Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy

Abstract

Fluorescence correlation spectroscopy (FCS) can be used to investigate the photobleaching properties of fluorophores in solution. The advantage with this method is that in addition to the photobleaching rate the formation and decay rates of the triplet state can be measured. In this way, it is possible to calculate the photodestruction quantum yield and relate the photostability of a fluorescent compound in a certain environment to the photodynamical behaviour of the singlet‐triplet transitions. This is likely to contribute to a better understanding of the mechanisms of photobleaching given the central importance of dye triplet states in photobleaching processes. The approach was applied to the measurement and characterization of the photobleaching of Rh6G in aqueous solution and FITC in 1 mM sodium carbonate buffer (pH 9). The photobleaching yields measured are discussed in view of the simultaneous triplet properties at different excitation intensities, oxygen concentrations as well as in the presence or absence of quencher molecules. This study suggests that FCS is likely to provide a valuable tool for the elucidation of the mechanisms of photobleaching, which are far from understood in all their details.

Iodide as a Fluorescence Quencher and Promoter—Mechanisms and Possible Implications

“…Due to the relatively high irradiances that can be used in FCS experiments, effects of excitation to higher singlet and triplet states (S n and T n states) can sometimes be noticed. In particular, from these states, photooxidation and photo-bleaching is typically strongly enhanced 7,[11][12][13] . To describe the generation of photo-oxidized states in the FCS experiments it is therefore motivated to include higher excited states (S n and T n ) into the electronic state model, as outlined in Figure …”

“…The usefulness of a certain added compound depends on what dye it will act on, the concentration of the dye in the sample, as well as on a range of environmental factors, including viscosity, local concentrations of oxygen, of naturally occurring scavengers and quenchers, and the local accessibility to the dye molecules. Also the excitation conditions can have a substantial influence [11][12][13][14] . High excitation rates, as often used in SMD or FCS experiments, or in other applications where high read-out rates or high sensitivities are required, can strongly increase the generation of several long-lived, photo-induced states of the dyes, such as triplet states, photo-isomerised states, photo-oxidized states or other states generated by photo-induced charge transfer 8,[12][13][14][15] .…”

“…Also the excitation conditions can have a substantial influence [11][12][13][14] . High excitation rates, as often used in SMD or FCS experiments, or in other applications where high read-out rates or high sensitivities are required, can strongly increase the generation of several long-lived, photo-induced states of the dyes, such as triplet states, photo-isomerised states, photo-oxidized states or other states generated by photo-induced charge transfer 8,[12][13][14][15] . The multitude of states generated, and that the effect of the added photo-stabilizers and quenchers may lead to the formation of additional intermediate states increases the complexity.…”

“…The experimental setup for the FCS studies has been described before 12,13,23,24 . The fluorophore molecules under investigation were illuminated by a multiline argon-ion laser (488nm or 514 nm, Lasos LGK 7812 ML, Jena, Germany), a helium-neon laser (594nm, Laser2000 model 30572, Munich, Germany) or a multi-line argon-krypton ion laser (568nm or 647 nm, Melles Griot 643-RYB-A02, Carlsbad, USA), reflected by a dichroic mirror (Z488RDC, 525DCLP, Z467/568RPC, 600DCLP), and focused by a cover glass corrected water-immersion objective (Zeiss, PlanNeofluar, 63x, 1.2 NA, 160 mm tube length) to a diameter of 0.6 m.…”

Spatial Organization of Bacteriorhodopsin in Model Membranes

“…First of all, a common source of errors in fluorescence correlation spectroscopy is the photobleaching of the dye molecules. This would artificially decrease the dwell time of the particles in the probe volume and, therefore, result in an apparent increase in mobility (33). To eliminate this possibility, we measured the mobility of BR at different excitation powers, determined the threshold below which the mobility was constant, and set the laser power for our measurements below that threshold (data not shown; see "Experimental Procedures").…”