Fluorescein CAS 2321-07-5

Fluorescein isothiocyanate, referred to as “FITC”. It is a small molecule fluorescein, and its efficiency depends on the pH value of the solution. Therefore, when using FITC, you should pay attention to the pH value of the solution. The molecular weight of FITC is 389.4, the maximum absorption wavelength is 490~495nm, and the maximum emission wavelength is 520~530nm, showing bright yellow-green fluorescence.

FITC can be stored in a cool, dark and dry place for many years and is currently the most widely used fluorescein. Its main advantage is that the human eye is more sensitive to yellow-green, and usually the green fluorescence in sectioned specimens is less than red.

Phycoerythrin, referred to as “PE”. The relative molecular mass is relatively large, about 240kD, and the maximum absorption peak is 564nm. When excited by a 488nm laser, its emission fluorescence peak is about 576nm, so it may cause steric hindrance to other large probes.

However, the chemical structure of PE is very stable, has high fluorescence efficiency, and is easy to combine with antibody molecules. It should be noted that PE, as a natural dye, may cause slight differences in the structure of fluorescein due to different sources, resulting in inconsistencies in its characteristics.

PI and EB. Both have the specificity to embed into and bind to base pairs of double-stranded DNA and RNA. In order to obtain specific DNA distribution, cells must be treated with RNase before staining to eliminate interference from double-stranded RNA.

PI and EB cannot enter the intact cell membrane, therefore, they can be used to detect dead and alive cells. PI and EB have similar physical and chemical properties, but the emission spectrum peak of PI is shifted to the longer wavelength direction than that of EB. Therefore, when performing dual-parameter measurement of DNA and protein, the red fluorescence of PI and the green fluorescence of FITC are easier to distinguish and measure. In addition, PI has a lower variation system (CV value) than the DNA distribution measured by EB, so PI is more widely used.

Single laser beam three-color fluorescence analysis requires single laser excitation, and the emission wavelengths of the three selected fluorophores should be different. In addition to FITC (emitting green light) and PE (emitting orange light), you should also choose phycoerythrin and cyanidinPC5 (phycoerythrin and cyanidinPC5), chlorophyll protein (peridinin chorophyll protein, PerCP) or Phycoerythrin-Texas Red (phycoerythrin and Texas Red tandem, ECD). Because these fluorophores all emit red light or deep red emission light after being excited by 488nm blue light.

1. PC5 and ECD are composed of two fluorescein molecules that are complementary in spatial structure and are covalently bonded to form one fluorescent molecule. PC5 is composed of PE and cyanidin 5, and ECD is composed of PE and Texas Red. The emission spectrum of their previous molecule coincides with the excitation light spectrum of the latter molecule. In this way, after the former molecule is excited by laser, the emitted light can directly excite the latter molecule, and finally the emission light of the latter molecule is reflected. Fluorescent properties of the entire combination. Therefore, this composition is said to be two molecules, but it exhibits the physical properties of one molecule.
2. PerCP is a pigment found in a flagellate that lives in the deep sea. Its function is to transmit falling light that can penetrate into the deep sea to the chlorophyll chromophore of the flagellate, thereby emitting red light. It should be noted that PerCP is a single molecule.
3. The excitation wavelength of the two fluorophores, allophycocyanin (APC) and cyanidin 5 (Cy5), is required to be around 630nm, and a second laser is required for excitation.