells transfected with luciferase, and a batch of cells with a relatively high transfection rate and stability is selected for in vivo experiments. For the usual cell detection, the luminescence intensity of the living cells can be specifically detected by a living imaging device, and the luminescence intensity of the cell lysate can also be detected by an in vitro chemiluminescence and a fluorescence detector.
When an in vitro chemiluminescence and fluorescence detector is used, the luminescence value and the more stable output luminescence value can be more accurately captured. For bioluminescence, the instrument required for the experiment is a microplate chemiluminescence detector such as Berthold Centro LB 960. For fluorescence mode, the experimental apparatus is required microplate fluorescence detector, such as Berthold Twinkle TM LB 970. (Figure 11-7)
Figure 11-7 on the left is Berthold Centro LB 960 and on the right is Berthold TwinkleTM LB 970
3. Living imaging
The experimental animals are anesthetized first, and the experimental animals can be anesthetized with a mixture of isoflurane or ketamine/xylazine. If gas anesthesia is used, the substrate can be injected first. The advantage of gas anesthesia is that the animal can quickly enter the anesthesia state, and once the anesthetic gas supply is stopped, the animal will wake up within a few minutes.
For bioluminescence experiments, the substrate fluorescein was then injected. The optimal detection time is between 15 and 35 minutes after the injection. However, it should be noted that for different animal models, the luminescence dynamics process is not completely consistent. It is best to conduct a preliminary experiment to determine when the luminescence signal is the strongest.
Imaging, for bioluminescence, the detection time is generally 1 minute to 5 minutes, if the signal is particularly weak, it can be extended to 10 minutes, if the signal is particularly strong, it can also be within 1 minute. For fluorescence, the detection time is within 1 second. To save time and detect bioluminescence, up to 5 mice can be tested simultaneously. For fluorescence experiments, anesthesia is good and can be detected immediately. Since the angle of the excitation light affects the detected signal value, the fluorescence experiment recommends that only one animal be tested at a time.
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Operation Room Equipment

Yingmed offered all of the basic equipment in operation room, from Surgical operation table, Electronic Obstetric Bed , Orthopedics Tractor Rack, Chair -Mounted Dental Unit, Shadowless Operation Lamps to medical machine like X-Ray Machine , Suction Machine , Stomach Cleaning Machine.

Other related devices which may be stored in operation room like ECG Machine , Patient monitoring, Disposable Gynecological Examination, Fetal Doppler will be found in the category of Yingmed Medical Instruments .

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