Spectroscopic Study and Evaluation of Red-Absorbing Fluorescent Dyes

Abstract

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.

The enzyme mechanism of nitrite reductase studied at single-molecule level

“…Similar traces were observed with the active L93C NiR (no ascorbate/PES, no nitrite) and with glass slides spin-coated with an ATTO 665 solution in 1% polyvinyl alcohol. Periods of high fluorescence alternate on a time scale of seconds with dark periods in which only the background signal is observed; we ascribe this to dye blinking (21)(22)(23)(24). The steady nature of the observed fluorescence during the ''on'' periods is consistent with crystallographic and NMR evidence, indicating absence of largescale conformational fluctuations (18,25).…”

Photoswitching microscopy with standard fluorophores

“…1a). Although MEA at lower millimolar concentrations shows only weak quenching of the S 1 state of the fluorophore (here ATTO 655 a fluorophore with photophysical characteristics similar to those of the oxazine derivative MR121 [24]), it is very likely that it promotes the formation of long-lived radical anion states generated via reduction of the T 1 state [38] even in the presence of oxygen. Thus, the lifetime of the off state can be adjusted by the MEA concentration (compare Figs.…”

“…Here, typically 500 to 1000 photons were typically detected for a single ATTO 655 or ATTO 680 fluorophore in its on-state. With a standard deviation of the PSF of ∼250 nm for the emission of ATTO 655 (absorption and emission maxima are centered at ∼660 and 680 nm, respectively [24]), we can expect a localization precision down to ∼10 nm. Under these imaging conditions, the fluorophores remain in the active state for several frames after spontaneous activation resulting in several thousand photons detected per fluorophore and switching cycle.…”

Quantitative analysis of the formation and diffusion of A 1 -adenosine receptor-antagonist complexes in single living cells

“…The choice of XAC as the basis of this fluorescent probe was based on structure-activity relationships that show that substituents on the C8 position of the xanthine ring are tolerated for A 1 -AR binding (19,(24)(25)(26). The fluorophore BODIPY 630͞650 was chosen because it is pharmacologically inactive at the A 1 -AR and has photophysical properties suitable for single-cell applications (27). The affinities of DPCPX and XAC for the A 1 -AR in both CHO-A1 and -A1Tpz cells were similar and consistent with previously reported values (1,3,18).…”