Improving certainty in single molecule imaging

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ScienceDirect Improving certainty in single molecule imaging John SH Danial1,2 and Ana J Garcı´a-Sa´ez1,2 The spatiotemporal organization of biological entities and the complex network of interactions they sponsor has proven challenging to visualize. Modern biophysics has brought a wealth of techniques for probing cellular structure and dynamics of which fluorescence-based single molecule detection has emerged as a powerful tool. In this review, we summarize notable breakthroughs in biological research based on single molecule imaging, identify prevailing shortcomings in single molecule detection and present current opinion on ameliorating some of its limitations for wider applicability.

Addresses 1 Max Planck Institute for Intelligent Systems, Heisenbergstraße 3, 70569 Stuttgart, Germany 2 Interfaculty Institute of Biochemistry, University of Tu¨bingen, Hoppe-Seyler-Straße 4, 72076 Tu¨bingen, Germany Corresponding author: Garcı´a-Sa´ez, Ana J ([email protected])

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For instance, localizing single molecules has proven detrimental in resolving the structure of the nuclear pore scaffold [2], periodicity of the axonal cytoskeleton [3], folding of chromatin [4], architecture of apoptotic protein assemblies [5], clustering of membrane proteins [6] and spatiotemporal dynamics of the endoplasmic reticulum [7]. Counting single molecules could describe the stoichiometry of pore-forming toxins [8–10], kinetochore complex [11], stator component of the bacterial flagella motor [12] and numerous channels and receptors [13,14]. Tracking single molecule has revealed the molecular organization of cellular membranes [15,16], step-wise motion of molecular motors [17,18], confined motion of membrane proteins in the periodic neural cytoskeleton, dynamics of cargo transport in micro tubular networks [19,20] and nanoscale organization of mitochondrial micro compartments [21]. Monitoring conformational changes in single molecules has, so far, identified rate-determining intermediates in ribosomal translocation, catalytic motions in ATP synthase, transport dynamics in a glutamate transporter and the unsynchronized motion of subunits in an aspartate transporter [22–28].

This review comes from a themed issue on Biophysical methods Edited by Carol Robinson and Carla Schmidt

http://dx.doi.org/10.1016/j.sbi.2017.04.007 0959-440X/ã 2017 Elsevier Ltd. All rights reserved.

Introduction Introducing their 1989 seminal findings, Moerner and Kador suggested that “the detection of single absorbers in a solid would provide an important new tool for the study of local absorber-host interactions that would be uncomplicated by the normal averaging over as many as 104 to 1016 similar absorbers” [1]. Prompted by this claim, chemists, physicists, and, engineers embarked on developing brighter labels, better optical microscopes, and, more sensitive detectors to detect single molecules under physiological conditions. It is almost two decades and immense progress has already been achieved in that respect. Today, several laboratories afford the detection of single fluorescent molecules—a surge that has led to a breakthrough in our understanding of the behavior of many biological molecules and their assemblies (Figure 1). Current Opinion in Structural Biology 2017, 46:24–30

In spite of these advances, the detection of single molecules remains limited in application. A number of factors cap the resolution across the three axes of detection: length, time and intensity. With these limits, distances closer than 5 nm cannot be resolved, events faster than 1 ms are missed, and, intensity changes comparable in magnitude to the amplitude of noise are misjudged. The dependence of different single molecule imaging modalities on more than one of those factors limit localization to a spatial resolution of 10 nm, monitoring to a temporal resolution of 1 ms, tracking to 10 nm ms 1 and counting to 7–30 copies per oligomer (Figure 1). With these numbers in scope, one can think of numerous situations in biology where imaging smaller and faster is needed. To satisfy those needs, certainty in single molecule imaging has to be improved (Figure 2). In this review, we identify the exclusive requirements for each single molecule modality and present current opinion on diminishing, reducing or, perhaps, compromising the associated sources of error for better and wider applicability across the biological spectrum.

Super-resolving macromolecular structures using single molecule localization microscopy Stochastic Optical Reconstruction Microscopy (STORM) [29], Photoactivatable Localization Microscopy (PALM) [30] and ground state depletion (GSD) [31] are examples of single molecule localization microscopy (SMLM) www.sciencedirect.com

Improving certainty in single molecule imaging Danial and Garcı´a-Sa´ez 25

Figure 1

Channels & transporters Gating dynamics stoichiometry

Receptors Binding dynamics organization movement ATP synthase stoichiometry

Endocytic pits Coat assembly structure

Mitochondrial proteins Assembly

Dynamics

Cross section

Pore-forming toxins Assembly

Flagella motor Mot B stoichiometry Motor proteins Rotation Translation

Structure Nuclear pore complex Transport dynamics pore structure

Kinases Folding dynamics

Ribosome Translocation dynamics

TFs Target search dynamic

Filamentous proteins Structure

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Chromatin folding Polymerase Organization dynamics

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Endoplasmic Reticulum Structure

Localizing

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d(t) Photobleaching trace is analyzed to obtain the number of monomers (copies) in a single oligomer. Noise limits precision in this method to 7 - 10 copies

The position of mobile molecules is record in ‘real’ time to measure their diffusion in different environment. Technique is limited by noise, temporal resolution, dye chemistry and photophysical roperties. Generally, the position of a single fluorophore can be precisely determined down to 10 nm at a 1 ms temporal resolution

HIV-1 Organization Dynamics

The ration of donor-to-acceptor emission is recorded to deduce the distance between two molecules in ‘real’ time. Technique is limited by noise and temporal resolutioin of currently available imaging technologies. As rule of thumb, dynamics faster than 1 ms and closer than 0.5 nm are not accessible to this technique

X0,y0

X1,y1

X2,y2

X3,y3

Random fluorophores are sequentially activated and localized to resolve a nanosized structure. Noise and dye chemistry limit precision to 5 - 10 nm

Current Opinion in Structural Biology

Upper semi-circle represents an atlas of single molecule experiments performed on cellular systems to date. Lower semi-circle describes the readout from the processes colored in the upper semi-circle and sets their current limitations. The experiments shown in map, together with the limitations of the techniques explained underneath, aim at highlighting areas in cellular biology that have already benefited from the application of single molecule methods, and implicitly point to others where a reduction in the technical limitations would permit improved applicability.

techniques which rely on the sequential activation and localization of random fluorophore subsets for overcoming the diffraction limit of light. The resolution of SMLM images is defined by the accuracy and precision of its localizations [32] and can range from 10 to 50 nm. Localization accuracy improves when the size of a label, and its target-linking moiety, get smaller. www.sciencedirect.com

Photo-Activatable Fluorescent Proteins (PA-FPs) can be genetically tagged to any target of interest but are large [33]. Smaller organic fluorophores can stain cellular targets, although this is achieved through large conventional antibodies [34]. ‘Nanobodies’ were developed to mitigate this problem [35], however, developing and validating them target-specific is costly and time consuming. One solution to this problem was to develop specific Current Opinion in Structural Biology 2017, 46:24–30

26 Biophysical methods

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(a) Radial tree map of the four single molecule experiments and their respective implementations. Sources of errors, in the 3rd level, are colorcoded corresponding to the errors and their solutions in (b). Thin color-coded radial sections correspond to conflicting solutions.

Current Opinion in Structural Biology 2017, 46:24–30

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Improving certainty in single molecule imaging Danial and Garcı´a-Sa´ez 27

GFP binding nanobodies. Targets linked to a GFP molecule can be readily tagged with nanobodies labeled with organic dyes. Although this renders binding generic, the presence of a large GFP molecule again deteriorates the localization accuracy. A third generation of nanobodies was recently reported to recognize a short linear epitope whose sequence can be genetically fused to any protein. The new class of nanobodies eliminates the need for expressing a large GFP tag but remain to be tested for compatibility with SMLM [36]. Localization accuracy is also improved in the absence of lateral drift. Bright nonblinking substrate-bound fiducial markers, such as gold nanoparticles and recently-reported nanodiamonds [37], can be used to track substrate drift with a single nanometer and frame precision. To avoid additional sample preparation efforts, localization-based cross correlation algorithms can be readily employed for image registration but at the expense of reduced spatiotemporal accuracy [38].

and particularly, where high-throughput imaging is required or the contribution of background noise cannot be neglected. Algorithms employing Maximum-Likelihood Estimation (MLE) and weighted Least Squares (LS) criterion were reported to computationally avoid the artificial effects of shot noise. The merits and applicability of those algorithms have been reviewed elsewhere [32,44].

Localization precision is determined by the photophysical properties of the label(s) used and the noise level of the detection system [39]. A suitable fluorophore would exhibit a high quantum yield and large number of shortlived switching cycles. PA-FPs can be used in SMLM modalities for their intrinsic ability to undergo ‘switching’ in the presence of ultraviolet illumination. Brighter organic fluorophores have been evaluated for suitability in SMLM and their switching dynamics were shown to improve in the presence of appropriate concentrations of molecular quenchers and thiols (see Refs. [40,41] for a complete list of switching dyes). Together with their high quantum yield and small size, these properties render them favorable for use in SMLM. In either case, the handful of photons emitted need to be efficiently recorded. Noise perturbs this process. The combination of shot noise, resulting from the stochastic arrival of photons on a detector, pixilation noise, resulting from the detection of photons on discrete pixels, and, background noise, resulting from the detection of unwanted photons represent the general term ‘noise’. The suppression of background noise correlates with improving the collection efficiency (i.e., improving optical circuitry design and implementation). Pixilation noise can be reduced by improving the detector’s fabrication process and increasing the number of accommodated pixels per unit area. sCMOS cameras offer the demonstrated possibility to image, up to, a 15 folds’ larger number of single molecules with a generous 20% higher signal-to-noise ratio compared to EMCCD cameras [42]. Shot noise can be reduced by spreading the signal (i.e., point spread function) onto a substantially large number of pixels to statistically ensure that each detects an average of less than a single photon per image [43]. This method, referred to as Ultrahigh Accuracy Imaging Modality (UAIM), eliminates the effects of shot noise almost entirely but might not be applicable in all situations

Probing the oligomeric state of biomolecules using single molecule counting methods

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Measuring the accuracy and precision of localizations in an experimental context is often complicated as it depends on the structure being imaged. Instead, the resolution of an image can be quantified. A new method was recently introduced based on the cross-correlation of Fourier-transformed sub-images each containing a subset of all localizations present in a single image [45]. The method known as Fourier Ring Correlation relies on sampling alone and, therefore, provides an unbiased measure of image quality [46].

In biology, knowledge about structure is often necessary for understanding function. In some situations, however, structural information is not always sufficient. This holds especially true for proteins that work as higher order assemblies. Single molecule counting methods offer the possibility to observe the dynamics of assembly formation by quantifying the underlying number of protein copies. To achieve this goal, either of the following strategies is used: a bottom-up approach, where molecules are counted one by one, or, a top-down one, where the number of molecules is deduced. ‘Step-wise photobleaching analysis’ is a bottom-up approach in which the time-dependent intensity trace of a single oligomer is filtered to detect discrete steps each corresponding to a monomer labeled with a single fluorophore [47]. In alternative top-down approaches, the brightness of individual oligomers can be related to the mean step size of a single monomer by division [48] or analyzed at the population level by calibration and fitting to a histogram [49] to obtain the total number of monomers. These methods are challenged by the incomplete maturation of fluorescent proteins, imperfect labeling efficiencies and spatial variation of the intensity of the excitation beam. Label-related uncertainties can be mitigated by quantifying the population of mature fluorescent proteins or partially labeled targets from bulk measurements and calculating true stoichiometry in retrospect [50]. Spatial intensity variations can be avoided through the use of adaptive optics to deliver homogenous illumination at the sample plane [51]. The accuracy with which the intensity of a single monomer can be appropriately quantified is limited by detection noise and would only permit counting of, up to, 7 copies by step detection, 10 by calibration and 30 by division [52]. Current Opinion in Structural Biology 2017, 46:24–30

28 Biophysical methods

The challenging requirement for precisely measuring the intensity of a single monomer is relieved in the ‘ratio comparison to fluorescent standards’ method where the intensity of an oligomer of unknown size is compared to the intensity of a ‘standard’ oligomer of known size [52]. For the comparison process to be valid, the standard must be distinguishable from, identically labeled as, and closely located to, the oligomer under study. The last requirement is difficult to fulfill in samples where the oligomer is randomly scattered across a field of view, and even more difficult to satisfy in organelles for which no standards have yet been engineered. The use of adaptive optics or image processing tools, as described above, can partially solve this problem, but the need for the standard to be located within the same focal plane of the oligomer has not been systematically satisfied. The use of step-wise photobleaching analysis and ratio comparison to fluorescent standards requires single oligomers to be resolvable. Many macromolecular assemblies do not fulfill this requirement, particularly in vivo, where they either are overexpressed or form anisotropic structures. Ratio comparison of oligomer-to-monomer blinking was reported to enable subunit counting in SMLM [53] but requires stoichiometric labeling of the protein of interest and the signal of the monomeric state as a reference. Although the method is capable of deriving insightful results on a population level, it is not suited for detecting numerical heterogeneities across oligomers. Other computational tools exist which have not been empirically verified [33,54,55] and are, thus, subject to further experimental scrutiny. On the biological side, stoichiometric labeling of proteins at their endogenous levels is key to avoiding overexpression artifacts. Direct knocking-in of fluorescent tags using CRISPR/Cas9 promises robust quantitative analysis of SMLM data in mammalian cells but remains to be fully characterized for this application.

Tracing the movement of single molecules using single particle tracking The movement of molecules is intrinsic to cellular life and, as such, its precise and accurate traceability is important for understanding their mechanisms of action. Single Particle Tracking (SPT) refers to the use of fluorescence and non-fluorescence-based methods for tracking the movement of a large number of single molecules in two and three dimensions with, ideally, single nanometer precision, microsecond temporal resolution and for long durations. The ultimate goal behind applying those methods is to uncover the structural organization of cellular structures and the spatiotemporal dynamics of their constituting molecules for providing insight into their molecular modes of action [56]. Tracking the displacement of a molecule is, alone, enough to detect confined movement and provide quantifiable measure of an environment’s spatial organization [20]. The Current Opinion in Structural Biology 2017, 46:24–30

Mean Squared-Displacement (MSD), and its dependent variable, the diffusion coefficient, can also be used to retrieve the hosting medium’s thermodynamic properties [57], where mobility is unrestricted, or, to discriminate between modes of diffusion [56], otherwise. The fidelity of both quantities in representing the underlying biological processes relies on maximizing the spatial and temporal resolution [58] along with satisfying the aforementioned requirements. Techniques for improving the localization precision through the suppression of noise have already been discussed although they might not be applicable here as they would compromise on the minimum achievable temporal resolution and, therefore, affect the accuracy of a measurement. On the other hand, increasing the signal requires high light levels that would lead to the shortening of trajectories. This problem can be mitigated using Quantum Dots (QDs, radius 5–10 nm), which can tolerate high light levels without photo blinking or bleaching [59]. Concern over the potential effects of the large size of QDs on the diffusion of small targets has, for long, been debated, however, evidence provided so far supports the independence of lateral diffusion from the size of the label.

Monitoring the dynamics of single biomolecules using single molecule Fo¨rster Resonance Energy Transfer Single molecule Fo¨rster Resonance Energy Transfer (smFRET) is the only tool available to measure intramolecular distances in real-time with angstrom spatial precision. It operates on the transfer of energy between closely located fluorophore pairs each containing a donor and an acceptor. Following excitation of the donor, energy is transferred from the donor to a closely located acceptor. The efficiency of energy transfer, or the ratio of donor-toacceptor emission, is distance-dependent; increasing with decreasing distance In spite of its merits, the utility of smFRET as a ‘nanoscopic ruler’ comes with plenty of challenges [60]. Of those, some are often circumventable, such as attaching fluorophore pairs to appropriate residues, identifying compaction due to crowding and accounting for spectral bleed. However, identifying equilibrium positions requires the conversion of an analog trace into a digital signal with steps corresponding to a biomolecule’s transition states. Noise affects the fidelity of this process. On the one hand, molecular quenching, resulting from energy transfer, can, in many experimental scenarios, decrease the intensity of one fluorophore in the pair to levels obscured by the time-independent average noise level. Even with a light-tight implementation, background noise, resulting from the incomplete rejection of the reflected excitation beam, might reach intolerable levels. On the other hand, time-dependent variations in www.sciencedirect.com

Improving certainty in single molecule imaging Danial and Garcı´a-Sa´ez 29

the background noise affect the detectability of transition states. One-step Hidden-Markov models [61] and change point analysis [62] were proposed to identify noise-deteriorated structural transitions provided that they are slower than the acquisition rate. Microsecond(s) transitions are over looked due to limitations in current imaging technology which can only image events as short as 1 ms without compromising the mean signal-to-noise ratio. Assuming a realistic situation in which the average lifetime of a transition is 5 times longer than the acquisition time, there is a significant probability of more than 15% that an event would occur between two consecutive acquisitions. In an attempt to reduce the resulting error in interpreting rapid signal fluctuations, a two-step analysis was recently proposed [63]. The methodology exploits Monte Carlo simulations to match ideal smFRET traces with those experimentally obtained. Implementing this additional step can result in a fourfold drop of the error incurred.

Conclusion The structural uniqueness of each biomolecule dictates cellular actions—from life to death. Counting, monitoring, localizing and tracking single molecules have become feasible, shedding new light on the structure and function of biological systems. To improve the accuracy of retrievable information, here we surveyed specific and common sources of errors in single molecule experiments and discussed modern means for reducing them. We stress on the requirement for accompanying experimental data by appropriate computational evidence to support, if applicable, minor, but important, observations. The upcoming challenge is to simultaneously combine single molecule methods to consolidate temporal and spatial scales, recapitulate the richness of cellular biology and firmly identify the distinct role of single molecules in biological activities.

Acknowledgments J.S.H.D is funded by a Max Planck Society postdoctoral fellowship (MaxPlanck-Gesellschaft). A.J.G.S is funded by Deutsche Forschungsgemeinschaft (DFG) grants FOR 2036 GA 164/2-1 and 3-1.

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