The difference between the main excitation light of the fluorescence and the main peak of the emission spectrum is called the stokes shift, and the common stokes shift does not exceed tens of nanometers. Molecules fluoresce in the experiment: First, light energy must be absorbed to make electrons transition to a higher energy level or excited state. When the electrons release part or all of the light energy, they will emit fluorescence. The lost excitation energy is non-radiative and the energy lost is lower than the energy absorbed.
There are many cyanine dyes, rhodamine, BDP, AFDye, fluorescein, near-infrared zone 1/2 dyes, fluorescent probes, fluorescently labeled proteins/sugars, etc. on the market. So what is the basis for choosing a suitable fluorescent dye:
1. To determine the suitable flow cytometer for the fluorescent dye itself, it is necessary to know the excitation wavelength and emission wavelength of the dye, the detection filter and the excitation light source prepared by the instrument.
2. Understand the relative fluorescence intensity of fluorescein: the relative fluorescence intensity reflects the brightness of the fluorescein-mAb conjugate, and its judgment standard is the staining index.
Factors that affect the relative fluorescence intensity include: different laser and filter combinations of different instruments, and the relative fluorescence intensity of fluorescein is different; the number of fluorescein molecules on the antibody will affect the relative fluorescence intensity.
3. Selection principle:
(1) Consider the density of antigens: for high-density expressed antigens, almost all fluoresceins can be considered; for low-density expressed antigens, fluoresceins that can provide a higher S/N ratio, such as PE/APC, are required.
(2) Autofluorescence: Each cell population has different levels of autofluorescence, and all fluorescence channels can observe autofluorescence, but the intensity of autofluorescence decreases rapidly at longer wavelengths. For cells with strong autofluorescence, choose fluorescein (APC) with long emission wavelength to get a better S/N ratio. For cells with weak autofluorescence intensity, fluorescein with long emission wavelength has no obvious effect on S/N improvement. Improvement, FITC can be selected.
(3) Non-specific binding complex dyes: avoid signal degradation. The complex dye will cause signal decay due to light, fixative or temperature rise, making the complex dye emit light at the upper dye.
This problem can be largely avoided by reducing sample exposure to light, heat, or fixatives. If the sample needs to be fixed, a stable fixative should be selected, which can effectively prevent the signal decay of the complex dye. Minimize the interference between signals. The more colors you detect, the more signal interference you face. When selecting reagents, minimize the overlap between fluorescence emission wavelengths.
APDye Fluors is the equivalent fluorescent dye of alexa fluors developed by axispharm. APDye Fluors series fluorescent dyes are bright in color, optically stable, very good in light stability of dyeing brightness, and very competitive in price. It can be applied to the labeling and localization of tissues, cells and biomolecules in biomedical research. Its excitation and emission spectra cover most of the visible and part of the infrared spectral region and are suitable for most fluorescence microscopes.
You may be interested in these products provided by Axispharm:
Af647, Cy7, Rhodamine dye, Rox dye, Tamra fluorophore, etc.