An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments

Abstract

The application of single-molecule fluorescence techniques to complex biological systems places demands on the performance of single fluorophores. We present an enzymatic oxygen scavenging system for improved dye stability in single-molecule experiments. We compared the previously described protocatechuic acid/protocatechuate-3,4-dioxygenase system to the currently employed glucose oxidase/catalase system. Under standardized conditions, we observed lower dissolved oxygen concentrations with the protocatechuic acid/protocatechuate-3,4-dioxygenase system. Furthermore, we observed increased initial lifetimes of single Cy3, Cy5, and Alexa488 fluorophores. We further tested the effects of chemical additives in this system. We found that biological reducing agents increase both the frequency and duration of blinking events of Cy5, an effect that scales with reducing potential. We observed increased stability of Cy3 and Alexa488 in the presence of the antioxidants ascorbic acid and n-propyl gallate. This new O(2)-scavenging system should have wide application for single-molecule fluorescence experiments.

Figure 1

Comparison of GODCAT and PCD O₂-scavenging systems. (A and B) Schematics of chemical reactions in GODCAT and PCD systems, respectively. (C) Dissolved oxygen time courses for GODCAT (solid) and standardized PCD (shaded) systems. Conditions for each reaction are as follows: GODCAT ∼100 nM glucose oxidase, ∼1.5 μM catalase, 56 mM glucose; PCD ∼50 nM PCD, 2.5 mM PCA. (C inset) Time courses of PCA consumption as monitored by absorbance at 290 nm. Each reaction contains 200 μM PCA and either 50 nM (solid) or 10 nM (shaded) PCD.

Figure 2

Analysis of single fluorophores. (A) Representative color overlay of all fluorophores observed in one acquisition movie. (B) Representative fluorescence versus time trace for a single dye. Initial events (1), off-blink (2), and on-blink events (3) were segregated before lifetime analysis; events limited by acquisition length (4) were discarded. (C) Initial, off-blink, and on-blink event distributions were fit, as described, to an exponential probability function to determine mean lifetimes, here Cy5 initial lifetimes with PCD.

Figure 3

Characterization of dye stability in the GODCAT and PCD systems. (A) Table of mean lifetimes and event frequencies (per molecule) in the GODCAT system. (B) Table of mean lifetimes and event frequencies (per molecule) in the standardized PCD system. (C) Comparison of initial dye lifetimes in the standardized PCD (open), dilute PCD (light shaded), GODCAT (shaded), and GODCAT+PCA (dark shaded) systems.

Figure 4

Cy5 dye stability in the presence of biological reducing agents. (A) Table of event frequencies and lifetimes for initial, on-blink, and off-blink events (per molecule) in the absence or presence of 10 mM BME, DTT, and TCEP. Lifetime distributions are fit as described in Materials and Methods. (B) Bar plot of the ratios of on-blink/initial lifetimes (open) and off-blink/initial lifetimes (shaded) for Cy5 in the presence of reducing agents. (C) Bar plot of the signal/noise ratio for Cy5, determined on a per molecule basis.

Figure 5

Effects of additives on initial lifetimes of Cy3, Cy5, and Alexa488. (A) Chemical structures of additives: MEA, DABCO, ascorbic acid, and nPG. (B) Bar plot of the fractional change in initial lifetimes of Cy3 (green), Cy5 (red), and Alexa488 (blue) in the PCD system with respective additives.