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The absorption spectrum is primarily determined [2] [3] [4] by the atomic and molecular composition of the material. Radiation is more likely to be absorbed at frequencies that match the energy difference between two quantum mechanical states of the molecules .
For measuring excitation spectra, the wavelength passing through the emission filter or monochromator is kept constant and the excitation monochromator is scanning. The excitation spectrum generally is identical to the absorption spectrum as the fluorescence intensity is proportional to the absorption. [5]
The anionic bound form of the dye which is held together by hydrophobic and ionic interactions, has an absorption spectrum maximum historically held to be at 595 nm. [5] The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.
Four protein mutations from the wild-type GFP found in Aequorea Victoria jellyfish were needed to create the YFP mutant. The most important mutation was the replacement of threonine with tyrosine at residue position 203 [1] (the substitution is denoted by T203Y, where T and Y represent the single letter code for the amino acids threonine and tyrosine, respectively).
Wavelengths of maximum absorption (≈ excitation) and emission (for example, Absorption/Emission = 485 nm/517 nm) are the typical terms used to refer to a given fluorophore, but the whole spectrum may be important to consider. The excitation wavelength spectrum may be a very narrow or broader band, or it may be all beyond a cutoff level.
Green fluorescent protein may be used as a reporter gene. [49] [50] For example, GFP can be used as a reporter for environmental toxicity levels. This protein has been shown to be an effective way to measure the toxicity levels of various chemicals including ethanol, p-formaldehyde, phenol, triclosan, and paraben. GFP is great as a reporter ...
In astronomy, the term spectrophotometry refers to the measurement of the spectrum of a celestial object in which the flux scale of the spectrum is calibrated as a function of wavelength, usually by comparison with an observation of a spectrophotometric standard star, and corrected for the absorption of light by the Earth's atmosphere.
The difference in the excitation and emission wavelengths is called the Stokes shift, and the time that an excited electron takes to emit the photon is called a lifetime. The quantum yield is an indicator of the efficiency of the dye (it is the ratio of emitted photons per absorbed photon), and the extinction coefficient is the amount of light ...