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Published in final edited form as: Curr Opin Chem Biol. 2014 Jun 21;0:103–111. doi: 10.1016/j.cbpa.2014.05.010

The bright future of single-molecule fluorescence imaging

Manuel F Juette a, Daniel S Terry a, Michael R Wasserman a, Zhou Zhou a, Roger B Altman a, Qinsi Zheng a,b, Scott C Blanchard a,b,*
PMCID: PMC4123530  NIHMSID: NIHMS607726  PMID: 24956235

Abstract

Single-molecule Förster resonance energy transfer (smFRET) is an essential and maturing tool to probe biomolecular interactions and conformational dynamics in vitro and, increasingly, in living cells. Multi-color smFRET enables the correlation of multiple such events and the precise dissection of their order and timing. However, the requirements for good spectral separation, high time resolution, and extended observation times place extraordinary demands on the fluorescent labels used in such experiments. Together with advanced experimental designs and data analysis, the development of long-lasting, non-fluctuating fluorophores is therefore proving key to progress in the field. Recently developed strategies for obtaining ultra-stable organic fluorophores spanning the visible spectrum are underway that will enable multi-color smFRET studies to deliver on their promise of previously unachievable biological insights.

Introduction

The 20th century saw the identification and characterization of the macromolecular constituents of life. A plethora of powerful techniques was developed to study these molecules at the ensemble level to understand their behavior and function and, perhaps most importantly, their “malfunction” due to disease. When the optical detection and spectroscopy of individual molecules in condensed matter became a reality in the late 1980s [1], it was soon recognized that this breakthrough would help researchers gain an entirely new perspective on the inner workings of biological systems [2,3].

While typical biochemical bulk experiments provide ensemble-averaged measurements of molecular properties, single-molecule approaches reveal not only the full probability distribution functions and their time dependence, but also enable the identification of sub-populations and transient intermediates. The resulting insights into heterogeneities and time-dependent fluctuations are fundamental for an accurate mechanistic description of bio-molecular function [3].

Among the possible far-field, optical readout modes for single molecules [1], fluorescence is notable for its simplicity of implementation, molecular specificity, contrast, and compatibility with multi-color and live-cell imaging [2,4]. Within the past two decades, single-molecule fluorescence techniques have proven their potential and are now routinely used in many biological investigations [5]. A key limitation that has been noted, however, is the need to broaden the range of imaging time scales that can be achieved to probe processes gain deeper insights into both rapid and slow time scale processes [6,7].

Another key challenge of contemporary single-molecule fluorescence imaging relates to the growing need to correlate multiple events in space and time. The function of many – if not most – complex biological systems entails both time-dependent changes in conformation and composition. If the goal is to dissect the macromolecular machinery in all of its complexity, the observation of only one molecular species or the interaction of just one pair of species at a time, providing only a partial view, is vastly insufficient. Fortunately, fluorescence techniques readily lend themselves to the simultaneous observation of multiple processes through the use of spectrally distinct fluorophores. Due to instabilities of the available fluorophores, however, their selection is often a performance-limiting factor [8].

In this review, we focus on single-molecule Förster resonance energy transfer (smFRET) using small-molecule organic fluorophores, a technique that is widely used to probe macromolecular binding and conformational dynamics [5]. While multi-color smFRET for the correlation of multiple events was introduced nearly a decade ago [9], it has only recently gained traction as a tool to solve important biological problems (reviewed in [10,11]).

The mainstream use of multi-color smFRET has been substantially held back by the paucity of bright and long-lasting complementary fluorescent probes, which are required to enable the imaging of complex systems at biologically relevant timescales [8]. In this review, we therefore highlight recent innovations in the design of organic fluorophores that have the potential to expand the palette of bright and stable fluorescent probes spanning the visible spectrum. Particular emphasis is placed on “self-healing” dyes developed in our lab, where undesirable dark states are quenched intrinsically through an incorporated protective moiety [12,13]. We further consider how the combination of ultra-stable dyes with other emerging technologies, including faster detectors and high-throughput imaging platforms, will expand the scope of smFRET experiments to new physical and kinetic regimes currently beyond reach.

The power of multi-color smFRET

FRET (Förster resonance energy transfer) is a powerful tool to investigate the dynamics of macromolecular machines by detecting nanoscale conformational changes as well as ligand binding events. FRET is based on an interaction occurring between two fluorophores in close proximity (10-90 Ångstrom) [14] (Figure 1a). Excited-state energy from a donor fluorophore is partially transferred to the nearby acceptor through non-radiative dipole-dipole coupling, leading to fluorescence emission of the acceptor accompanied by (partial) quenching of the donor. The transfer efficiency is strongly distance-dependent (following an inverse sixth-power law), thus allowing the experimentalist to infer changes in the relative position of the two fluorophores from the ratio of donor and acceptor fluorescence emissions [15] (Figure 1a). For excellent practical introductions to smFRET we refer to [15] and [16].

Figure 1.

Figure 1

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Principles of multi-color smFRET. (a) Left: illustration of energy transfer from a donor fluorophore (here: Cy3) to an acceptor fluorophore (here: Cy5). Right: distance dependence of energy transfer. (b) Illustration of the correlation of conformational events by multi-color smFRET. Grey panels: two independent structural perspectives monitored by standard two-color smFRET experiments. Blue panel: three-color smFRET experiments distinguishing between fully coupled (left) and partially uncoupled (right) modes of ribosomal motions.(c) Possible FRET interactions between three and four fluorophores. (d) Emission spectra of common cyanine dyes frequently used in smFRET imaging, along with typical excitation laser wavelengths (dashed lines) and detection windows (shaded areas).

Since the first observation of a single donor-acceptor pair [17], the use of smFRET has grown rapidly [5]. It has enabled important insights into biological systems [18], including protein folding and binding [19], RNA folding and catalysis [20], transcription [21], translation [6,22], and membrane transporters [23-25].

The observation of a single FRET pair, however, yields only a single distance vector. Thus, multiple FRET measurements from distinct structural perspectives are typically required to gain the insights required to deduce the origin of the observed motion [26-28]. This limitation leaves open the possibility that separate measurements may be difficult to compare due to experimental variability. Multicolor-FRET imaging has the potential to circumnavigate such challenges to reveal whether conformation events are correlated, uncorrelated or partially coordinated in nature. Figure 1b illustrates how multi-color smFRET may help differentiate between alternative biological models. In our example, multiple conformational events are required to achieve an “unlocked” (i.e. activated transient intermediate) configuration of the ribosome on path to translocation of the mRNA and tRNAs [27]. Probing the interplay of three fluorophores allows distinguishing whether these events proceed in a coordinated or partially uncoupled fashion.

The first implementation of multi-color smFRET was demonstrated using a combination of one donor (Cy3) with two acceptors (Cy5 and Cy5.5) [9]. A key shortcoming of these early experiments related to the considerable spectral overlap of the Cy5 and Cy5.5 acceptor fluorophores. This was significantly improved by the introduction of the dye combination Cy3/Cy5/Cy7 [29] (Figure 1c/d), which exploits the previously unoccupied near-infrared spectral region and remains in common use for three-color smFRET imaging [30,31]. The Cy7 fluorophore is, however, particularly labile under the intense illumination that is required for single-molecule imaging [12].

Three-color smFRET, while expanding the scope of measurable phenomena, is not capable of probing two fully independent molecular interactions. This capability was recently introduced with the advent of four-color smFRET [32,33], where Cy2 was used as the additional donor fluorophore (Figure 1c/d). While four-color smFRET is capable of probing complex biological systems by exploiting the multitude of pairwise interactions that can occur between four distinct fluorophores (Figure 1c), the increased demands associated with spectral separation and the use of multiple high-frequency illumination tends to severely limit the signal-to-noise ratio – and thus the interpretation – of such experiments.

Advances in organic fluorophores for single-molecule imaging

Detecting single molecules requires bright, long-lasting fluorophores. This requirement is amplified in multicolor techniques, where several such fluorophores are used simultaneously. Like most fluorophores, organic dyes are compromised by the prevalence of non-emissive “dark states” that limit the brightness and duration of fluorescence emission [7,8,34]. Hence, the choice of suitable fluorophores, as well as specifically optimized buffer conditions to maximize photon emission, are indispensable for a successful experimental outcome

Phenomenologically, fluorophore dark states can be divided into intermittent transitions (blinking) and permanent damage to the fluorophore, rendering it non-emissive (photobleaching). Photophysically, such transitions disrupt the excitation-induced cycling of a fluorophore between its electronic ground state S0 and the first excited singlet state, S1. Figure 2a illustrates some of the most important processes. Intersystem crossing (ISC) of a fluorophore from S1 to a non-fluorescent triplet state, T1, has been identified as a key transition determining fluorophore performance [7]. While ISC is a rare event for typical fluorophores (quantum yield < 0.01), triplet lifetimes are orders of magnitude longer (10-6 to 10-4 seconds) compared to those of singlet excited states (10-10 to 10-9 seconds). In addition to directly causing blinking, triplet excursions also mediate downstream photobleaching due to the highly reactive nature of the triplet state.

Figure 2.

Figure 2

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Fluorophores for single-molecule imaging. (a) Schematic view of key photophysical processes related to fluorophore instabilities and their mitigation (S0 – ground state; S1 – first excited singlet state; T1 – triplet state; R± - radical ion; ISC – intersystem crossing; ROX – reduction/oxidation). (b) Left: generic structure of cyanine fluorophores, characterized by the central polymethine chain, whose length is a key determinant of spectral properties. Right: NHS ester of a protective agent (PA)-conjugated self-healing Cy5 molecule. (c) Comparison of total photon emission and brightness (as represented by the signal-to-noise ratio) of conventional and COT-conjugated cyanine dyes across the visible spectrum.

Although molecular oxygen is a strong triplet state quencher that tends to suppress triplet-induced blinking events, the oxidation of the fluorophore by molecular oxygen is particularly detrimental to single-molecule imaging [35,36], as it generates a highly reactive superoxide radical (O2-) along with a cationic radical form of the fluorophore that are susceptible to photobleaching The common practice of removing molecular oxygen from the imaging buffer through enzymatic oxygen scavenging systems [37-40] therefore has the ambivalent result of improving overall fluorophore lifetimes at the expense of increased triplet-induced blinking.

To further improve the emission rate and lifetime of organic fluorophores, a variety of soluble small-molecule protective agents [41] have been used in many single-molecule fluorescence studies. For an in-depth discussion of these, mostly redox-active compounds, we refer to [7]. An especially elegant and successful strategy to control the blinking behavior of fluorophores via solution additives is the use of a combined, carefully balanced reducing and oxidizing system (ROXS) [42,43]. This has proven to be of particular importance in localization-based super-resolution microscopy, where finely tuned control of both bright and dark states is desirable [8].

While solution-based protective agents have afforded remarkable improvements in fluorophore performance, several factors limit their effective use in biological studies. The millimolar concentrations required to provide sufficient collision frequencies with dye molecules are often near the solubility limit of these substances. In addition, their use may interfere with the integrity of biological systems [44]. Finally, the effect of a given protective agent on different fluorophores is not universal [42,43]: what benefits one fluorophore, may be detrimental to another. This is especially problematic for multicolor approaches, which require combinations of fluorophores that all perform well under the same environmental conditions.

In an effort to generate intrinsically stabilized fluorophores, we have recently synthesized derivatives of commonly used cyanine fluorophores [45] with protective agents covalently attached to the fluorogenic center (Figure 2b) [12] [13]. The goal of this strategy was to achieve the maximum “local concentration” of the single, specific protective agents, 1,3,5,6-cyclooctatetraene (COT), 4-nitrobenzylalcohol (NBA), and Trolox. These engineered fluorophores [8] were shown to exhibit markedly reduced blinking and lower photobleaching rates than the parent compounds.

Recent studies [35,46,47] have provided further insights into the mechanisms of photostabilization of these so-called “self-healing dyes”, showing that COT operates through a triplet-triplet energy transfer mechanism while NBA and Trolox appear to function through a less controllable charge transfer mechanism. Focus was therefore given to the generation of COT-linked cyanine fluorophore derivatives where substantial benefits were observed in fluorophore performance across the visible spectrum (Figure 2c). Recent studies have shown that COT-linked derivatives of Cy5 exhibit negligible saturation of fluorescence excitation under increasing illumination intensities- a finding compatible with single-molecule FRET imaging at millisecond time resolution [7]. By extension, such efforts may also apply to multicolor techniques. To ultimately achieve such a goal, synergistic strategies are likely to be required to reduce propensities of cyanine fluorophores to undergo cis-trans isomerization of the polymethine chain [48]. Considerations related to solvent shell effects may also need to be included as water itself can be a quenching determinant [49,50].

While cyanine dyes remain the most commonly used fluorophores in single-molecule studies [8,45], a variety of alternative dye combinations have been used for multicolor smFRET, including dyes from the Alexa [51-53] and ATTO [54] families of commercially available dyes. Although, it remains to be determined whether analogous self-healing strategies can be applied to these rhodamine, oxazine, and carbopyronine dyes, such results are anticipated given the universally negative impact of triplet states on fluorophore performance [7]. Achievements in this area are expected to broadly impact diverse imaging modalities and focus areas.

Expanding the scope of smFRET measurements

While fluorophore development is only one aspect of the multifaceted challenge to increase the scope and depth of information accessible by smFRET imaging. [10,11], the spectral versatility, long lifetimes, and high photon emission rates exhibited by the emerging new generation of fluorophores has the potential to enable single-molecule investigations of unprecedented spatiotemporal resolution. Important biological events occur in nature at timescales that typically exceed the rates of photon emission that can be achieved with commercially available dyes to saturation of triplet states or photobleaching under intense illumination. Observing fast timescales of milliseconds to microseconds through fluorescence has mostly been limited to the analysis of short emission bursts, e.g. in FCS [55] or smFRET implemented on confocal microscopes [56].

Wide-field approaches that facilitate the generation of robust statistics over short time periods and that enable pulse-chase type, pre-steady state measurements [15] are typically limited in temporal resolution by the readout speed of electron-multiplying charge-coupled device (EM-CCD) cameras. Recently developed, scientific complementary metal-oxide semiconductor (sCMOS) cameras with much faster readout and larger field of view have, however, been shown to have sufficient performance to increasingly replace EM-CCDs for single-molecule imaging at fast time resolutions (milliseconds and below) [57,58]. Combined with sophisticated microfluidic systems for millisecond scale mixing of reagents [59], these novel technologies will enable fast pre-steady state kinetic measurements in vitro. For live-cell studies, combining smFRET with fast, active single-particle tracking [60] and tracking FCS [61] technologies may enable the investigation of conformational processes in an undisturbed functional context.

The ability to perform experiments over a vast range of timescales is essential for investigations of an increasing number of molecular machines, whose different functions dictate dynamics in entirely different temporal regimes. Even processes within the same machine may be orders of magnitude apart in timescale, as different functional units perform individual steps of a complex process. In the case of the translating ribosome, the selection of aminoacyl-tRNA occurs on the sub-millisecond to millisecond timescale [28], while the synthesis of an entire protein may take many minutes (Figure 3a). The development of ultra-stable fluorophores will therefore be paramount to gaining direct insights into the mechanistic features of processive reactions spanning all timescales relevant to function.

Figure 3.

Figure 3

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Temporal regimes accessible in smFRET imaging. (a) Overview of relevant timescales for the photophysics of organic fluorophores (blue) and for translation (green). (b) Fluctuations in ribosomal pre-translocation complexesobserved using site-specifically labeled tRNA molecules as previously described [62](see cartoon) imaged at 2 ms and 0.5 stime resolution (1.4 W and 8 mW laser excitation at 532 nm, respectively). Data were collected in Tris-polymix buffer using ribosomal complexes and a wide-field TIRF illumination system as previously described [62].

Self-healing cyanine fluorophores, in combination with current detector technology, already enable investigations covering a wide range of timescales. To illustrate this, we imaged dynamic processes within ribosomal pre-translocation complex at both 2 ms and 0.5 s temporal resolution using sCMOS technologies (Hamamatsu Flash 4.0) (Figure 3b). While the fast measurements highlight the occurrence of non-accumulating, transient species related to activated reaction intermediates [26,62,63] (red shaded area), the longer timescale measurements demonstrate that the same system exhibits signatures of dynamic disorder that may be of unknown importance to ribosome function.

These data are meant to emphasize the importance of both fine-grained and coarse-grained observations to obtain a comprehensive picture of biological function. As dye performances tend to be broadly distributed, the study of complex, processive reactions of this kind benefit from large numbers of single-molecule observations. High-throughput, potentially automated instrumentation has the potential to enable highly parallelized observations. An example of such instrumentation has been achieved with the recent adaptation of a commercially available DNA sequencing machine into a widely applicable high-throughput platform for up to four-color single-molecule experiments [64].

Biological impact of multi-color smFRET

As exemplified by the photophysical shortcomings discussed above, smFRET imaging with multiple fluorophores remains remarkably challenging. Proof-of-principle and other early studies have often focused on oligonucleotides [9,54,65], which are photophysically favorable [34] but enable only a limited scope of investigations.

Owing to the advent of optimized dyes and imaging conditions, three-color smFRET has successfully been applied to a variety of biological systems and made an impact on the study of ribosomal dynamics and assembly [27,30] as well as protein folding and binding (reviewed in [66]). Recent studies have investigated elastic deformations of the rotary motor of a bacterial ATP synthase [67], the quarternary structure of RecR oligomers [68], the activity and folding of 10-23 deoxyribozyme [52], conformational dynamics and ligand binding of the maltose-binding protein [31] and dynamics of intrinsically disordered proteins [53]. Four-color smFRET, by contrast, has yet to prove its potential outside of oligonucleotide systems [32,33,69].

While the scarcity of suitable combinations of fluorophores, as well as the challenges associated with the potential need for orthogonal biochemical labeling strategies are serious impediments, the demand for fluorophore performance in these kinds of experiments is an especially limiting aspect of three- and four-color single-molecule fluorescence imaging. We therefore expect developments in the area of highly stabilized fluorophores that span the visible spectrum will have a major impact in this field by improving signal-to-noise ratios and extended observation times.

To illustrate this point, we have used the dye conjugates Cy5-COT and Cy7-COT, in combination with Cy3B [48], to perform three-color smFRET imaging of intrinsic dynamic processes in the E.coli ribosome with high photon counts (Figure 4). As we have highlighted in our initial discussion (Figure 1b), measurements of this kind are able to differentiate between correlated and uncorrelated motion, in contrast to standard smFRET experiments, where individual structural perspectives are observed in isolation. In our multicolor example, ribosomal domain motions and tRNA fluctuations appear as two partially uncoupled processes that contribute to the formation of an activated reaction intermediate, consistent with previous observations [27].

Figure 4.

Figure 4

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Multi-color smFRET with self-healing fluorophores. Ribosomal pre-translocation complexes were labeled as shown (cartoon) on tRNA molecules and ribosomal protein L1 and imaged as described previously [27]. Fluorescence (top) and FRET traces (bottom) of labeled pre-translocation complex imaged at 40 ms time resolution during continuous laser excitation (120 mW, 532 nm wavelength).

Conclusions

More than a decade into the 21st century, the significance of single-molecule biology is no longer a matter of contention (compare [2],[5]). However, despite their ubiquity, single-molecule fluorescence studies have mostly focused on certain “sweet spots” on the map of spatiotemporal regimes relevant to biology [70].

As we have outlined in this review, some of the key obstacles to be overcome in cartographing new terrain include the photon emission rate and longevity of fluorescent dyes [7], the concentration limit imposed by extended observation volumes [71], the challenge to combine sufficient experimental throughput with high time resolution [19,64], the development of bio-orthogonal labeling strategies [72], and the application of single-molecule techniques to living cells [73]. Important inroads have been achieved to all of these aspects. While no technology can transcend its basic physical limitations, it is important to approach further development from the perspective of what is required to enable practical investigations, rather than purely focusing on improving technology for its own sake.

Multi-color smFRET has just begun to find its stride as a viable tool for insightful biological investigations. A synthesis of efforts in dye and detector development, labeling chemistry, data analysis, and advanced instrumental design will enable multi-color smFRET imaging to leverage its unique potential to elucidate correlated conformational events in vitro and in living cells.

Acknowledgments

Fluorophore and ribosome research in the Blanchard lab are supported by the National Institutes of Health (GM098859 and GM079238, respectively to S.C.B.). M.R.W. is supported by a graduate training fellowship from the National Institutes of Health. M.F.J. is supported by a postdoctoral fellowship from the German Academic Exchange Service (DAAD).

Footnotes

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References

References of special interest (•):

References of outstanding interest (••):

  • 1.Moerner WE. A Dozen Years of Single-Molecule Spectroscopy in Physics, Chemistry, and Biophysics. The Journal of Physical Chemistry B. 2002;106:910–927. [Google Scholar]
  • 2.Weiss S. Fluorescence spectroscopy of single biomolecules. Science. 1999;283:1676–1683. doi: 10.1126/science.283.5408.1676. [DOI] [PubMed] [Google Scholar]
  • 3.Xie XS, Lu HP. Single-molecule enzymology. J Biol Chem. 1999;274:15967–15970. doi: 10.1074/jbc.274.23.15967. [DOI] [PubMed] [Google Scholar]
  • 4.Giepmans BNG, Adams SR, Ellisman MH, Tsien RY. The Fluorescent Toolbox for Assessing Protein Location and Function. Science. 2006;312:217–224. doi: 10.1126/science.1124618. [DOI] [PubMed] [Google Scholar]
  • 5.Joo C, Balci H, Ishitsuka Y, Buranachai C, Ha T. Advances in Single-Molecule Fluorescence Methods for Molecular Biology. Annual Review of Biochemistry. 2008;77:51–76. doi: 10.1146/annurev.biochem.77.070606.101543. [DOI] [PubMed] [Google Scholar]
  • 6.Blanchard SC. Single-molecule observations of ribosome function. Curr Opin Struct Biol. 2009;19:103–109. doi: 10.1016/j.sbi.2009.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7••.Zheng Q, Juette MF, Jockusch S, Wasserman MR, Zhou Z, Altman RB, Blanchard SC. Ultra-stable organic fluorophores for single-molecule research. Chemical Society Reviews. 2014 doi: 10.1039/c3cs60237k. An in-depth review of the photophysical mechanisms of fluorophore instabilities and different approaches for their mitigation. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8•.Ha T, Tinnefeld P. Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging. Annual Review of Physical Chemistry. 2012;63:595–617. doi: 10.1146/annurev-physchem-032210-103340. An outstanding review of the photophysics and photochemistry of fluorophores used for single-molecule and super-resolution imaging. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hohng S, Joo C, Ha T. Single-molecule three-color FRET. Biophys J. 2004;87:1328–1337. doi: 10.1529/biophysj.104.043935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10••.Hohng S, Lee S, Lee J, Jo MH. Maximizing information content of single-molecule FRET experiments: multi-color FRET and FRET combined with force or torque. Chemical Society Reviews. 2014;43:1007–1013. doi: 10.1039/c3cs60184f. An up-to-date review of multi-color smFRET and approaches combining smFRET with measurements of orthogonal properties like force or torque. [DOI] [PubMed] [Google Scholar]
  • 11.Kim H, Ha T. Single-molecule nanometry for biological physics. Reports on Progress in Physics. 2013;76 doi: 10.1088/0034-4885/76/1/016601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12••.Altman RB, Terry DS, Zhou Z, Zheng Q, Geggier P, Kolster RA, Zhao Y, Javitch JA, Warren JD, Blanchard SC. Cyanine fluorophore derivatives with enhanced photostability. Nature Methods. 2012;9:68–71. doi: 10.1038/nmeth.1774. A strategy to improve the stability of organic fluorophores by covalent attachment of protective agents (“self-healing dyes”) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Altman RB, Zheng Q, Zhou Z, Terry DS, Warren JD, Blanchard SC. Enhanced photostability of cyanine fluorophores across the visible spectrum. Nature Methods. 2012;9:428–429. doi: 10.1038/nmeth.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Förster T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Annalen der Physik. 1948;437:55–75. [Google Scholar]
  • 15.Roy R, Hohng S, Ha T. A practical guide to single-molecule FRET. Nat Methods. 2008;5:507–516. doi: 10.1038/nmeth.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Schuler B. Single-molecule FRET of protein structure and dynamics - a primer. Journal of Nanobiotechnology. 2013;11 doi: 10.1186/1477-3155-11-S1-S2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ha T, Enderle T, Ogletree DF, Chemla DS, Selvin PR, Weiss S. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc Natl Acad Sci U S A. 1996;93:6264–6268. doi: 10.1073/pnas.93.13.6264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Arai Y, Nagai T. Extensive use of FRET in biological imaging. Microscopy. 2013;62:419–428. doi: 10.1093/jmicro/dft037. [DOI] [PubMed] [Google Scholar]
  • 19•.Schuler B, Hofmann H. Single-molecule spectroscopy of protein folding dynamics—expanding scope and timescales. Current Opinion in Structural Biology. 2013;23:36–47. doi: 10.1016/j.sbi.2012.10.008. Focusing on protein folding dynamics, this review lays out how fluorescence techniques can cover different timescales over many orders of magnitudes. [DOI] [PubMed] [Google Scholar]
  • 20.Zhuang X. Single-molecule RNA science. Annu Rev Biophys Biomol Struct. 2005;34:399–414. doi: 10.1146/annurev.biophys.34.040204.144641. [DOI] [PubMed] [Google Scholar]
  • 21.Wang F, Greene EC. Single-Molecule Studies of Transcription: From One RNA Polymerase at a Time to the Gene Expression Profile of a Cell. Journal of Molecular Biology. 2011;412:814–831. doi: 10.1016/j.jmb.2011.01.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Petrov A, Chen J, O'Leary S, Tsai A, Puglisi JD. Single-Molecule Analysis of Translational Dynamics. Cold Spring Harbor Perspectives in Biology. 2012;4 doi: 10.1101/cshperspect.a011551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Zhao Y, Terry D, Shi L, Weinstein H, Blanchard SC, Javitch JA. Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature. 2010;465:188–193. doi: 10.1038/nature09057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zhao Y, Terry DS, Shi L, Quick M, Weinstein H, Blanchard SC, Javitch JA. Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature. 2011;474:109–113. doi: 10.1038/nature09971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Akyuz N, Altman RB, Blanchard SC, Boudker O. Transport dynamics in a glutamate transporter homologue. Nature. 2013;502:114–118. doi: 10.1038/nature12265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Munro JB, Altman RB, Tung CS, Sanbonmatsu KY, Blanchard SC. A fast dynamic mode of the EF-G-bound ribosome. Embo J. 2010;29:770–781. doi: 10.1038/emboj.2009.384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Munro JB, Altman RB, Tung CS, Cate JH, Sanbonmatsu KY, Blanchard SC. Spontaneous formation of the unlocked state of the ribosome is a multistep process. Proc Natl Acad Sci U S A. 2010;107:709–714. doi: 10.1073/pnas.0908597107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Geggier P, Dave R, Feldman MB, Terry DS, Altman RB, Munro JB, Blanchard SC. Conformational sampling of aminoacyl-tRNA during selection on the bacterial ribosome. J Mol Biol. 2010;399:576–595. doi: 10.1016/j.jmb.2010.04.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lee S, Lee J, Hohng S. Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time. PLoS ONE. 2010;5:e12270. doi: 10.1371/journal.pone.0012270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30••.Kim H, Abeysirigunawarden SC, Chen K, Mayerle M, Ragunathan K, Luthey-Schulten Z, Ha T, Woodson SA. Protein-guided RNA dynamics during early ribosome assembly. Nature. 2014;506:334–338. doi: 10.1038/nature13039. Application of three-color smFRET to protein-RNA interactions during ribosome assembly. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kim E, Lee S, Jeon A, Choi JM, Lee HS, Hohng S, Kim HS. A single-molecule dissection of ligand binding to a protein with intrinsic dynamics. Nature Chemical Biology. 2013 doi: 10.1038/nchembio.1213. advance online publication. [DOI] [PubMed] [Google Scholar]
  • 32••.Lee J, Lee S, Ragunathan K, Joo C, Ha T, Hohng S. Single-Molecule Four-Color FRET. Angewandte Chemie International Edition. 2010;49:9922–9925. doi: 10.1002/anie.201005402. First demonstration of four-color smFRET imaging, with application to DNA Holliday junctions. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.DeRocco V, Anderson T, Piehler J, Erie DA, Weninger K. Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes. BioTechniques. 2010;49:807–816. doi: 10.2144/000113551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Stennett EMS, Ciuba MA, Levitus M. Photophysical processes in single molecule organic fluorescent probes. Chemical Society Reviews. 2013 doi: 10.1039/c3cs60211g. [DOI] [PubMed] [Google Scholar]
  • 35.Zheng Q, Jockusch S, Zhou Z, Blanchard SC. The Contribution of Reactive Oxygen Species to the Photobleaching of Organic Fluorophores. Photochemistry and Photobiology. 2013 doi: 10.1111/php.12204. n/a-n/a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.van de Linde S, Krstic I, Prisner T, Doose S, Heilemann M, Sauer M. Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging. Photochemical & Photobiological Sciences. 2011;10:499–506. doi: 10.1039/c0pp00317d. [DOI] [PubMed] [Google Scholar]
  • 37.Benesch RE, Benesch R. Enzymatic Removal of Oxygen for Polarography and Related Methods. Science. 1953;118:447–448. doi: 10.1126/science.118.3068.447. [DOI] [PubMed] [Google Scholar]
  • 38.Aitken CE, Marshall RA, Puglisi JD. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J. 2008;94:1826–1835. doi: 10.1529/biophysj.107.117689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Swoboda M, Henig J, Cheng HM, Brugger D, Haltrich D, Plumeré N, Schlierf M. Enzymatic Oxygen Scavenging for Photostability without pH Drop in Single-Molecule Experiments. ACS Nano. 2012;6:6364–6369. doi: 10.1021/nn301895c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Schäfer P, van de Linde S, Lehmann J, Sauer M, Doose S. Methylene Blue- and Thiol-Based Oxygen Depletion for Super-Resolution Imaging. Analytical Chemistry. 2013;85:3393–3400. doi: 10.1021/ac400035k. [DOI] [PubMed] [Google Scholar]
  • 41.Eggeling C, Widengren J, Rigler R, Seidel CAM. Applied Fluorescence in Chemistry, Biology and Medicine. Springer; Berlin Heidelberg: 1999. Photostability of Fluorescent Dyes for Single-Molecule Spectroscopy: Mechanisms and Experimental Methods for Estimating Photobleaching in Aqueous Solution; pp. 193–240. [Google Scholar]
  • 42.Dave R, Terry DS, Munro JB, Blanchard SC. Mitigating Unwanted Photophysical Processes for Improved Single-Molecule Fluorescence Imaging. Biophys J. 2009;96:2371–2381. doi: 10.1016/j.bpj.2008.11.061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Vogelsang J, Kasper R, Steinhauer C, Person B, Heilemann M, Sauer M, Tinnefeld P. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed Engl. 2008;47:5465–5469. doi: 10.1002/anie.200801518. [DOI] [PubMed] [Google Scholar]
  • 44.Alejo Jose L, Blanchard Scott C, Andersen Olaf S. Small-Molecule Photostabilizing Agents are Modifiers of Lipid Bilayer Properties. Biophysical Journal. 2013;104:2410–2418. doi: 10.1016/j.bpj.2013.04.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45•.Levitus M, Ranjit S. Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments. Q Rev Biophys. 2011;44:123–151. doi: 10.1017/S0033583510000247. An outstanding review of the photophysics and photochemistry of cyanine dyes. [DOI] [PubMed] [Google Scholar]
  • 46.Zheng Q, Jockusch S, Zhou Z, Altman RB, Warren JD, Turro NJ, Blanchard SC. On the Mechanisms of Cyanine Fluorophore Photostabilization. The Journal of Physical Chemistry Letters. 2012;3:2200–2203. doi: 10.1021/jz300670p. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.van der Velde JHM, Ploetz E, Hiermaier M, Oelerich J, de Vries JW, Roelfes G, Cordes T. Mechanism of Intramolecular Photostabilization in Self-Healing Cyanine Fluorophores. ChemPhysChem. 2013;14:4084–4093. doi: 10.1002/cphc.201300785. [DOI] [PubMed] [Google Scholar]
  • 48.Cooper M, Ebner A, Briggs M, Burrows M, Gardner N, Richardson R, West R. Cy3B™: Improving the Performance of Cyanine Dyes. Journal of Fluorescence. 2004;14:145–150. doi: 10.1023/b:jofl.0000016286.62641.59. [DOI] [PubMed] [Google Scholar]
  • 49.Klehs K, Spahn C, Endesfelder U, Lee SF, Fürstenberg A, Heilemann M. Increasing the Brightness of Cyanine Fluorophores for Single-Molecule and Superresolution Imaging. ChemPhysChem. 2014;15:637–641. doi: 10.1002/cphc.201300874. [DOI] [PubMed] [Google Scholar]
  • 50.Lee SF, Vérolet Q, Fürstenberg A. Improved Super-Resolution Microscopy with Oxazine Fluorophores in Heavy Water. Angewandte Chemie International Edition. 2013;52:8948–8951. doi: 10.1002/anie.201302341. [DOI] [PubMed] [Google Scholar]
  • 51.Clamme JP, Deniz AA. Three-Color Single-Molecule Fluorescence Resonance Energy Transfer. ChemPhysChem. 2005;6:74–77. doi: 10.1002/cphc.200400261. [DOI] [PubMed] [Google Scholar]
  • 52.Jung J, Han KY, Koh HR, Lee J, Choi YM, Kim C, Kim SK. Effect of Single-Base Mutation on Activity and Folding of 10-23 Deoxyribozyme Studied by Three-Color Single-Molecule ALEX FRET. The Journal of Physical Chemistry B. 2012;116:3007–3012. doi: 10.1021/jp2117196. [DOI] [PubMed] [Google Scholar]
  • 53.Milles S, Koehler C, Gambin Y, Deniz AA, Lemke EA. Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range. Molecular BioSystems. 2012;8:2531–2534. doi: 10.1039/c2mb25135c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Person B, Stein IH, Steinhauer C, Vogelsang J, Tinnefeld P. Correlated Movement and Bending of Nucleic Acid Structures Visualized by Multicolor Single-Molecule Spectroscopy. ChemPhysChem. 2009;10:1455–1460. doi: 10.1002/cphc.200900109. [DOI] [PubMed] [Google Scholar]
  • 55.Haustein E, Schwille P. Fluorescence correlation spectroscopy: novel variations of an established technique. Annu Rev Biophys Biomol Struct. 2007;36:151–169. doi: 10.1146/annurev.biophys.36.040306.132612. [DOI] [PubMed] [Google Scholar]
  • 56.Campos LA, Liu J, Wang X, Ramanathan R, English DS, Munoz V. A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy. Nat Meth. 2011;8:143–146. doi: 10.1038/nmeth.1553. [DOI] [PubMed] [Google Scholar]
  • 57.Saurabh S, Maji S, Bruchez MP. Evaluation of sCMOS cameras for detection and localization of single Cy5 molecules. Optics Express. 2012;20:7338–7349. doi: 10.1364/OE.20.007338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58•.Huang F, Hartwich TMP, Rivera-Molina FE, Lin Y, Duim WC, Long JJ, Uchil PD, Myers JR, Baird MA, Mothes W, et al. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat Meth. 2013;10:653–658. doi: 10.1038/nmeth.2488. Video-rate super-resolution microscopy demonstrates the use of sCMOS cameras for fast single-molecule imaging. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59•.Wunderlich B, Nettels D, Benke S, Clark J, Weidner S, Hofmann H, Pfeil SH, Schuler B. Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes. Nature Protocols. 2013;8:1459–1474. doi: 10.1038/nprot.2013.082. Detailed description of a microfluidic mixing platform supporting single-molecule experiments over a wide range of timescales (milliseconds to minutes) [DOI] [PubMed] [Google Scholar]
  • 60.Juette MF, Rivera-Molina FE, Toomre DK, Bewersdorf J. Adaptive optics enables three-dimensional single particle tracking at the sub-millisecond scale. Applied Physics Letters. 2013;102 [Google Scholar]
  • 61.Berglund A, Mabuchi H. Tracking-FCS: Fluorescence correlation spectroscopy of individual particles. Optics Express. 2005;13:8069–8082. doi: 10.1364/opex.13.008069. [DOI] [PubMed] [Google Scholar]
  • 62.Munro JB, Altman RB, O'Connor N, Blanchard SC. Identification of two distinct hybrid state intermediates on the ribosome. Mol Cell. 2007;25:505–517. doi: 10.1016/j.molcel.2007.01.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Wang L, Altman RB, Blanchard SC. Insights into the molecular determinants of EF-G catalyzed translocation. RNA. 17:2189–2200. doi: 10.1261/rna.029033.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64••.Chen J, Dalal RV, Petrov AN, Tsai A, O'Leary SE, Chapin K, Cheng J, Ewan M, Hsiung PL, Lundquist P, et al. High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence. Proceedings of the National Academy of Sciences. 2014;111:664–669. doi: 10.1073/pnas.1315735111. Adaptation of a commercial zero-mode waveguide based DNA sequencer as a high-throughput platform for multi-color single-molecule imaging of processive reactions. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Lee NK, Kapanidis AN, Koh HR, Korlann Y, Ho SO, Kim Y, Gassman N, Kim SK, Weiss S. Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances. Biophys J. 2007;92:303–312. doi: 10.1529/biophysj.106.093211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Gambin Y, Deniz AA. Multicolor single-molecule FRET to explore protein folding and binding. Molecular BioSystems. 2010;6 doi: 10.1039/c003024d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Ernst S, Düser MG, Zarrabi N, Börsch M. Three-color Förster resonance energy transfer within single FOF1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time. Journal of Biomedical Optics. 2012;17:0110041–0110049. doi: 10.1117/1.JBO.17.1.011004. [DOI] [PubMed] [Google Scholar]
  • 68.Kim C, Kim JY, Kim SH, Lee BI, Lee NK. Direct characterization of protein oligomers and their quaternary structures by single-molecule FRET. Chemical Communications. 2012;48:1138–1140. doi: 10.1039/c2cc16528g. [DOI] [PubMed] [Google Scholar]
  • 69.Stein IH, Steinhauer C, Tinnefeld P. Single-Molecule Four-Color FRET Visualizes Energy-Transfer Paths on DNA Origami. Journal of the American Chemical Society. 2011;133:4193–4195. doi: 10.1021/ja1105464. [DOI] [PubMed] [Google Scholar]
  • 70.Henzler-Wildman K, Kern D. Dynamic personalities of proteins. Nature. 2007;450:964–972. doi: 10.1038/nature06522. [DOI] [PubMed] [Google Scholar]
  • 71.Holzmeister P, Acuna GP, Grohmann D, Tinnefeld P. Breaking the concentration limit of optical single-molecule detection. Chemical Society Reviews. 2013 doi: 10.1039/c3cs60207a. [DOI] [PubMed] [Google Scholar]
  • 72.Grohmann D, Werner F, Tinnefeld P. Making connections — strategies for single molecule fluorescence biophysics. Current Opinion in Chemical Biology. 2013 doi: 10.1016/j.cbpa.2013.05.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73•.Persson F, Barkefors I, Elf J. Single molecule methods with applications in living cells. Current Opinion in Biotechnology. 2013;24:1–8. doi: 10.1016/j.copbio.2013.03.013. Excellent review of the emerging application of single-molecule techniques to living cells. [DOI] [PubMed] [Google Scholar]

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