Fluorescence Quench and Photobleach

Photobleaching is the reduced output of a fluorophore due to irreversible damage to the molecule in the presence of molecular oxygen. This is a permanent loss of the 

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In choosing a fluorescent tag, the most important factors to consider are good adsorption (high extinction coefficient), stable excitation without photobleaching, and efficient, high quantum yield of fluorescence. Some fluorophores, such as fluorescein, exhibit rapid fluorescent quenching which lowers the quantum yield over time. Up to 50 percent of the fluorescent intensity observed on a fluorescein-stained slide can be lost within 1 month in storage. AMCA and... 

Since the heterocyclic-substituted platinum 1,2-enedithiolates are dual emitters with only one emission that is oxygen quenched, ratiometric oxygen analysis is possible (see Fig. 3) (21, 29). While fluorescence and phosphorescence intensities vary with changes in optical clarity, fluctuations in the source and detector, and photobleaching of the emitter, the intensity ratio of a dual emitter (phosphorescence-fluorescence) does not. As such, the 3I/1I intensity ratio can be used in place of I, in Eq. 3 to generate Eq. 7 and 8 (29-31, 69). 

Common to most fluorescence-based single-molecule methods, photobleaching limits the observation time window using the fluorescence quenching via energy transfer approach. In addition, this approach only obtains the fluorescence intensity from one probe thus, fluorescence intensity fluctuations due to probe photophysics, such as fluorescence blinking, can complicate the results and data analyses. Triplet quenchers such as Trolox 95 can reduce fluorescence blinking. Careful control experiments are in any case necessary. 

In this section we review a number of important concepts that affect the choice of dyes for single molecule fluorescence applications. We introduce some concepts such as quantum yield, quenching, photobleaching, and blinking, many of which have already been highlighted in the context of single molecule fluorescence experiments and analysis in Chapters 2 and 3. We also refer the reader to a number of sources that provide further information on some or all of these topics [ 1-4]. 

Compared to fluorescence, SERRS has some significant advantages. Unlike fluorescence-based methods, SERRS measurements are unaffected by quenching by oxygen or other species, are less sensitive to photobleaching, and are inherently... 

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