What is life? There is hardly a more fundamental question raised by aspiring researchers, and one less prone to ever be answered in a scientifically satisfying way. In the long, productive and highly influential period of research following his Nobel-recognised work on relaxation kinetics, Manfred Eigen made seminal contributions towards a quantifiable definition of life, with a strong focus on its evolutionary character. In the last years of his time as an active researcher, however, he devoted himself to another, purely experimental topic: the detection and analysis of single biomolecules in aqueous solution. In this short review, I will give an overview of the groundbreaking contributions to the field of single molecule research made by Eigen and coworkers, and show that both, in its intrinsic motivation, and in its consequences, single molecule research strongly relates to the question of the physical–chemical essence of life. In fact, research on living systems with single molecule sensitivity will always refer the researcher to the question of the simplest possible representation, and thus the origin, of any biological phenomenon. Keywords Fluorescence correlation spectroscopy · Single molecule detection · Molecular evolution · Synthetic biology Introduction out this obvious discrepancy, but the scientific community would have suffered a great loss if it had not appeared as In 1994, a review paper appeared that greatly inspired a gen- such. Briefly, in this paper, the authors express their excite - eration of researchers at the interface of chemistry, physics, ment about a new technical breakthrough that Rigler and his and biology. To date, it has been cited more than 1000 times, coworkers had previously accomplished: the direct detection and a wealth of research projects, patents, and even compa- of single fluorescently labeled molecules diffusing freely in nies have followed in its wake. “Sorting single molecules: aqueous solution. Application to diagnostics and evolutionary biotechnology” That this has become possible was primarily due to three (Eigen and Rigler 1994) is a hallmark of scientific commu - important accomplishments, two of them being purely tech- nication co-authored by National Academy member Man- nical—the availability of stable and sufficiently strong lasers fred Eigen, a grand scientific authority and Nobel laureate. on one hand, and of sensitive-enough detectors to record Remarkably, in spite of his many outstanding contributions single photons on the other hand. The most important and to science, this late one turned out to become his third high- ingenious accomplishment, however, was the optical setup of est cited journal paper. Even more remarkable, however, is the new single molecule device presented in the framework its scientific content, which is only moderately review-like in of a method called FCS—Fluorescence Correlation Spec- character, instead rather serving the purpose of a visionary troscopy. It consisted of a confocal optical pathway, in which perspective. If submitted by less eminent authors, editors the laser was focused down to the resolution limit by an and reviewers would have potentially requested to straighten objective with high numerical aperture (NA), and the focal spot so created projected through an aperture with similarly small orifice in the image plane, a so-called pinhole, which Special Issue: Chemical Kinetics, Biological Mechanisms and limited the detection volume additionally in axial dimen- Molecular Evolution. sion. This produced open volume elements in the femtoliter * Petra Schwille range—nine orders of magnitude below what could be pipet- email@example.com ted in the lab, and close to the size of single bacterial cells. FCS was at that time already a twenty-year-old concept, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Vol.:(0123456789) 1 3 494 European Biophysics Journal (2018) 47:493–498 originally designed as a technique to analyze spontaneous the ability of actually identifying them individually, or even fluctuations around a thermodynamic equilibrium—and of finding them in a volume nine or more orders of magni- thus, in fact, a relaxation technique such as the ones that tude larger than the confocal probe volume. With respect to Eigen had pioneered, but with the important difference that evolutionary biotechnology, the challenge was in fact even no external disturbances needed to be induced, such as a greater, as detection was only the first step towards any pos- pressure or temperature jump (Magde et al. 1972; Ehren- sible cycle of variation, selection and amplification. How to berg and Rigler 1974). The history of how FCS was origi- hold on to a single molecule in solution? Although, intrigu- nally conceived by the consequent extrapolation of Eigen’s ingly, the following decades indeed brought forward practi- own methods is nicely set out in another article in this issue cal realizations of molecular traps (Fields and Cohen 2010), (Rigler and Widengren, this issue). To make FCS work, Eigen and Rigler already then sketched some solutions, however, the ability to resolve random thermodynamic fluc- based on a combination of microfluidic sample handling and tuations on the molecular level was indispensable, and this electrical fields, that were remarkably visionary. Microsys- first became convincingly possible by the new confocal illu - tems technology had at that time just started to provide mination and detection (Rigler and Widengren 1990; Rigler attractive miniaturization perspectives for the biosciences. et al. 1993). Once being able to detect the presence of single Indeed, only a couple of years after the release of this article, molecules, and their fluctuating number in an open volume researchers accomplished the detection and tracking of sin- element, through random bursts of fluorescence, temporal gle fluorescent molecules (Schmidt et al. 1996), and aston- autocorrelation analysis yields statistically relevant informa- ishingly, there are now even commercial solutions for single tion about all kinds of processes that influence the dynamics molecule DNA sequencing (Eid et al. 2009). However, the of these molecules, from simple three-dimensional diffusion original idea of sorting single molecules for evolutionary to inter- and intramolecular reactions. biotechnology had been dropped by Eigen and Rigler after a The presentation of FCS and the breakthrough accom- relatively short time, due to the many obvious experimental plishment of its confocal representation by Rigler and his difficulties presented, and has to my knowledge not been colleagues to a broader audience was, however, only one followed up on by other groups to date. Instead, Eigen and motivation of the 1994 PNAS paper. Another one was the coworkers pioneered many other influential applications in implication that single molecule detection obviously had to the following decade, with a strong focus on molecular diag- the other big scientific topic of Eigen’s research: evolution- nostics, which I will briefly review in the next section. ary biotechnology. The recognition that evolutionary mecha- nisms manifest themselves at much simpler levels than the ones of living organisms, which Eigen had greatly supported Applications and variations of FCS by his quantitative concepts, such as the molecular quasispe- in the 1990s cies theory, made it extremely exciting to speculate about the possibility of directly evolving molecules by rounds of In the years following the 1994 PNAS paper, there were mutation and selection, i.e., without having to go through the two technical breakthroughs in particular that greatly tedious process of amplifying them by the genotype–pheno- helped FCS to receive the attention that Eigen and Rigler type coupling of producing organisms. Being able to observe had envisioned. This was the combination of FCS detec- functional differences in single molecules, and to sort them tion with microfluidic sample handling on one side (Brink - according to these differences, would open up the possibil- meier et al. 1999; Dörre et al. 1997), and the establish- ity for the researcher to artificially establish any kind of fit- ment of dual-color cross-correlation on the other side ness measure for selection that he/she would have in mind. (Schwille et al. 1997). Playing to the strengths of the ris- Besides that, a detection sensitivity that high would have ing microsystems technology, advocating the “lab on the exciting consequences for biomedicine in a much broader chip”, it became possible to sample volumes much larger sense: the possibility to detect traces of infectious agents, than the confocal spot in a relatively short time, and by such as single viruses, in blood serum, would open up a this overcome the limits of passive diffusion, which is fast completely new chapter of diagnostics. The last vision that on small scales, but painstakingly slow on large scales. Eigen and Rigler brought forward in this seminal article The theory of fluctuation correlation analysis of flowing sounded even more far-fetched: the possibility of single mol- samples (Magde et al. 1978) was expanded, and it could ecule DNA sequencing, based on the successive detection of be shown that by implementing spatial correlation between fluorescently labeled single bases or base-pairs. two or more different volume elements, flow directions Of course, the ability to optically detect the transits of could be determined (Brinkmeier et al. 1999), and cali- single u fl orescent molecules through the light cone of a laser bration-free diffusion measurements without knowing the focused into an aqueous environment, although a technical exact shape and size of the measurement volumes were breakthrough at that time, was still a long stretch away from possible (Dittrich and Schwille 2002). 1 3 European Biophysics Journal (2018) 47:493–498 495 The impact of dual-color cross-correlation spectroscopy particles, supposed to induce the misfolding, aggregation (FCCS) realized by Schwille et al. (1997) received even and cytotoxicity of cellular proteins, resulting in severe dam- more widespread attention, as it provided, for the first time, age and degeneration of neurons in the brain, and in fatal a direct way to measure molecular interactions between two diseases such as “Mad Cow” and Creutzfeldt-Jakob. Due species of molecules in any transparent environment, and to the excellent features of FCS to access protein aggrega- with single molecule sensitivity. In contrast to the analysis tion through the change of molecular brightness and diffu- of irreversible reactions through the change of diffusion of sion, which had been outlined very early as a key applica- one, usually small, reaction partner upon binding to a large tion (Meyer and Schindler 1988), prion protein diagnostics or immobile target, which had up to then been the most became the latest but definitely not least successful line of prominent FCS application to biochemical kinetics (Kinjo active research in Manfred Eigen’s department. Initiated and Rigler 1995), FCCS now induced no more constraints to by a theoretical article about the potential mechanisms of molecular sizes. First demonstrated with a differently labeled infectivity on the molecular level (Eigen 1996), the group pair of complementary oligonucleotides and later expanded combined FCS and particularly FCCS with strategies for to protein–protein interactions in binary or even ternary sys- large sample handling: identifying rare species of early tems (Heinze et al. 2004), FCCS has been established as infectious aggregates in a large background of soluble mono- a very powerful technical advance to FCS that later even mers was apparently the greatest challenge for diagnostics. found commercial realization. In FCCS, the shape of the Small traces of double-colored early aggregates having been cross-correlation curve compiled from two separate fluctu- formed by differently labeled monomeric precursors could ating fluorescence signals, resulting from different species be best identified by scanning the laser focus through the of molecules diffusing through a focal spot illuminated by sample, or by flowing the sample through microfluidic chan- two different laser beams, is no longer the primary source nels crossing the detection volume (Bieschke et al. 2000). of information. Instead, the focus of FCCS analysis is on In these dual-color applications, it became increasingly the amplitude of the curve, reflecting directly on the pres- evident that the mathematical correlation procedure which ence of dual-colored molecules, usually the reaction prod- gave FCS and FCCS its name was not really relevant any- uct from two species of single-colored species, supposed to more. Rare events, such as the occurrence of aggregates or interact with each other. However, also the reverse reaction, of other sparse infectious targets could be evidenced through the cleavage or breaking of bonds between two molecules, the simultaneous occurrence of fluorescence bursts, a so- or two subunits of a single molecule, each bearing a separate called coincidence, in two separate measurement channels. fluorescent label, can be precisely quantified by FCCS. This This recognition was also the starting point to the late truly could be demonstrated by the analysis of enzymatic cleav- biotechnological applications of FCS-related methods in the age reactions, and thus, specifically, the activity of endonu- Eigen group. They continued with the concept of character- cleases and proteases on double-labeled DNA templates or izing enzymatic activity through the formation or breakage proteins (Kettling et al. 1998; Kohl et al. 2005). of bonds in target molecules, but now optimized the perfor- With respect to the diagnostic potential of FCS, exciting mance of the method towards true large number screening applications with fluorescently labeled short DNA prim- (Koltermann et al. 1998; Winkler et al. 1999; Heinze et al. ers targeted to large DNA or RNA targets from pathogens, 2002). Scanning the beam or flowing the sample, in addition such as HIV, could be demonstrated (Walter et al. 1996; to more quickly helping to find rare species like needles in a Oehlenschläger et al. 1996). Already small traces of patho- haystack, also greatly helped to decrease the time required to genic DNA, when amplified by PCR, resulted in a notice- compile statistically relevant signals from diluted solutions. able difference in the average diffusion behavior of small To analyze enzymatic activity, the perspective of recording probes, evidenced by a shift of the autocorrelation function meaningful data from very small amounts of enzyme and to larger decay times. The kinetics of these recognition reac- template in a short amount of time laid the foundation for tions depended critically on the number of corresponding screening routines, where thousands of different types of accessible base-pairs and could thus even be used to assess enzymes, e.g., evolutionary optimized ones, could be tested secondary structures of targets (Schwille et al. 1996). Also, in fractions of a second each. Thus, this late streak of appli- the onset of probe elongation allowed the total number of cations published by the Eigen group to some extent con- pathogenic nucleic acid molecules to be inferred, and posi- firmed the visions laid out in the 1994 PNAS paper, although tive samples to be distinguished from false positives. the direct detection and sorting of evolved molecules them- In the mid-90s, another alarming class of pathogens selves was no longer a goal, but all the more the smart adap- had just started to surface: the prions—infectious protein tation of FCS-related routines for commercial applications. 1 3 496 European Biophysics Journal (2018) 47:493–498 reduced by employing fluorescence two-photon excitation Continuation towards cells and organisms (Schwille et al. 1999a, b). Herein, the excitation laser is in the 2000s tuned to approximately twice the absorption wavelength of the respective dyes. Using pulsed excitation with high pho- With respect to basic science, it soon appeared that FCS and ton flux during the pulses, there is a significant probability FCCS, realized in a very similar instrumental setup as used of simultaneous absorption of two photons, which add their for confocal laser scanning microscopes, which now became energy to make the required transition to the excited state of very widespread in the biosciences, would be a fantastic com- the fluorophore. The resulting square dependence of fluores- plement to imaging for quantitatively targeting processes in cence on intensity shows a very strong spatial decay away live cells or even organisms. The very fundamental informa- from the focal plane, significantly reducing out-of-focus tion about fluorescently labeled molecules that FCS could photobleaching. In addition, the much broader excitation deliver—concentrations and mobilities, turned out to be spectra of standard dyes for two-photon excitation allow the important for the characterization of biochemical processes simultaneous excitation of spectrally distinct labels, as used occurring in living cells. In the postgenomic era, the new for dual-color FCCS, with one beam (Heinze et al. 2000; challenge of proteomics has been to yield detailed informa- Kim et al. 2004). tion about the amount of expressed proteins in any cell, as Another instrumental solution to minimize dynamic and well as their multiple interactions. To access these data sep- cumulative photobleaching is to use beam-scanning, which aration-free in an unperturbed system has been a great prom- had already been successfully applied in the diagnostic ise by FCS. Thus, cellular applications of FCS and FCCS applications outlined above. The scan pathway can either became increasingly popular in the 2000s, aided by the com- be a closed circle sampled at constant scan speed, enabling mercial instrumental solutions that were often integrated into data recording throughout the scan (Petrásek and Schwille laser scanning microscopes (Schwille 2001). Early cellular 2008), or a straight line, which is scanned repeatedly. Here, FCS yielded diffusion constants in the cytoplasm, the nucleus due to the requirements of turning around, constant record- and the membrane (Brock et al. 1998; Schwille et al. 1999a, ing cannot be carried out, and smart data processing has to b; Wachsmuth et al. 2000), as well as transport processes in be applied to identify and separate the right signal periods tubular structures (Köhler et al. 2000; Gennerich and Schild (Ries et al. 2009a, b). Utilizing these sophisticated scanning 2000). With regard to molecular interactions, dual-color and data recording procedures, FCS was enabled in cells FCCS allowed the determination of cellular enzyme activity and organisms with optically quite precluding conditions, (Kohl et al. 2005), to characterize various mechanisms of such as bacteria and yeast, as well as C. elegans and D. endocytosis (Bacia et al. 2002), and to study protein–protein rerio embryos (Meacci et al. 2006; Petrásek et al. 2008; Ries interactions to the point that even reactions with variable et al. 2009a, b). FCCS in early zebrafish embryos allowed stoichiometry could be quantified (Kim et al. 2005, 2007). the quantitative mapping of the establishment of morphogen However, it soon became apparent that there are two gradients (Yu et al. 2009) and quantification of the affin- major practical challenges which render FCS measure- ity of receptor–morphogen interactions in situ (Ries et al. ments in cells particularly complicated, or even preclude 2009a, b). them: The first one is photobleaching, which always occurs at high light intensities as used for confocal illumination. It is a nuisance for all fluorescence applications, including Towards minimal biological systems imaging, but particularly harmful for FCS, as it introduces additional time-scales to the dynamic analysis. The second In spite of the great success stories of FCS and related single challenge is more fundamental in nature. The theoretical molecule techniques when applied to cells, it became in the concept of FCS is targeted towards analyzing fluctuations last years even more evident that such sensitive methods around thermodynamic equilibrium. The physiological situ- will be subject to all kinds of nonidealities and disturbances ation in cells, however, is far away from being equilibrated. occurring in living systems. Parameters that can be deter- In addition to that, the requirement of a small open probe mined with very high statistical accuracy in well-defined volume in a much larger sample pool is often not fulfilled, solutions will suffer from great tolerances and huge error due to the cellular ultrastructure, retaining many molecules bars when measured in the much less well-defined and con- in compartments close to or even far below the resolution trolled cell interior. Even worse, it will hardly be possible to limit. This requires very careful calibration of the optical unequivocally assign the effect of any change in condition on setup and preparation of the biological system, as well as a cellular process to a particular variable, as indirect effects the use of smart routines for data processing. or the involvement of unknown other factors can never be It has been demonstrated that the cumulative photobleach- fully ruled out. Thus, meaningful cellular applications of ing due to small reservoirs in cells can be dramatically 1 3 European Biophysics Journal (2018) 47:493–498 497 FCS have increasingly been complemented by applications and if so, could we reconstitute a biochemical system that in reconstituted systems, retaining the essential features indeed allows to witness a transition from chemistry into and variables, but otherwise greatly simplifying the land- biology? In particular, which ones would be the first consti- scape of relevant parameters. The most popular platform tutive processes? Eigen mainly focused on the emergence for such cell mimetic studies are giant unilamellar vesicles of information and, in particular, of evolutionary mecha- (GUVs) that provide a perfect environment for investigating nisms, but there are other fundamental features, such as processes taking place on cell membranes (Korlach et al. metabolism and compartmentation, whose functional ori- 1999; Schwille et al. 1999a, b). They have been particularly gins also deserve great attention (Schwille 2017). From our influential in characterizing the effect of local membrane own recent work on the emergence of self-organization and structure and fluidity on the diffusion and interactions of pattern formation in minimal protein–membrane systems lipids and proteins (Bacia et al. 2004; Kahya and Schwille (Loose et al. 2008; Zieske and Schwille 2014), we can con- 2006). In these greatly simplified systems, it became appar - clude that single molecule methods such as FCS are uniquely ent that physical phenomena, such as lipid phase separa- suited to characterize and understand these fundamental tion, or mechanical constraints due to cytoskeletal filaments processes. This now finally closes a circle: single molecule attached (Heinemann et al. 2013) influence key biochemical analysis had been proposed by Eigen and Rigler to aid the processes and thus, dynamics as measured by FCS. understanding of molecular evolution. And after many years Reconstitution of biological functions in minimal systems, of single molecule research, we are referred back to the ini- i.e., systems with a minimal number of species and/or free tial questions of the origin of biology, when searching for parameters, has generally become very attractive also for the optimal system to apply our single molecule methods to. investigations by single molecule imaging. The most promi- Acknowledgements Open access funding provided by Max Planck nent scenario is the analysis of single molecules of motor Society. I am greatly indebted to Manfred Eigen, for creating an atmos- proteins, e.g., when moving along filaments (Vale et al. phere of true scientific interdisciplinarity and independent thinking 1996). It has led to new mechanistic insight or confirmed during my own doctoral training, and for many inspirations before and after. existing hypotheses, such as the recognition of rotary move- ment of the ATP synthase through an attached actin filament Open Access This article is distributed under the terms of the Crea- (Noji et al. 1997). For biophysics in general, the concept of tive Commons Attribution 4.0 International License (http://creat iveco assembling biological functionality from the bottom-up, in mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- a synthetic approach, seems very appropriate and rewarding tion, and reproduction in any medium, provided you give appropriate when aiming for reproducible and quantitatively meaningful credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. results (Schwille and Diez 2009). On the other hand, this can only be achieved by at least partly sacrificing the “physiologi- cal relevance” as cherished by most biologists. That leads to References a certain dichotomy, nowadays observed with respect to the understanding of biology. Do we understand biology by tak- Bacia K, Majoul IV, Schwille P (2002) Probing the endocytic pathway ing into account each and every aspect of its compositional in live cells using dual-color fluorescence cross-correlation analy - and dynamic makeup? Or do we understand biology by trying sis. Biophys J 83:1184–1193 to separate underlying fundamental principles from specific, Bacia K, Scherfeld D, Kahya N, Schwille P (2004) Fluorescence cor- relation spectroscopy relates rafts in model and native membranes. presumably exchangeable, compositional representations? In Biophys J 87:1034–1043 other words: is today’s obvious physiological complexity an Bieschke J et al (2000) Ultrasensitive detection of pathological prion attribute of life that was acquired through evolution, or is it protein aggregates by dual-color scanning for intensely fluorescent the essence of life as we know it? If the former is true, it is targets. PNAS 97(10):5468–5473 Brinkmeier M, Dörre K, Stephan J, Eigen M (1999) Two-beam cross- exciting to speculate about greatly simplified models of living correlation, a method to characterize transport phenomena in entities, which would be the ideal study objects for all bio- micrometer-sized structures. Anal Chem 71:609–616 physical techniques, but first and foremost for the exquisitely Brock R, Hink MA, Jovin TM (1998) Fluorescence correlation micros- sensitive single molecule methods. copy of cells in the presence of autofluorescence. Biophys J 75(5):2547–2557 Dittrich PS, Schwille P (2002) Spatial two-photon fluorescence cross- correlation spectroscopy for controlling molecular transport in Conclusions and outlook: back to the origin microfluidic structures. Anal Chem 74:4472–4479 of life Dörre K et al (1997) Techniques for single molecule sequencing. 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Published: Mar 22, 2018
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