fluorescence Principle and Dyes

Principle of Fluorescence

Fluorescent dyes have wavelengths of light absorbed by each dye.
If you shine light in the corresponding wavelength range of light, you can excite electrons in fluorescent dye.
Excited electrons go to a higher energy state, an excited state, and then go back to a lower energy state, a ground state.
At this time, the energy emitted is emitted in the form of photons.
Through this, the fluorescent dye emits unique fluorescence that suits it, and the emitted fluorescence allows it to observe whether the protein is expressed.


Light emission methods include 'fluorescence' and 'phosphorescence'.
Both are phenomena that emit light again when absorbed, but it varies depending on whether there is afterglow.
Fluorescence absorbs light and emits it immediately, so removing light leaves no afterglow,
Phosphorescence does not immediately emit absorbed light, so there is a difference in that afterglow continues to remain.
Also, when fluorescent dyes absorb light and emit it again, they emit light from short to long wavelengths.
This wavelength shift is called 'stokes shift'.

The data sheet of the product labeled with fluorescent dye shows the excitation and emission wavelength of the labeled fluorescent dye.
Excitation refers to the wavelength of light that stimulates fluorescent dyes, and Emission refers to the wavelength of light emitted by fluorescent dyes.

There are many fluorescent dyes, but there are three common fluorescent dyes (FITC, TRITC, DAPI).
In general, when classifying fluorescent dyes with blue and green wavelengths, FITC series,
When classifying dyes with red wavelengths, they're also called the TRITC family.


1) FITC(Fluorescein Isothiocyanate)

FITC is a fluorescent dye that's used in the ultraviolet and blue light ranges because it has a broad spectrum, as you can tell from the wavelength range.
It mainly emits bright green light fluorescence, and it is widely used as a fluorescent marker for target proteins because it is visible when observing a fluorescence microscope.


2) TRITC(Tetramethylrhodamine)

TRITC or rhodamine absorbs green light and emits organoleptic to red light fluorescence.
In some cases, it is used alone, but it is mainly used with FITC when you want to identify different antigens in double staining.
In addition, when performing fluorescent antibody methods in cells, Actin filament (fine fiber) is counterstained to understand the structure well.
It is used by combining Rhodamine-based fluorescent dye with "Phalloidin," a substance used at this time.


3) DAPI(4′,6-diamidino-2-phenylindole)

DAPI is a fluorescent dye that stains DNA (binding to adenine and thymine) in cells by nuclear counterstaining.
It emits blue light fluorescence and counterstaining allows you to approximate the location of tissues and cells.