Fluorescent labeling technology: It is a technology that connects the covalent bonds of fluorescent groups to proteins, nucleic acids, etc. that can identify molecular substances. This technology reacts with specific groups of fluorescent signal substances and recognizes specific groups of molecular substances to complete the labeling process, and uses the fluorescent properties of the markers to provide the signal of the detected object. The fluorescent labeling method has advantages such as no radioactivity, simple operation, high sensitivity and good selectivity. There are many types of fluorescent markers and flexible labeling methods, which can be applied to the detection of various biological macromolecules and drugs.
Small animal in vivo fluorescence imaging technology has been more and more popular and applied at home and abroad. More and more researchers hope to use this technology to track and observe the growth of tumor cells in living animals and the response to drug treatment for a long time. The distribution and metabolism of fluorescently labeled polypeptides, antibodies, and small-molecule drugs in vivo were observed.
Compared with traditional technology, in vivo fluorescence imaging technology does not need to kill animals, and can perform long-term repeated tracking imaging of the same animal, which can not only improve the comparability of data, avoid the influence of individual differences on the test results; but also can understand the markers. The distribution and metabolism in animals avoid many shortcomings of traditional in vitro experimental methods; in particular, the original ecological method can also be used to study problems, that is, the research objects do not need to be labeled first, and then use fluorescent markers to study their behavior. The observations are true and reliable.
There are three main labeling methods for in vivo fluorescence imaging: fluorescent protein labeling, fluorescent dye labeling and quantum dot labeling. Fluorescent proteins are suitable for labeling tumor cells, viruses, genes, etc. Commonly used are GFP, EGFP, RFP (DsRed), etc. Fluorescent dye labeling is the same as in vitro labeling. Cy3, Cy5, Cy5.5 and Cy7 are commonly used, which can label antibodies, peptides, and small molecule drugs. As a new labeling method, quantum dot labeling is 20 times the emission intensity of organic fluorescent dyes, more than 100 times more stable, with narrow fluorescence emission spectrum, high quantum yield, not easy to bleach, wide excitation spectrum, color Adjustable, and high photochemical stability, not easy to decompose and many other advantages. Quantum dots are a kind of semiconductor nanocrystals that can emit fluorescence with a size of less than 100 nm. They can withstand repeated excitations and are not prone to fluorescence quenching like organic fluorescent dyes.
However, the tissue penetration power of different fluorescence wavelengths is different, and the transmittance of light of various wavelengths to various organs of mice increases significantly when the wavelength is >600nm. In the near-infrared range of 650nm-900nm, the absorption of light of these wavelengths by hemoglobin, fat and water is kept at a relatively low level.
The relatively common fluorescent labeling method is to label control samples with Cy3-dUTP (green fluorescence) and laboratory samples with Cy5-dUTP (red fluorescence).
Introduction of fluorescent dyes:
APDye Fluor 647 (Alexa Fluor 647 equivalent; AF647) is a bright green-fluorescent dye optimal for use with the 633, 650 nm Argon laser. The dye is water soluble and pH-insensitive from pH 4 to pH 10. The dye has 4 sulfonate groups make it high water soluble and less aggregation in the aqueous solution. APDye Fluor 647 is used for protein and antibody labeling, or nucleic acid applications with high labeling density.
APDye Fluor 647 is structurally similar to Alexa Fluor 647, and spectrally is almost identical to Cy5 Dye, Alexa Fluor 647, CF 647 Dye, or any other Cyanine5 based fluorescent dyes.