Sometimes quenching results from the transfer of energy to other acceptor molecules residing physically close to the excited fluorochromes, a phenomenon known as resonance energy transfer. This particular phenomenon has become the basis for a newer technique of measuring distances far below the lateral resolution of the light microscope. Impurities in the chromophore may also reduce fluorescence intensity either by photobleaching or quenching. Neutral density filters can be placed in the light path before the light reaches the excitation filter, thus diminishing the excitation light intensity. Chemicals that are capable of quenching singlet oxygen can also be employed to reduce the effects of photobleaching.
Examples are the amino acid histidine and diphenylisobenzofuran.
Fading effects can also be reduced, in some instances, by changing the pH concentration of the mounting medium. The occurrence of photobleaching has led to a technique known as FRAP , Fluorescence Recovery After Photobleaching. This technique is based upon bleaching by short laser bursts and subsequent observation of the recovery of fluorescence caused by the diffusion of fluorochromes into the bleached area. Authors Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747. All Rights Reserved .