Visible light in vivo imaging technology includes two technologies: bioluminescence and fluorescence.
Bioluminescence is labeled with luciferase (Luciferase) gene or DNA, while fluorescence technique is labeled with fluorescent reporter groups (GFP, RFP,Cyt, dyes, etc.).
Sensitive optical detection instruments are needed to allow researchers to directly monitor cellular activity and gene behavior in living organisms.
Application
Through this system, biological processes such as the growth and metastasis of tumors, the development of infectious diseases and the expression of specific genes in living animals can be observed. Traditional animal experimental methods need to kill experimental animals at different time points to obtain data and get experimental results at multiple time points. In contrast, by recording the same group of subjects at different time points and tracking the movement and changes of the same observation target (labeled cells and genes), the data obtained by visible light imaging is more real and reliable.
In addition, this technology has extremely high sensitivity for detecting tumor micro-metastasis, does not involve radioactive substances and methods, and is very safe. Because of its extremely simple operation, intuitive results, and high sensitivity, it has been widely used in life sciences, medical research, and pharmaceutical development within a few years of its development.
Optical principle
Light is scattered and absorbed when propagating within mammalian tissues. Photons are refracted when they encounter cell membranes and cytoplasm. Moreover, different types of cells and tissues have different photon absorption characteristics.
In the reddish light region, a large amount of light can pass through tissue and skin and be detected.At the same depth, there is a very good linear relationship between the detected luminescence intensity and the number of cells. The basic principle of visible light in vivo imaging technology is that light can penetrate the tissue of experimental animals and the detected light intensity can be quantified by the instrument, while reflecting the number of cells.
Experimental process
Through the molecular biological cloning technology and the screening of monoclonal cell technology, the luciferase gene was stably integrated into the chromosomes of the expected cells, and the cell lines that could stably express luciferase protein were cultured.
Typical imaging process: mice are anesthetized and put into the imaging dark box platform, the software controls the platform to rise and fall to a suitable field of vision, and automatically turns on the lights to take the first background picture.
Next, the lights are automatically turned off and the light emitted by the mouse is photographed without an external light source, which is called bioluminescence imaging.Superimposed with the first background image, the position of the light source in the animal can be clearly displayed and the imaging operation can be completed.After that, the software completes the image analysis process. Users can easily select the area of interest for measurement and data processing and preservation work.When the area to be measured is selected, the software can calculate the number of photons emitted from this area and obtain the experimental data. The data processing and saving function of the software is very powerful, which can speed up the experiment and facilitate mass experiments.
Fluorescence
Fluorescent luminescence is through the excitation of light to excite the fluorescent groups to reach a high energy state, and then produce emitted light. Green fluorescent protein (GFP), red fluorescent protein DsRed and other fluorescence reporter groups are commonly used, and the labeling method is similar to that of in vitro 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 much better than that of blue-green light, and near-infrared fluorescence is the best choice for observing physiological indexes.
Bioluminescence
Although the fluorescence signal is much stronger than bioluminescence, the background noise produced by non-specific fluorescence makes its signal-to-noise ratio much 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 result in low sensitivity of fluorescence imaging.
Most of the high-level articles still use bioluminescence to study in vivo imaging of living animals. However, fluorescence imaging has the advantages of convenient, cheap, intuitive, diverse labeling targets and easy to be accepted by most researchers. it has also been used in the study of plant molecular biology and the observation of metabolism of small molecules. For different studies, appropriate methods can be chosen according to their characteristics and experimental requirements. Recently, in many experiments reported in the literature, green fluorescent protein and luciferase were used to double label cells or animals, mature fluorescence imaging techniques were used for in vitro detection, and molecular biology and cell biology studies were carried out. Then bioluminescence technology was used for in vivo detection and in vivo research.
We can provide
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