The full understanding of cellular functions requires information about protein numbers for various biomolecular assemblies and their dynamics, which can be partly accessed by super-resolution fluorescence microscopy. Yet, many protein assemblies and cellular structures remain below the accessible resolution on the order of tens of nanometers thereby evading direct observation of processes, like self-association or oligomerization, that are crucial for many cellular functions. Over the recent years, several approaches have been developed addressing concentrations and copy numbers of biomolecules in cellular samples for specific applications. This has been achieved by new labeling strategies and improved sample preparation as well as advancements in superresolution and single-molecule fluorescence microscopy. So far, none of the methods has reached a level of general and versatile usability due to individual advantages and limitations. In this article, important requirements of an ideal quantitative microscopy approach of general usability are outlined and discussed in the context of existing methods including sample preparation and labeling quality which are essential for the robustness and reliability of the methods and future applications in cell biology.
To quantify spatial protein-protein proximity (colocalization) in fluorescence microscopic images, cross-correlation and autocorrelation functions were decomposed into fast and slowly decaying components. The fast component results from clusters of proteins specifically labeled and the slow one from background/image heterogeneity. We show that the calculation of the protein-protein proximity index and the correlation coefficient are more reliably determined by extracting the fast-decaying component.
Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interference scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. Here, we present a protocol for combining IRM with a commercial TIRF microscope for the simult…
Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at different wavelengths. We applied MA to spectral data of purified Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of dioleoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two extrinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Forster res…
There remains a need for techniques to monitor thermal processes at high spatiotemporal resolution, with myriad potential applications in chemistry, biology, and engineering. Measurement of temperature from nanoscale molecular phenomena are particularly promising due to their general compatibility with biological systems. Among these phenomena, fluorescence polarization anisotropy is particularly attractive due to its high sensitivity to thermal and photophysical information, and its general utility with a wide spectrum of fluorophores. In this work, we propose that the measurement of the intr…
In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, th…
Diffusion processes in biological membranes are of interest to understand the macromolecular organisation and function of several molecules. Fluorescence Recovery After Photobleaching (FRAP) has been widely used as a method to analyse this processes using classical Brownian diffusion model. In the first part of this work, the analytical expression of the fluorescence recovery as a function of time has been established for anomalous diffusion due to long waiting times. Then, experimental fluorescence recoveries recorded in living cells on a membrane-bound protein have been analysed using three …
Single-molecule fluorescence techniques have revolutionized our ability to study proteins. However, the presence of a fluorescent label can alter the protein structure and/or modify its reaction with other species. To avoid the need for a fluorescent label, the intrinsic autofluorescence of proteins in the ultraviolet offers the benefits of fluorescence techniques without introducing the labelling drawbacks. Unfortunately, the low autofluorescence brightness of proteins has greatly challenged single molecule detection so far. Here we introduce optical horn antennas, a dedicated nanophotonic pl…
In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spe…
Amyloid proteins are associated with a broad spectrum of neurodegenerative diseases. However, it remains a grand challenge to extract molecular structure information from intracellular amyloid proteins in their native cellular environment. To address this challenge, we developed a computational chemical microscope integrating 3D mid-infrared photothermal imaging with fluorescence imaging, termed Fluorescence-guided Bond-Selective Intensity Diffraction Tomography (FBS-IDT). Based on a low-cost and simple optical design, FBS-IDT enables chemical-specific volumetric imaging and 3D site-specific m…
The poor photostability and low brightness of protein autofluorescence have been major limitations preventing the detection of label-free proteins at the single molecule level. Overcoming these issues, we report here a strategy to promote the photostability of proteins and use their natural tryptophan autofluorescence in the ultraviolet (UV) for fluorescence correlation spectroscopy (FCS). Combining enzymatic oxygen scavengers with antioxidants and triplet state quenchers greatly promotes the protein photostability, reduces the photobleaching probability and improves the net autofluorescence d…
Watt-level all polarization-maintaining femtosecond fiber laser source at 1100 nm for multicolor two-photon fluorescence excitation of fluorescent proteins
Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used a…
Conformational changes of single proteins are monitored in real time by Forster-type resonance energy transfer, FRET. Two different fluorophores have to be attached to those protein domains, which move during function. The distance between the fluorophores is measured by relative fluorescence intensity changes of FRET donor and acceptor fluorophore, or by fluorescence lifetime changes of the FRET donor. The fluorescence spectrum of a single FRET donor fluorophore is influenced by local protein environment dynamics causing apparent fluorescence intensity changes on the FRET donor and acceptor d…
More than twenty years ago, scientists succeeded in pushing the limits of optical detection to single molecules using fluorescence. This breakthrough has revolutionized biophysical measurements, but restrictions in photophysics and labeling protocols have motivated many efforts to achieve fluorescence-free single-molecule sensitivity in biological studies. Although several interesting mechanisms using vibrational spectroscopy, photothermal detection, plasmonics or microcavities have been proposed for biosensing at the single-protein level, no method has succeeded in direct label-free detection…
Using the ultraviolet autofluorescence of tryptophan aminoacids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labelling. However, the low autofluorescence signals have so far limited the UV detection to large proteins containing several tens of tryptophan residues. This limit is not compatible with the vast majority of proteins which contain only a few tryptophans. Here we push the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. Our results show how the combination of nano…
The plasma membrane of living cells is compartmentalized at multiple spatial scales ranging from the nano- to the meso-scale. This non-random organization is crucial for a large number of cellular functions. At the nanoscale, cell membranes organize into dynamic nano-assemblies enriched by cholesterol, sphingolipids and certain types of proteins. Investigating these nano-assemblies known as lipid rafts is of paramount interest in fundamental cell biology. However, this goal requires simultaneous nanometer spatial precision and microsecond temporal resolution which is beyond the reach of common…
Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput is necessary to answer the question of "who eats what, when, and where" in complex microbial communities. Here, we present a mid-infrared photothermal-fluorescence in situ hybridization (MIP-FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically-labelled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labelling w…
We present a novel method for real-time monitoring and kinetic analysis of fluorescence in situ hybridization (FISH). We implement the method using a vertical microfluidic probe containing a microstructure designed for rapid switching between a probe solution and a non-fluorescent imaging buffer. The FISH signal is monitored in real time during the imaging buffer wash, during which signal associated with unbound probes is removed. We provide a theoretical description of the method as well as a demonstration of its applicability using a model system of centromeric probes (Cen17). We demonstrate…
Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended c…
Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies which rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report a novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. …
The presynaptic free calcium dynamics of neurons due to various stimulation protocols is investigated in a mathematical model. The model includes calcium influx through voltage-dependent calcium channels, calcium buffering by endogenous and exogenous buffers as well as calcium efflux through ATP-driven plasma membrane calcium pumps and sodium/calcium exchangers. We want to support a specific way of modeling which starts on the level of single proteins. Each protein is represented by characteristics that are determined by single protein experiments and that are considered to be widely independe…
The fluorescence microscope is one of the most important tools in modern clinical diagnosis and biological science. However, its expense, size and limited field-of-view (FOV) are becoming bottlenecks in key applications such as large-scale phenotyping and low-resource-setting diagnostics. Here we report a low-cost, compact chip-scale fluorescence-imaging platform, termed the Fluorescence Talbot Microscopy (FTM), which utilizes the Talbot self-imaging effect to enable efficient fluorescence imaging over a large and directly-scalable FOV. The FTM prototype has a resolution of 1.2 microns and an …
DNA-interacting proteins have roles multiple processes, many operating as molecular machines which undergo dynamic metastable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner fast to sample the molecular transitions across the free energy landscape. Progress has been made over the past decade in utilising cutting-edge tools of the physical sciences to address challenging biological que…
Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here we combine Atomic Force Microscopy based force spectroscopy with Fluorescence Microscopy to produce systematic, high-resolution elasticity maps for three different types of live neuronal cells: cortical (embryonic rat), embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem cells) neurons. We measure how the stiffness of neurons chan…
Super-resolution imaging techniques have largely improved our capabilities to visualize nanometric structures in biological systems. Their application further enables one to potentially quantitate relevant parameters to determine the molecular organization and stoichiometry in cells. However, the inherently stochastic nature of the fluorescence emission and labeling strategies imposes the use of dedicated methods to accurately measure these parameters. Here, we describe a Bayesian approach to precisely quantitate the relative abundance of molecular oligomers from segmented images. The distribu…
It is crucial to measure position and conformational changes of a membrane-interacting protein relative to the membrane surface. This is however challenging because the thickness of a membrane is usually only about 4 nm. We developed a fluorescence method which makes use of the principle of FRET between a fluorophore and a cloud of quenchers encapsulated in a liposome, hence the name LipoFRET. LipoFRET can readily locate a fluorophore in different depths inside and at different heights above the membrane. We applied LipoFRET to study α-synuclein, a key player in the pathology of Parkinson's di…
One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response.We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precis…
Understanding the pathways by which viral capsid proteins assemble around their genomes could identify key intermediates as potential drug targets. In this work we use computer simulations to characterize assembly over a wide range of capsid protein-protein interaction strengths and solution ionic strengths. We find that assembly pathways can be categorized into two classes, in which intermediates are either predominantly ordered or disordered. Our results suggest that estimating the protein-protein and the protein-genome binding affinities may be sufficient to predict which pathway occurs. Fu…
Roughly half of a cells proteins are located at or near the plasma membrane. In this restricted space the cell senses its environment, signals to its neighbors and ex-changes cargo through exo- and endocytotic mechanisms. Ligands bind to receptors, ions flow across channel pores, and transmitters and metabolites are transported against con-centration gradients. Receptors, ion channels, pumps and transporters are the molecular substrates of these biological processes and they constitute important targets for drug discovery. Total internal reflection fluorescence microscopy suppresses background…
Plasmids are extra-chromosomal DNA molecules which code for their own replication. We previously reported a setup using genes coding for fluorescent proteins of two colors that allowed us, using a simple model, to extract the plasmid copy number noise in a monoclonal population of bacteria [J. Wong Ng et al., Phys. Rev. E, 81, 011909 (2010)]. Here we present a detailed calculation relating this noise to the measured levels of fluorescence, taking into account all sources of fluorescence fluctuations: the fluctuation of gene expression as in the simple model, but also the growth and division of…
Liquid-liquid phase separation occurs not only in bulk liquid, but also on surfaces. In physiology, the nature and function of condensates on cellular structures remain unexplored. Here, we study how the condensed protein TPX2 behaves on microtubules to initiate branching microtubule nucleation, which is critical for spindle assembly in eukaryotic cells. Using fluorescence, electron, and atomic force microscopies and hydrodynamic theory, we show that TPX2 on a microtubule reorganizes according to the Rayleigh-Plateau instability, like dew droplets patterning a spider web. After uniformly coati…
Quantitative fluorescence analysis is often used to derive chemical properties, including stoichiometries, of biomolecular complexes. One fundamental underlying assumption in the analysis of fluorescence data -- whether it be the determination of protein complex stoichiometry by super-resolution, or step-counting by photobleaching, or the determination of RNA counts in diffraction-limited spots in RNA fluorescence {\it in situ} hybridization (RNA-FISH) experiments -- is that fluorophores behave identically and do not interact. However, recent experiments on fluorophore-labeled DNA-origami stru…
Superresolution fluorescence microscopy techniques beat the diffraction limit, enabling ultra-high resolution imaging in biological physics and nanoscience. In all cases that have been studied experimentally, the resolution scales inversely with the square root of some parameter that measures the number of photons used. However, this ubiquitous limit arises from very distinct mechanisms in different approaches, raising the question of whether it is a fundamental limit that cannot be exceeded, or merely a coincidence of the techniques studied thus far. We demonstrate that, under very general as…
We present a novel experimental setup in which magnetic and optical tweezers are combined for torque and force transduction onto single filamentous molecules in a transverse configuration to allow simultaneous mechanical measurement and manipulation. Previously we have developed a super-resolution imaging module which in conjunction with advanced imaging techniques such as Blinking assisted Localisation Microscopy (BaLM) achieves localisation precision of single fluorescent dye molecules bound to DNA of ~30 nm along the contour of the molecule; our work here describes developments in producing…
The interactions of a protein, its phase behavior, and ultimately, its ability to function, are all influenced by the interactions between the protein and its hydration waters. Here we study proteins with a variety of sizes, shapes, chemistries, and biological functions, and characterize their interactions with their hydration waters using molecular simulation and enhanced sampling techniques. We find that akin to extended hydrophobic surfaces, proteins situate their hydration waters at the edge of a dewetting transition, making them susceptible to unfavorable perturbations. We also find that …
Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process, but also to quantify interactions, for instance within molecular complexes. We combined Fluorescence Correlation Spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutio…
