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Improving the Photostability of Red- and Green-Emissive SingleMolecule Fluorophores via Ni Mediated Excited Triplet-State Quenching 2+
Viktorija Glembockyte, Junan Lin, and Gonzalo Cosa J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10725 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 5, 2016
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The Journal of Physical Chemistry
Improving the Photostability of Red- and Green-emissive Singlemolecule Fluorophores via Ni2+ Mediated Excited Triplet-state Quenching Viktorija Glembockyte, Junan Lin, and Gonzalo Cosa* Department of Chemistry and Center for Self-Assembled Chemical Structures (CSACS/CRMAA), McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 0B8, Canada. ABSTRACT: Methods to improve the photostability/photon output of fluorophores without compromising their signal stability are of paramount importance in single-molecule fluorescence (SMF) imaging applications. We show herein that Ni2+ provides a suitable photostabilizing agent for three green-emissive (Cy3, ATTO532, Alexa532) and three red-emissive (Cy5, Alexa647, ATTO647N) fluorophores, four of which are regularly utilized in SMF studies. Ni2+ works via photophysical quenching of the triplet excited state eliminating the potential for reactive intermediates being formed. Measurements of survival time, average intensity, and mean number of photons collected for the six fluorophores show that Ni 2+ increased their photostability 10- to 45-fold, comparable to photochemically-based systems, without compromising the signal intensity or stability. Comparative studies with existing photostabilizing strategies enabled us to score different photochemical and photophysical stabilizing systems, based on their intended application. The realization that Ni 2+ allowed achieving a significant increase in photon output both for green- and red-emissive fluorophores positions Ni2+ as a universal tool to mitigate photobleaching, most suitable for multicolor single-molecule fluorescence studies.
INTRODUCTION Single-molecule fluorescence (SMF) methodologies have become routinely used toward exploring key biophysical and biochemical pathways shedding light on cellular structure and dynamics.1-3 Detecting a fluorescently tagged single biological molecule of interest in a complex biological setting, however, is a challenging task requiring bright, frequently synthetic, fluorophores to maximize signal to background ratios.4-5 Under the high duty cycle characteristic of SMF imaging applications the inherent photophysical processes associated with these fluorophores make photobleaching a limiting factor.6-7 In this regard the fluorophore triplet excited state is often a key intermediate in the photodegradation as it is prone to undergo energy transfer or electron transfer reactions with molecular oxygen leading, respectively, to the generation of singlet oxygen (1O2) or superoxide radical anion (O2•-) and additional downstream reactive oxygen species (ROS).4, 8 These nascent ROS may next react with the precursor fluorophore destroying the chromophore unit.9 To maximize fluorophore photostability/photon output many strategies have been adopted over the years relaying both on depleting molecular oxygen via an enzymatic pathway, and on quenching the fluorophore excited triplet state, see Scheme 1.6, 10 In the latter case both photochemical and photophysical quenching pathways have been explored. Photochemical strategies employ a cocktail of reducing and oxidizing systems (ROXS).11-13 Here the reducing agents (e.g. ascorbic acid (AA), Trolox (TX),
n-propyl gallate) and oxidizing agents (e.g. methyl viologen (MV), Trolox quinone (TQ)) quench the triplet excited state forming a radical ion intermediate, regenerating next the fluorophore ground state following a pingpong redox reaction scheme. ROXS cocktails may, however, react with biomacromoelcules via e.g. electron transfer or nucleophilic attack, to name a few, precluding their universal application. Moreover, in designing SMF experiments requiring dual14-15 or multiple labelling schemes it may be challenging to find a ROXS system that works with different fluorophores (e.g. green-emissive and red-emissive dyes). 11, 13, 16-19;20 Fluorophore excited triplet state quenching via energy transfer 21-22 is an attractive physical alternative to photostabilization not involving reactive intermediates. We recently showed that Ni2+ was an efficient excited triplet state quencher for Cy3 leading to a 13-fold improvement in its photostability.16 Herein we illustrate that Ni2+ provides an ample spectrum photophysical quencher for triplet excited states. Solutions containing Ni2+ in submilimollar concentrations increase 10- to 45-fold the photon output of a number of dyes including three redemissive (Cy5, Alexa647, and ATTO647N) and three green-emissive (ATTO532, Alexa532, and Cy3) fluorophores, see Fig. S1.7 Overall Ni2+ resulted in similar or improved photostability and survival lifetime for green and red dyes compared to ROXS systems. Ours is thus a “universal photostabilizing method” applicable to two-color experiments such as SM-FRET studies or photobleaching
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analysis. Ni2+ additionally offers an alternative for imaging
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substrates that are redox sensitive.
Figure 1. Quantitative evaluation of the effect of different triplet quenchers on dye photostability exemplified with data obtained with an Alexa647-DNA duplex in the presence of 0.4 mM Ni2+ and the GODCAT oxygen scavenging system. a. The survival time of each single molecule was found as illustrated in the representative SMF intensity-time trajectory; b. Survival histogram constructed from the survival times recorded for hundreds of molecules. The indicated mean survival time was derived from the single exponential fit of the distribution (red curve); c. Histogram of the total number of photons collected per molecule, integrated from the SMF intensity-time trajectories of hundreds of molecules. The mean number of photons detected per molecule before photobleaching was obtained by fitting the distribution to a single exponential decay function (red curve).
Our results, including measurements of survival time, average intensity, and mean number of photons collected for the six fluorophores provide a practical guide toward choosing the best combination of fluorophore and photostabilization system. The advantages and limitations of a particular combination in the context of a specific application, such as SM Forster resonance energy transfer (SMFRET),23 protein-induced fluorescence enhancement (PIFE),24 or single-particle tracking are discussed.25 Scheme 1: Illustration of mechanistic strategies employed toward quenching the triplet excited state of fluorophores in SMF imaging applications
RESULTS AND DISCUSSION To examine the performance of fluorophores in SMF measurements, we employed double-stranded DNA duplexes (Table S1) containing the fluorophore of interest on one strand and a biotin moiety on the other strand allowing for specific immobilization onto glass coverslips (Fig. S2)15, 26-27 and imaging using a total internal reflection fluorescence (TIRF) microscope (see Table S2 for details on imaging conditions). Photostability was evaluated from SMF trajectories by determining the total number of photons collected per molecule before photobleaching. Next
we calculated the average number of photons collected. In an analogous manner an average survival time was also computed from the individual survival times recorded for the hundreds of single molecules imaged in any given TIRF experiment. Briefly, the photo bleaching time and total number of photons detected for each molecule were extracted from the SMF trajectories (Fig. 1a) and histograms were constructed from all survival times and total photons detected from several hundred molecules. Fitting the distributions to single exponential decay functions allowed us to extract the mean survival time and the mean number of detected photons per molecule for different imaging conditions. We initially evaluated the survival time and the mean number of photons collected for each fluorophore in the presence and absence of 0.4 mM Ni2+ under oxygen depleted conditions using glucose oxidase/catalase (GODCAT) enzymatic oxygen scavenging system (see Table S3). This Ni2+ concentration allowed for efficient quenching of the triplet excited states -based on increased survival times observed- without significantly quenching the singlet excited state for the fluorophores studied. 28 In the presence of Ni2+ a 10- to 45-fold increase in the number of photons collected was observed for the six fluorophores studied compared to experiments recorded under identical conditions but lacking Ni2+ (Fig. 2). We note that even though Ni2+ increased the photon yield for all dyes investigated here to a similar extent, the average number of photons collected for each dye until photobleaching varied significantly, indicating that some fluorophores might be better suited for extended SMF imaging than others. Consistent with previous studies reported in the literature,11 the number of photons collected for Cy3, Cy5, Alexa647, and ATTO647N was relatively high (in the range of 0.5 x 106 to 3 x 106), whereas those for ATTO532 and Alexa532 were at least one order of magnitude smaller 0.8 x 105 to 1.3 x 105, respectively, as they are less photostable (photobleaching occurs much faster under otherwise identical conditions, resulting in a shorter survival time).
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Figure 2: Normalized photon output measured for different SM dyes in the presence of 0.4 mM Ni2+ in combination with the GODCAT oxygen scavenging system. The mean number of detected photons in the presence of Ni2+ was normalized to the mean number of photons detected in the presence of GODCAT alone.
0.4 mM Ni2+. The photostability of Cy3 could not be evaluated in the presence of a ROXS system consisting of MV and AA due to fluorescent impurities observed when a combination of these two reagents were added to the SM imaging buffer (Fig. S4).17 On the other hand, a ROXS system consisting of TX/TQ could still be used to increase the photostability of Cy3; however, the average number of photons collected in the presence of TX/TQ was lower compared to imaging conditions with either Ni2+ or β-ME. No pronounced blinking dynamics were observed in the SMF intensity time trajectories of Cy3 for all triplet quenchers studied here (see Fig. S3). GODCAT + 2 mM TX/TQ + 143 mM -ME 2+ + 0.4 mM Ni 2+ + 1 mM Ni + 1 mM MV + 1 mM AA
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We next compared the antifading properties of Ni2+ to those of other commonly used photostabilizing systems comprising β-ME (geminate recombination mechanism17), TX/TQ quinone (ROXS mechanism13), and AA in combination with MV (ROXS mechanism11). We evaluated the photostability (photon output) and dynamics observed in SMF intensity-time trajectories (signal stability) for all the conditions explored. These triplet quenchers were assessed at the concentrations reported in the literature11-12 in the presence of the GODCAT oxygen scavenging system (see also Table S3). Ni2+ was in turn assessed at a 0.4 mM concentration as well as a higher 1.0 mM concentration to determine whether, despite a measurable fluorescence quenching in the latter case, further improvements in photon output could be achieved as a result of an increased fluorophore duty cycle27 in SMF experiments. We focused our investigations on the four most stable fluorophores, namely green-emissive Cy3 and the three redemissive dyes Cy5, Alexa647, and ATTO647N as they were the most promising for extended SMF imaging applications. Fig. 3 (as well as Table S3) summarizes the mean number of photons detected for each fluorophore in the presence of the different triplet quenchers, including the two different concentrations of Ni2+ utilized. The green-emissive fluorophore Cy3 generated the highest photon output in the presence of either 0.4 mM Ni2+ or 143 mM β-ME computing a 29- and 34.6-fold increase in photons collected, respectively, compared to oxygen scavenger alone. As shown in ensemble intensity histograms assembled from all events in SMF intensity-time trajectories (Fig. 4a-b) the increase of Ni2+ concentration to 1 mM lead to significant singlet excited state quenching. This manifested in dimmer SMF trajectories (Fig. S3). Since there was no lengthening of the survival time associated with the larger Ni2+ content the net result was fewer photons collected (Table S3) compared to experiments with
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Figure 3: Mean number of photons per molecule collected for four different fluorophores in the presence of the GODCAT oxygen scavenging system and in combination with commonly used triplet state quenchers including either MV/AA, TX/TQ, or β-ME. Also shown are the results for Ni2+.
The combination of Ni2+ and oxygen scavenger also proved to be very efficient in improving the photon output of all three red-emissive fluorophores. The photostability of Cy5, Alexa647, and ATTO647N were improved 45-, 41-, and 22-fold, respectively, in the presence of 1 mM Ni2+ (Fig. 4, Table S3). Here the total number of collected photons was higher when 1 mM rather than 0.4 mM Ni2+ was utilized (Fig. 3, also Table S3). Although increasing the Ni2+ concentration to 1 mM led to a dimmer fluorescence signal from single-emitters (Fig. 4 f, j, n) suggesting considerable singlet state quenching at this concentration, the larger Ni2+ concentration in turn resulted in a very significant lengthening of the survival time. The net result was that the photon gain arising from enhanced survival time, arguably due to better quenching of triplet excited states at higher concentrations of Ni2+, amply outweighed the reduced frequency of photon detection due to quenching of fluorescence. Thus while the average intensities for Cy5, Alexa647, and ATTO647N dropped by ca. 1.2, 1.1 and 1.3 fold, the survival time in turn increased by 1.4, 1.9 and 2.7 fold, respectively (Table S3). For Cy3 the balance between enhanced survival time (due to efficient triplet state quenching) and minimal singlet state (fluorescence) quenching is achieved at lower Ni2+ concentrations.
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Figure 4: Ensemble intensity histograms constructed from individual events in the SMF intensity time trajectories obtained for Cy3 (a-d), Cy5 (e-h), Alexa647 (i-l), and ATTO6476N (m-p) in the presence of GODCAT oxygen scavenging and different triplet quenchers: 0.4 mM Ni2+ (blue), 1 mM Ni2+ (green), 2 mM TX/TQ (red), 143 mM β-ME (yellow), and 1 mM MV + 1 mM AA (magenta). Insets report the means of the fitted distribution for the largest/brightest population.
Importantly, blinking dynamics with short-lived off states (1 to 2 frames when imaging at 10Hz) while not pronounced, were observed in SMF transients of Cy5, Alexa647, and ATTO647N (Fig. 5 a-c, also Fig. S7-9). We propose that this blinking is due to the formation of reactive radical intermediates that are formed due to photoionization or other redox pathways such as photoinduced electron transfer with DNA bases.6, 29-30 We note, however, that the extent of this “redox” blinking is reduced at the higher Ni2+ concentration of 1 mM (Fig. 5 d-f) further underscoring this as the optimal concentration to be utilized in SMF experiments. For ATTO647N-DNA duplex, besides short time scale blinking, we have also observed fluctuations between multiple intensity states (Fig. 5 right panel and Fig. S9). This type of photoswithcing was not unique to an imaging cocktail containing Ni2+, but was also observed for all the other photostabilizing systems evaluated here and is consistent to previous reports by Vogelsang et al.11 and by Zheng et al.31
ing of MV and AA (Fig. 3, Table S3). We did not observe fluorescent impurities when imaging upon excitation with a 641 nm laser. However, a dim fluorescent state that was recorded in SMF intensity time trajectories (Fig. 5 g-i, Fig. S5) as well as in ensemble intensity histograms assembled from all SMF trajectories (Fig. 4h, 4l, and 4p). Ensemble fluorescence quenching studies of either Cy5-DNA or Alexa647-DNA duplexes with MV show that approximately 7% and 11%, respectively, of the initial fluorescence is quenched at 1 mM MV concentration (Fig. S6). Possibly, the dim fluorescent state observed for the three fluorophores may be related to cationic MV binding to the DNA duplex and partially quenching the singlet excited state of the fluorophore. In the case of ATTO647N DNA duplex the multiple intensity states observed (Fig. 4p and 5i) might be the result of both – the photoswitching nature of this fluorophore, discussed above, and the presence of MV/AA in the imaging cocktail.
The red-emissive fluorophores delivered the largest number of photons in the presence of a ROXS system consist-
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Figure 5: SMF transients obtained for 3 red-emissive dyes: Cy5 (left panel), Alexa647 (middle) and ATTO647N (right panel) in the presence of GODCAT oxygen scavenger and different photostabilizing systems selected to demonstrate different dynamics that are observed in each case: a-c) 0.4 mM Ni2+; d-f) 1.0 mM Ni2+; g-i) 1 mM MV and 1 mM AA; j-l) 2 mM TX/TQ; m-o) 143 mM β-ME. Note: the fluorescence transients in g-i are not representative of the overall behavior, but are rather shown to demonstrate that dim and bright states are observed under these imaging conditions.
The observed increase in photon count for the redemissive fluorophores was lower with the 2 mM TX/TQ ROXS-based system (8- to 22-fold) compared to conditions when Ni2+ was used as a triplet quencher (Fig. 4, Table S3). Noticeably, with TX/TQ the blinking in the SMF intensity-time trajectories was completely suppressed (Fig. 5 j-l and Fig. S7-9). Data acquired with β-ME in the imaging buffer displayed blinking in the form of long-lived (tens of seconds) off states for the two cyanine fluorophores, Cy5 and Alexa647 (Fig. 5m and 5n, Fig. S78) consistent with previous reports.18 This type of photoinduced switching has been successfully employed in localization-based super-resolution imaging,19 however, it is not suited for quantitative SMF studies that rely on fluorophore intensity changes. Overall, the dynamics observed in Cy5 and Alexa647 SMF transients were very similar, in accordance with their structural similarity. Altogether our experiments show that Ni2+ ions can be used as an efficient excited triplet state quencher for a wide range of fluorophores typically utilized in SMF imaging including cyanine dyes (Cy3, Cy5, Alexa647), carbopy-
ronine dyes (ATTO647N) and rhodamine dyes (ATTO532 and Alexa532). The effect of Ni2+ on photon output (10- to 45-fold increase in photons collected) varied from dye to dye. The differences recorded between the dyes will depend on the excited triplet vs. singlet state quenching efficiency by Ni2+. This efficiency will be sensitive to their respective excited state decay rate constants, to the energy transfer rate constants that in turn depend on the triplet energy levels of each fluorophore, and to the rates of competing photochemical processes from the triplet excited state, such as redox reactions or photoxidation.32 Furthermore, due to structural differences between fluorophores, their conformational dynamics when attached to a DNA duplex may affect the encounter rate between the fluorophore and the Ni2+ ions hopping on the DNA duplex. Dyes that are excited at shorter wavelengths (higher energies) have higher propensity to undergo destruction pathways through higher excited singlet states. In such cases the photophysical quenching of triplet states might not be the an efficient photostabilization strategy. In fact,
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for blue-emissive dye Alexa488, we did not observe any increased photostability in the presence of 0.1 mM Ni 2+ (concentration that resulted in pronounced increase in
photostability for the green- and red-emissive dyes mentioned above (see Fig. S10 for comparative SMF trajectories)).
Table 1: Scoring of the different photostabilizing systems for SMF imaging based on photostability and stable fluorescence signal
1.0 mM Ni2+
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MV+AA (1mM)
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Signal stabilityb
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Photostability (photon output)a
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Cy3 Cy5
++ ++
+ ++
+ +
++ -
n/a +++
++ +
++ +
++ ++
++ -
n/a -
Alexa647 ATT0647N
+ +
++ +
+ +
-
++ +++
+ -
+ -
++ -
-
-
aThe conditions were assigned a “+++”, “++”, “+”, and “-“ when the fold increase in photon output was >50-fold, 50- to 25-fold, 25- to 5-fold, and
