Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux

Abstract

SummaryExposure to light can destroy the ability of a molecule to fluoresce. Such photobleaching limits the use of fluorescence and confocal microscopy in biological studies. Loss of fluorescence decreases the signal-to-noise ratio and so image resolution; it also prevents the acquisition of meaningful data late during repeated scanning (e.g. when collecting three-dimensional images). The aim of this work was to investigate the role of oxygen in the photobleaching of fluorophores bound to DNA in fixed cells, and to explore whether anoxia could minimize such bleaching. Anoxia significantly reduced bleaching rates and changed the order of reaction of both propidium iodide (an intercalator) and chromomycin A 3 (a minor-groove binder) bound to DNA; it afforded the greatest protection at low photon fluxes. However, it had no effect on the bleaching of the green fluorescent protein (GFP) covalently attached to a histone and so bound to DNA, probably because the protein shielded the chromophore from oxygen. Bleaching of all three fluorophores depended on photon flux. Practical ways of minimizing bleaching were examined, and examples of three-dimensional images of DNA marked by propidium and GFP (collected under standard and optimized conditions) are presented.

Cell death in grape berries: varietal differences linked to xylem pressure and berry weight loss

“…1; Tyerman et al 2004). Bondada et al (2005) qualitatively confirmed that xylem hydraulic conductance continued in post veraison berries by demonstrating that dye uptake could still occur provided the appropriate driving force on water flow to the berry could be sustained. Tyerman et al (2004) also showed that the xylem pressure changed from being negative to slightly positive during veraison, supporting the view that the nature of the driving force for water movement to the berry in the xylem changes during phase 2 (Fig.…”

“…There has been much interest recently in the water budget of the developing grape berry (Bondada et al 2005;Keller et al 2006;Rogiers et al 2006; Thomas et al 2006;Tilbrook and Tyerman 2006;Zhang et al 2006b). This is for two reasons.…”

“…It binds non-covalently, intercalating in a stoichiometric manner with single and double stranded DNA and RNA. When intercalated it fluoresces red (Cosa et al 2001;Bernas et al 2004;Kral et al 2005). The loss of cell membrane integrity in plants is considered to mark the end of homeostasis and indicates cell death (Noodén 2004).…”

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 …”

Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching

“…It has been shown that excited fluorophores interact with dissolved oxygen producing reactive oxygen species, such as super oxide radicals (Wright et al, 2002). These can oxidize and destroy a variety of components, such as proteins and their associated fluorophores, leading to a loss of fluorescence signal (Greenbaum et al, 2000;Bernas et al, 2004). For this to happen, oxygen must collide with the excited dye.…”