1. Quenching of fluorescence:
Fluorescence quenching refers to the irreversible damage of fluorescent molecules caused by the simultaneous action of internal and external factors. The internal factor is mainly that the molecule releases energy in the form of non-radiative transitions from the excited state back to the ground state. External factors include many aspects, mainly including:
①Light irradiation is the most common cause of fluorescence quenching. The generation of fluorescence requires light irradiation, but at the same time, light irradiation will also promote the interaction of excited molecules with other molecules, causing collisions and quenching fluorescence;
②Molecules of fluorescent substances and external molecules (or ions) form non-fluorescent compounds;
③ transfer of resonance energy;
④ Environmental conditions such as solvent type, pH value and temperature.
Substances that can cause fluorescence quenching are called quenchers. Such as halogen ions, heavy metal ions, oxidizing organic compounds (nitro compounds, diazo compounds, carbonyl compounds and hydroxyl compounds) and oxygen molecules.
2. Anti-quenching of fluorescence:
Fluorescence quenching of labeled samples is a major problem encountered in fluorescence microscopy and confocal microscopy. Compared with ordinary fluorescence microscopy, the photobleaching of the specimen is more obvious due to the stronger power and more accurate focusing of the laser scanning confocal microscope, and the fluorescence of fluorescein can gradually weaken or disappear during the continuous observation process.
Therefore, the use of anti-fluorescence quenchers (anti-fluorescence attenuators) should be considered. Commonly used anti-fluorescence quenchers include P-phenylene diamine (PPD), n-propyl gallate (NPG), 1,4-diazobicyclo[2,2,2) -Octane (1,4-diazobicyelo(2,2,2)-octane, DABCO) and the like. Among them, p-phenylenediamine is the most effective anti-quenching agent, but its application in vivo is limited due to its strong sensitivity to light and heat and its toxicity.
The ideal PPD anti-quencher mixture formula is: 9 parts of glycerol, 1 part of PBS and PPl with a concentration of 2 to 7 retool/L), and the final pH is 8.5 to 9.0. n-Propyl gallate is nontoxic, stable to light and heat, but not as effective against fluorescent bleaching as PPl) and can be used for in vivo studies.
The recommended concentration is 3 to 9 retool/L, and the effect is better when formulated with glycerol. l,4-DiazobicycloC2,2,2)-octane is a stable nonionic anti-quenching agent that is inexpensive and easy to use for in vivo studies.
In addition, there are other anti-fluorescence quenching methods that can also enhance the fluorescence intensity of living cells or non-viable specimens, such as the use of intermediate density filters, the use of high numerical aperture objectives and relatively low magnifications.