Perspectives on Fluorescence

Professor Gregorio Weber was an outstanding, dedicated scientist, versed in a great variety of scientific subjects. But he was also an exceptional human being: of a reserved nature, he never placed himself above or before others, never put people down. He was a cultivated man with a brilliant intellect, amicable, generous, modest, and prone to listen. He had a real concern with the underlying social issues in all the countries where he lived. His childhood, youth, and university training up to his first doctoral degree took place in his home country, Argentina, and these periods of his life had a deep impact on the shaping of his persona, his cultural habits, and his scientific interests. His great mind and avid quest for knowledge in all spheres of life were undoubtedly nourished by the high standards of the educational system in Argentina at that time but were also fired by the crucial influence of his family and cultural environment, his circle of talented friends, and the informal training that he received in Buenos Aires.

The trajectory of Gregorio Weber from his childhood to scientific eminence is examined in the context of the major personages and other influences that he encountered on the way. In the process, unique aspects of his personality, intellect, and philosophical outlook become apparent.

This is a personal account of my postdoctoral research period in 1976–1977 with Professor Gregorio Weber at the University of Illinois at Urbana-Champaign (USA). For me it was a year of enlightenment that had a large impact on my own academic career. Since we worked on (high-pressure) fluorescence research on flavins and flavoproteins in Urbana, I describe in somewhat larger detail our own completed research on this topic at Wageningen University (The Netherlands). Because of increasing research interest in cellular biochemistry and biophysics we have founded the Microspectroscopy Centre in Wageningen harboring sophisticated fluorescence microscopy setups.

Among the many contributions of Gregorio Weber to science and technology, the development of frequency domain technology in his lab in 1969 has caused a deep controversy, dividing scientists that will refuse using anything but the frequency domain approach to measure the fluorescence decay from the other camp that simply refuses anything but the time-correlated single photon counting (TCSPC) approach. Although at the time of the major contribution of Gregorio Weber and Richard Spencer in 1969, the TCSPC method was not yet invented, the basic controversy “frequency domain vs time domain” in the field continues today. We have made progress both in the scientific understanding and in describing the technical differences between the two approaches; still it is interesting how scientists continue to be divided. As for many of the contributions of Gregorio Weber that stirred controversy, I would like to mention a common theme of my conversations with Dr. Weber about refusing to follow a “god’ and about the independence of the scientific thinking from “common beliefs” that ultimately slows scientific progress. In this chapter I would like to describe the scientific progress brought about by Weber’s ideas in this specific “technological” area that should be judged by “pure” scientific analysis rather than by beliefs.

The very fast fluorescence anisotropy decay of N-acetyl-l-tryptophanamide (NATA) in aqueous solutions has been measured with sub-picosecond laser excitation and detection with time-correlated single photon counting. By using global analysis of both parallel and perpendicular polarized fluorescence intensity decays involving deconvolution, the rotational correlation times of NATA in the tens of picosecond range are accurately recovered. Since rotational correlation times are directly proportional to viscosity, we have used these correlation times to derive the (relative) microscopic viscosity of increasing concentrations of guanidine hydrochloride (GuHCl) in buffered water. GuHCl is a well-known chaotropic agent of protein denaturation. We give a step-by-step description how to obtain the final results. Subsequently, we compare the obtained microscopic viscosities with macroscopic viscosity data reported half a century ago using capillary viscosimeters. From the results it is clear that GuHCl, present in molar concentration, associates with NATA making the apparent molecular volume larger.

Weber’s red-edge effect is formulated as follows: “In rigid and highly viscous environments the excited-state energy transfer producing depolarization of fluorescence emission in concentrated dye solutions stops to be observed when fluorescence is excited at the red (long wavelength) edge of absorption spectrum.” After its discovery, it led to finding of a number of new wavelength-selective effects in spectral shifts, quenching, anisotropy and lifetimes, and also in different excited-state reactions forming a new vision of structural disorder and molecular dynamics in condensed media. These effects were consistently explained based on a new paradigm that accounts for statistical distribution of fluorescence emitters on their interaction energy with the environment leading to static or dynamic inhomogeneous broadening of spectra and to directional excited-state energy homo-transfer. These phenomena can be modulated by the energy of the excitation quanta. Their description, optimal conditions for their observation, information that they carry, and overview of their different applications are the subject of this chapter.

This chapter discusses the critical contributions of Gregorio Weber to the development of techniques to measure fluorescence lifetimes. The fluorescence lifetime is the average time required for a population of fluorophores in the excited state to decay to the ground state. Events in a fluorophore’s environment that influence the excited state can alter the lifetime, and this is measured using fluorescence lifetime imaging microscopy (FLIM). This chapter describes the application of FLIM to quantify Förster resonance energy transfer (FRET) between labeled proteins inside living cells. FRET is a non-radiative pathway through which a donor fluorophore in the excited state transfers energy to nearby acceptor molecules. The transfer of energy reduces the donor’s fluorescence lifetime, and this can be quantified by FLIM. Since energy transfer occurs through near-field electromagnetic interactions, it can only occur over a distance of 80 angstroms or less. Thus, FRET microscopy has become a valuable tool for investigating biochemical networks inside living cells. In this regard, Gregorio Weber recognized the importance of measuring the biological and physical properties of proteins as integrated systems. Here, proteins labeled with the genetically encoded fluorescent proteins (FPs) are used to demonstrate how FRET-FLIM enables robust and sensitive measurements of protein interactions inside living cells.

The judicious use of traditional spectroscopy light sources throughout the postwar era led to the foundations of fluorescence spectroscopy, both theoretically and experimentally. Those principles provided many tools for understanding the structure and dynamics of macromolecules, cells, and even tissues. In the last four decades those tools have been supplemented and sometimes extended by the availability of novel light sources, advanced electronics, and burgeoning computing power. This chapter will chronicle the former – the impact of four decades of laser evolution upon biological fluorescence spectroscopy and microscopy. It is necessarily focused on only the systems that were most popular and influential (many other sources were of great value) and (for space concerns) it also summarizes only a few of the many linked technological advances.

Research highlights cited here illustrate some unconventional usage of fluorescent probes in biophysical studies on sterol superlattices in model membranes. The use of small sterol mole fraction increments over a wide range correctly delineates the global trend as well as the fine details of the effects of sterol content on membrane properties. An alternating variation of fluorescence signals and membrane properties with sterol content, with maxima or minima appeared at critical sterol mole fractions, was observed in many different membrane systems and can be explained by the sterol superlattice model. This model has been progressing over the last two decades. The current model links sterol superlattice formation with condensed complex formation, gives a deeper understanding of the liquid-ordered phase, and reveals two concentration-induced sharp phase transitions immediately below and above a critical sterol mole fraction for maximal superlattice formation. The density and size of membrane rafts isolated from model membranes as detergent resistant membrane fragments show characteristics typical for sterol superlattices, which suggests that membrane rafts and sterol superlattices are closely related. The concept of sterol superlattice formation can be used to optimize liposomal drug formulations and develop a method for a facile screening of lipid-soluble antioxidants for potency and toxicity.

As Gregorio Weber anticipated in his seminal 1979 article, 6-acyl-2-(dimethylamino)naphthalene probes became excellent tools to study nanosecond relaxation processes of biological systems. Examples are the use of PRODAN (or DANCA) to study relaxation of specific protein matrixes, or LAURDAN (as well as PRODAN) extensively used to study the extent of water dipolar relaxation processes in biological membranes. In this chapter a novel application for this family of molecules is presented and discussed. Specifically, we show how these fluorescent probes can be used to monitor intracellular water dipolar relaxation in living cells displaying oscillatory metabolism. Our experimental results show a strong coupling between metabolism and intracellular water dynamics, challenging the view that water in the interior of cells exists mostly as a medium whose global properties are comparable to the properties of dilute solutions. The observed results can be very well interpreted in light of the association-induction hypothesis postulated by Gilbert Ling in 1962.

In addition to his pioneering work utilizing quantitative fluorescence techniques to study biological macromolecules and assemblies, Gregorio Weber contributed substantially to our understanding of the thermodynamic relationships between different ligand-binding equilibria and subunit association equilibria in proteins. These ideas have proven to be very valuable in clarifying the basis for allosteric modification of enzyme behavior when allosteric ligands act predominantly to modify the affinity of an enzyme’s substrate. Dr. Weber’s influence, both scientific and personal, on the author’s efforts to understand the allosteric behavior of prokaryotic and eukaryotic phosphofructokinase (PFK) in particular is described. These observations not only serve to illuminate the regulatory properties of this important enzyme, but they also, in turn, serve to illustrate the validity of the principles that Weber originally described. Not surprisingly fluorescence techniques have played important roles in elucidating many of these insights.

Gregorio Weber’s legacy, in addition to seminal contributions in the application of fluorescence to the study of biological molecules, includes, as well, a profound understanding of how protein–protein interactions are intimately coupled to their interactions with ligands. Such energetic and structural coupling implies that protein sequences have evolved such that these interactions are finely tuned to the physiological habitat and state of the organisms in which these proteins function. The work of my group, in collaboration with a number of biologists and biochemists over the years, has sought to discover how protein–protein interactions, both homologous oligomerization and heterologous complex formation, are implicated in the regulation of gene expression. Herein are given several examples of how fluorescence can be applied to characterize the molecular and energetic basis for the role of protein interactions in the regulation of gene expression. Described are several fluorescence approaches, some quite basic and others more complex, how they were applied to specific gene expression regulatory systems both in vitro and in vivo, and what information could be extracted from the results. Apparent from these few examples is the central role played by protein–protein interactions in these regulatory mechanisms, and how any model for regulatory mechanisms must take into account these higher order protein interactions.

Absorption of light by solvatochromic dyes including tryptophan leads to formation of an excited state with a change in the magnitude and/or direction of its permanent electric dipole moment. The excited state can interact with surrounding atoms with formation of a relaxed state. The kinetics of this process can be measured by time/energy-resolved fluorescence spectroscopy. Time-dependent spectral shifts (time-resolved emission spectra) provide information about protein relaxation. Ground-state heterogeneity, two-state excited-state reactions, and dielectric relaxation all give rise to time-dependent spectral shifts. Ways to tell these processes apart are discussed. Examples are presented which illustrate how rates and extent of spectral shifts can differentiate between ordered and disordered parts of a protein molecule. Rates of spectral shifts can be related to nanosecond motions of specific protein residues.

The fluorescence polarization technique that Prof. Weber developed at Cambridge University between the late 1940s and early 1950s has had a tremendous impact on our understanding of the structure and dynamics of macromolecules and in the analysis of proteins interactions and detection of target proteins in biologically complex samples. His decision to develop dimethylaminonaphthalene sulfonyl chloride (Dansyl-Cl) as the first probe for fluorescence polarization studies was brilliant, as its long fluorescence lifetime and well-defined dipole are ideally suited to study protein conjugates as large as 100 kDa. Indeed, after almost 70 years, the Dansyl group is still the probe of choice for in vitro applications of fluorescence polarization. Unfortunately, Dansyl is not very suitable for related studies in living cells, primarily because it requires excitation in the near ultraviolet, while the in vivo labeling of a target protein with Dansyl group is challenging. We have developed a new class of genetically encoded fluorescent protein that may help to overcome these limitations. The lumazine-binding protein (LUMP) harbors a fluorescent probe with a cerulean-colored emission that like Dansyl has a long excited state lifetime (14 ns). Moreover, LUMP has a smaller mass than GFP that allows us to genetically append capture sequences as large as 20 kDa and still generate a fusion protein with sufficient dynamic range in the fluorescence polarization value to quantify the amounts of the free and target-bound states in an equilibrium. In this article, I will compare and contrast key features of Dansyl and LUMP as probes for fluorescence polarization studies and discuss the potential of using LUMP and related encoded proteins to advance the application of fluorescence polarization to analyze target proteins and protein interactions in living cells.

The fundamental unit of biology is unarguably the cell. Thus, as we move forward in our understanding of the processes occurring in the cell, it is crucial to reflect on how much of the cell biophysics remains unexplained or unknown. A ubiquitous observation in cell biology is that the translational motion of molecules within the intracellular environment is strongly suppressed as compared to that in dilute solutions. By contrast, molecular rotation is not affected by the same environment, indicating that the close proximity of the molecule must be aqueous. Theoretical models provide explanations for this apparent discrepancy pointing to the presence of macromolecular intracellular crowding, but with expectations that depend on the nanoscale organization assigned to crowding agents. A satisfactory experimental discrimination between possible scenarios has remained elusive due to the lack of techniques to explore molecular diffusion at the appropriate spatiotemporal scale in the 3D-intracellular environment. Here we discuss our recent experimental evidences for molecular diffusion in crowded biological media. By using monomeric GFP as a fluorescent tracer, and spatiotemporal fluorescence correlation spectroscopy (FCS) as main analytical tool, we reconstruct an imaginary journey, one molecule at a time, across intracellular compartments, such as cytoplasm and nucleoplasm, as well as within subcellular dynamic nanostructures, such as the nuclear pore complex. Results in cells are complemented by in vitro experiments where a variety of model systems mimic physiological crowding conditions. During this journey, Gregorio Weber intuitions on the nature of the cell protoplasm (see below) and on the intrinsic link between the spatial and temporal scales of diffusion processes both inspired our measurements and guided data interpretation. We do believe that the experimental observations on molecular diffusion collected in the interior of cells might influence the way biochemical reactions take place, with possible significant contributions to our understanding of crucial, still obscure phenomena, e.g., the biological benefit of anomalous transport, the regulation of protein folding/unfolding, intracellular signaling, target-search processes, and bimolecular reactions kinetics.

In the last decades, several techniques have been developed to push the spatial resolution of far-field fluorescence microscopy beyond the diffraction limit. Stimulated emission depletion (STED) microscopy is a super-resolution technique in which the targeted switching off of the fluorophores by a secondary laser beam results in an effective increase in optical resolution. However, to fully exploit the maximum performances of a STED microscope (effective spatial resolution achievable for a given STED beam’s intensity, versatility, live-cell imaging capability, etc.) several experimental precautions have to be considered. In this respect, the temporal dimension (at the pico- and nanosecond scale) has often a central role on the overall efficiency and versatility of a STED microscope, working in pulsed or continuous-wave mode.

In pulsed STED, temporal alignment between the excitation and STED pulses has direct consequences on the maximum spatial resolution achievable by the STED microscope. In a specific pulsed STED implementation, called single wavelength two-photon excitation STED, the modulation of the temporal width of the pulse results in the use of the very same laser for excitation and depletion of the fluorophores. In continuous-wave (CW)-STED, the analysis of nanosecond fluorescence dynamics allows one to preserve the effective resolution of a STED microscope, but with a significant reduction of the illumination intensity. In this respect, we discuss two different approaches for the analysis of nanosecond dynamics in CW-STED images, namely the so-called gated-STED microscopy and Separation of Photons by LIfetime Tuning (SPLIT)-STED microscopy. Overall, these examples show that concepts developed in time-resolved fluorescence spectroscopy are important for the advancement of optical super-resolution microscopy.

DC-SIGN (a single-pass transmembrane protein and C-type lectin) is a major receptor for a variety of pathogens on human dendritic cells including dengue virus (DENV), which has become a global health threat. DENV binds to cell-surface DC-SIGN and the virus/receptor complexes migrate to clathrin-coated pits where the complexes are endocytosed; during subsequent processing, the viral genome is released for replication. DC-SIGN exists on cellular plasma membranes in nanoclusters that may themselves be clustered on longer length scales that appear as microdomains in wide-field and confocal fluorescence microscopy. We have investigated the dynamic structure of these clusters using fluorescence and super-resolution imaging in addition to large-scale single particle tracking. While clusters themselves can be laterally mobile there appears to be little mobility of DC-SIGN within clusters or exchange of DC-SIGN between the clusters and the surroundings. We end this account with some outstanding issues that remain to be addressed with respect to the composition and architecture of DC-SIGN domains and some highly unusual aspects of their lateral mobility on the cell surface that may accompany and perhaps facilitate DENV infection.