Extracting, quantifying, and comparing dynamical and biomechanical properties of living matter through single particle tracking.

A panoply of new tools for tracking single particles and molecules has led to an explosion of experimental data, leading to novel insights into physical properties of living matter governing cellular development and function, health and disease. In this Perspective, we present tools to investigate the dynamics and mechanics of living systems from the molecular to cellular scale via single-particle techniques. In particular, we focus on methods to measure, interpret, and analyse complex data sets that are associated with forces, materials properties, transport, and emergent organisation phenomena within biological and soft-matter systems. Current approaches, challenges, and existing solutions in the associated fields are outlined in order to support the growing community of researchers at the interface of physics and the life sciences. Each section focuses not only on the general physical principles and the potential for understanding living matter, but also on details of practical data extraction and analysis, discussing limitations, interpretation, and comparison across different experimental realisations and theoretical frameworks. Particularly relevant results are introduced as examples. While this Perspective describes living matter from a physical perspective, highlighting experimental and theoretical physics techniques relevant for such systems, it is also meant to serve as a solid starting point for researchers in the life sciences interested in the implementation of biophysical methods.

Temperature Controls Onset and Period of NF-κB Oscillations and can Lead to Chaotic Dynamics.

The transcription factor NF-κB plays a vital role in the control of the immune system, and following stimulation with TNF-α its nuclear concentration shows oscillatory behaviour. How environmental factors, in particular temperature, can control the oscillations and thereby affect gene stimulation is still remains to be resolved question. In this work, we reveal that the period of the oscillations decreases with increasing temperature. We investigate this using a mathematical model, and by applying results from statistical physics, we introduce temperature dependency to all rates, resulting in a remarkable correspondence between model and experiments. Our model predicts how temperature affects downstream protein production and find a crossover, where high affinity genes upregulates at high temperatures. Finally, we show how or that oscillatory temperatures can entrain NF-κB oscillations and lead to chaotic dynamics presenting a simple path to chaotic conditions in cellular biology.

Thermoplasmonic nano-rupture of cells reveals annexin V function in plasma membrane repair.

Maintaining the integrity of the cell plasma membrane (PM) is critical for the survival of cells. While an efficient PM repair machinery can aid survival of healthy cells by preventing influx of extracellular calcium, it can also constitute an obstacle in drug delivery and photothermal therapy. We show how nanoscopic holes can be created in a controlled fashion to the cell's plasma membrane, thus allowing identification of molecular components which have a pivotal role in PM repair. Cells are punctured by laser induced local heating of gold nanostructures at the cell surface which causes nano-ruptures in cellular PMs. Recruitment of annexin V near the hole is found to locally reshape the ruptured plasma membrane. Experiments using model membranes, containing recombinant annexin V, provide further biophysical insight into the ability of annexin V to reshape edges surrounding a membrane hole. The thermoplasmonic method provides a general strategy to monitor the response to nanoscopic injuries to the cell surface which offer new insight into how cells respond to photothermal treatment.

Foregut organ progenitors and their niche display distinct viscoelastic properties in vivo during early morphogenesis stages.

Material properties of living matter play an important role for biological function and development. Yet, quantification of material properties of internal organs in vivo, without causing physiological damage, remains challenging. Here, we present a non-invasive approach based on modified optical tweezers for quantifying sub-cellular material properties deep inside living zebrafish embryos. Material properties of cells within the foregut region are quantified as deep as 150 µm into the biological tissue through measurements of the positions of an inert tracer. This yields an exponent, α, which characterizes the scaling behavior of the positional power spectra and the complex shear moduli. The measurements demonstrate differential mechanical properties: at the time when the developing organs undergo substantial displacements during morphogenesis, gut progenitors are more elastic (α = 0.57 ± 0.07) than the neighboring yolk (α = 0.73 ± 0.08), liver (α = 0.66 ± 0.06) and two mesodermal (α = 0.68 ± 0.06, α = 0.64 ± 0.06) progenitor cell populations. The higher elasticity of gut progenitors correlates with an increased cellular concentration of microtubules. The results infer a role of material properties during morphogenesis and the approach paves the way for quantitative material investigations in vivo of embryos, explants, or organoids.

Changes in Cell Morphology and Actin Organization in Embryonic Stem Cells Cultured under Different Conditions.

The cellular cytoskeleton provides the cell with a mechanical rigidity that allows mechanical interaction between cells and the extracellular environment. The actin structure plays a key role in mechanical events such as motility or the establishment of cell polarity. From the earliest stages of development, as represented by the ex vivo expansion of naïve embryonic stem cells (ESCs), the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. Naïve ESCs have a characteristic morphology, and the ultrastructure that underlies this condition remains to be further investigated. Here, we investigate the 3D actin cytoskeleton of naïve mouse ESCs using super-resolution optical reconstruction microscopy (STORM). We investigate the morphological, cytoskeletal, and mechanical changes in cells cultured in 2i or Serum/LIF media reflecting, respectively, a homogeneous preimplantation cell state and a state that is closer to embarking on differentiation. STORM imaging showed that the peripheral actin structure undergoes a dramatic change between the two culturing conditions. We also detected micro-rheological differences in the cell periphery between the cells cultured in these two media correlating well with the observed nano-architecture of the ESCs in the two different culture conditions. These results pave the way for linking physical properties and cytoskeletal architecture to cell morphology during early development.

Optical trapping reveals differences in dielectric and optical properties of copper nanoparticles compared to their oxides and ferrites.

In a nanoplasmonic context, copper (Cu) is a potential and interesting surrogate to less accessible metals such as gold, silver or platinum. We demonstrate optical trapping of individual Cu nanoparticles with diameters between 25 and 70 nm and of two ionic Cu nanoparticle species, CuFe2O4 and CuZnFe2O4, with diameters of 90 nm using a near infrared laser and quantify their interaction with the electromagnetic field experimentally and theoretically. We find that, despite the similarity in size, the trapping stiffness and polarizability of the ferrites are significantly lower than those of Cu nanoparticles, thus inferring a different light-particle interaction. One challenge with using Cu nanoparticles in practice is that upon exposure to the normal atmosphere, Cu is spontaneously passivated by an oxide layer, thus altering its physicochemical properties. We theoretically investigate how the presence of an oxide layer influences the optical properties of Cu nanoparticles. Comparisons to experimental observations infer that oxidation of CuNPs is minimal during optical trapping. By finite element modelling we map out the expected temperature increase of the plasmonic Cu nanoparticles during optical trapping and retrieve temperature increases high enough to change the catalytic properties of the particles.

Plasmonic Heating of Nanostructures.

The absorption of light by plasmonic nanostructures and their associated temperature increase are exquisitely sensitive to the shape and composition of the structure and to the wavelength of light. Therefore, much effort is put into synthesizing novel nanostructures for optimized interaction with the incident light. The successful synthesis and characterization of high quality and biocompatible plasmonic colloidal nanoparticles has fostered numerous and expanding applications, especially in biomedical contexts, where such particles are highly promising for general drug delivery and for tomorrow's cancer treatment. We review the thermoplasmonic properties of the most commonly used plasmonic nanoparticles, including solid or composite metallic nanoparticles of various dimensions and geometries. Common methods for synthesizing plasmonic particles are presented with the overall goal of providing the reader with a guide for designing or choosing nanostructures with optimal thermoplasmonic properties for a given application. Finally, the biocompatibility and biological tolerance of structures are critically discussed along with novel applications of plasmonic nanoparticles in the life sciences.

Quantification of Loading and Laser-Assisted Release of RNA from Single Gold Nanoparticles.

Novel RNA-based technologies provide an avenue of possibilities to control the regulation of gene expression in cells. To realize the full potential of small interfering RNA (siRNA)-based therapy, efficient delivery vehicles and novel strategies for triggering release from carrier vehicles have to be developed. Gold nanoparticles (AuNPs) with sizes of ∼50-150 nm have the ability to accumulate in tumor tissue and can be transported across the membrane by endocytosis. Therefore, a laser-controlled oligonucleotide release from such particles is of particular interest. Here, we quantify the loading of specifically attached microRNA oligonucleotides (miRNA) onto single gold nanoparticles with diameters of 80, 100, 150, and 200 nm. We show that AuNPs have a curvature-dependent density of miRNA loading: the higher the curvature, the higher the loading density. Moreover, we demonstrate how one sensing strand of an RNA duplex can be dehybridized and hence released from the AuNP by heating the AuNP by irradiation with a near-infrared (NIR) laser. Laser-induced release is also demonstrated inside living cells. Together, these findings show that plasmonic nanoparticles with high curvatures are ideal carriers of oligonucleotides into cells, and their cargo can be released in a controlled manner by a thermoplasmonic mechanism. Importantly, this remotely controlled release strategy can be applied to any cargo attached to a plasmonic nanocarrier, on either the single particle or ensemble level.

Cancer cells' ability to mechanically adjust to extracellular matrix stiffness correlates with their invasive potential.

Increased tissue stiffness is a classic characteristic of solid tumors. One of the major contributing factors is increased density of collagen fibers in the extracellular matrix (ECM). Here, we investigate how cancer cells biomechanically interact with and respond to the stiffness of the ECM. Probing the adaptability of cancer cells to altered ECM stiffness using optical tweezers-based microrheology and deformability cytometry, we find that only malignant cancer cells have the ability to adjust to collagen matrices of different densities. Employing microrheology on the biologically relevant spheroid invasion assay, we can furthermore demonstrate that, even within a cluster of cells of similar origin, there are differences in the intracellular biomechanical properties dependent on the cells' invasive behavior. We reveal a consistent increase of viscosity in cancer cells leading the invasion into the collagen matrices in comparison with cancer cells following in the stalk or remaining in the center of the spheroid. We hypothesize that this differential viscoelasticity might facilitate spheroid tip invasion through a dense matrix. These findings highlight the importance of the biomechanical interplay between cells and their microenvironment for tumor progression.

Gold Nanostars Coated with Mesoporous Silica Are Effective and Nontoxic Photothermal Agents Capable of Gate Keeping and Laser-Induced Drug Release.

Herein, a novel drug photorelease system based on gold nanostars (AuNSts), coated with a mesoporous silica shell and capped with paraffin as thermosensitive molecular gate, is reported. Direct measurements of the surface temperature of a single gold nanostar irradiated using a tightly focused laser beam are performed via a heat-sensitive biological matrix. The surface temperature of a AuNSt increases by hundreds of degrees (°C) even at low laser powers. AuNSts coated with a mesoporous silica shell using a surfactant-templated synthesis are used as chemotherapeutic nanocarriers. Synthetic parameters are optimized to avoid AuNSt reshaping, and thus to obtain nanoparticles with suitable and stable plasmonic properties for near-infrared (NIR) laser-triggered cargo delivery. The mesoporous silica-coated nanostars are loaded with doxorubicin (Dox) and coated with octadecyltrimethoxysilane and the paraffin heneicosane. The paraffin molecules formed a hydrophobic layer that blocks the pores, impeding the release of the cargo. This hybrid nanosystem exhibits a well-defined photodelivery profile using NIR radiation, even at low power density, whereas the nonirradiated sample shows a negligible payload release. Dox-loaded nanoparticles displayed no cytotoxicity toward HeLa cells, until they are irradiated with 808 nm laser, provoking paraffin melting and drug release. Hence, these novel, functional, and biocompatible nanoparticles display adequate plasmonic properties for NIR-triggered drug photorelease applications.

Platinum nanoparticles: a non-toxic, effective and thermally stable alternative plasmonic material for cancer therapy and bioengineering.

Absorption of near infrared (NIR) light by metallic nanoparticles can cause extreme heating and is of interest for instance in cancer treatment since NIR light has a relatively large penetration depth into biological tissue. Here, we quantify the extraordinary thermoplasmonic properties of platinum nanoparticles and demonstrate their efficiency in photothermal cancer therapy. Although platinum nanoparticles are extensively used for catalysis, they are much overlooked in a biological context. Via direct measurements based on a biological matrix we show that individual irradiated platinum nanoparticles with diameters of 50-70 nm can easily reach surface temperatures up to 900 K. In contrast to gold nanoshells, which are often used for photothermal purposes, we demonstrate that the platinum particles remain stable at these extreme temperatures. The experiments are paralleled by finite element modeling confirming the experimental results and establishing a theoretical understanding of the particles' thermoplasmonic properties. At extreme temperatures it is likely that a vapor layer will form around the plasmonic particle, and we show this scenario to be consistent with direct measurements and simulations. Viability studies demonstrate that platinum nanoparticles themselves are non-toxic at therapeutically relevant concentrations, however, upon laser irradiation we show that they efficiently kill human cancer cells. Therefore, platinum nanoparticles are highly promising candidates for thermoplasmonic applications in the life sciences, in nano-medicine, and for bio-medical engineering.

Cooke-Triplet tweezers: more compact, robust, and efficient optical tweezers.

We present a versatile three-lens optical design to improve the overall compactness, efficiency, and robustness for optical tweezers based applications. The design, inspired by the Cooke-Triplet configuration, allows for continuous beam magnifications of 2-10×, and axial as well as lateral focal shifts can be realized without switching lenses or introducing optical aberrations. We quantify the beam quality and trapping stiffness and compare the Cooke-Triplet design with the commonly used double Kepler design through simulations and direct experiments. Optical trapping of 1 and 2 µm beads shows that the Cooke-Triplet possesses an equally strong optical trap stiffness compared to the double Kepler lens design but reduces its lens system length by a factor of 2.6. Finally, we demonstrate how a Twyman-Green interferometer integrated in the Cooke-Triplet optical tweezers setup provides a fast and simple method to characterize the wavefront aberrations in the lens system and how it can help in aligning the optical components perfectly.

Remotely controlled fusion of selected vesicles and living cells: a key issue review.

Remote control over fusion of single cells and vesicles has a great potential in biological and chemical research allowing both transfer of genetic material between cells and transfer of molecular content between vesicles. Membrane fusion is a critical process in biology that facilitates molecular transport and mixing of cellular cytoplasms with potential formation of hybrid cells. Cells precisely regulate internal membrane fusions with the aid of specialized fusion complexes that physically provide the energy necessary for mediating fusion. Physical factors like membrane curvature, tension and temperature, affect biological membrane fusion by lowering the associated energy barrier. This has inspired the development of physical approaches to harness the fusion process at a single cell level by using remotely controlled electromagnetic fields to trigger membrane fusion. Here, we critically review various approaches, based on lasers or electric pulses, to control fusion between individual cells or between individual lipid vesicles and discuss their potential and limitations for present and future applications within biochemistry, biology and soft matter.

Optical manipulation of individual strongly absorbing platinum nanoparticles.

Nanostructures with exceptional absorption in the near infrared (NIR) regime are receiving significant attention due to their ability to promote controlled local heating in biological material upon irradiation. Also, such nano-structures have numerous applications in nano-electronics and for bio-exploration. Therefore, significant effort is being put into controlling and understanding plasmonic nanostructures. However, essentially all focus has been on NIR resonant gold nanoparticles and remarkably little attention has been given to nanoparticles of other materials that may have superior properties. Here, we demonstrate optical control and manipulation of individual strongly absorbing platinum nanoparticles in three dimensions using a single focused continuous wave NIR laser beam. Also, we quantify how the platinum nanoparticles interact with light and compare to similarly sized absorbing gold nanoparticles, both massive gold and gold nanoshells. By finite element modeling, we find the scattering and absorption cross sections and the polarizability of all particles. The trapping experiments allow for direct measurements of the interaction between the nanoparticles and NIR light which compares well to the theoretical predictions. In the NIR, platinum nanoparticles are stronger absorbers than similarly sized massive gold nanoparticles and scatter similarly. Compared to NIR resonant gold nanoshells, platinum nanoparticles absorb less, however, they also scatter significantly less, thus leading to more stable optical trapping. These results pave the way for nano-manipulation and positioning of platinum nanoparticles and for using these for to enhance spectroscopic signals, for localized heating, and for manipulation of biological systems.

18F-FDG PET/CT-based early treatment response evaluation of nanoparticle-assisted photothermal cancer therapy.

Within the field of nanoparticle-assisted photothermal cancer therapy, focus has mostly been on developing novel heat-generating nanoparticles with the right optical and dimensional properties. Comparison and evaluation of their performance in tumor-bearing animals are commonly assessed by changes in tumor volume; however, this is usually a late-occurring event. This study implements 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging to perform early evaluation of the treatment outcome of photothermal therapy. Silica-gold nanoshells (NS) are administered intravenously to nude mice bearing human neuroendocrine tumor xenografts and the tumors are irradiated by a near-infrared laser. The animals are positron emission tomography scanned with 2-deoxy-2-[F-18]fluoro-D-glucose one day before and one day after treatment. Using this setup, a significant decrease in tumor uptake of 2-deoxy-2-[F-18]fluoro-D-glucose is found already one day after therapy in the group receiving NS and laser treatment compared to control animals. At this time point no change in tumor volume can be detected. Moreover, the change in tumor uptake, is used to stratify the animals into responders and non-responders, where the responding group matched improved survival. Overall, these findings support the use of 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging for preclinical and clinical evaluation and optimization of photothermal therapy.

Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells.

The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the ß1 adrenergic receptor and the ß2 adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.

Chirality in microbial biofilms is mediated by close interactions between the cell surface and the substratum.

From microbial biofilms to human migrations, spatial competition is central to the evolutionary history of many species. The boundary between expanding populations is the focal point of competition for space and resources and is of particular interest in ecology. For all Escherichia coli strains studied here, these boundaries move in a counterclockwise direction even when the competing strains have the same fitness. We find that chiral growth of bacterial colonies is strongly suppressed by the expression of extracellular features such as adhesive structures and pili. Experiments with other microbial species show that chiral growth is found in other bacteria and exclude cell wall biosynthesis and anisotropic shape as the primary causes of chirality. Instead, intimate contact with the substratum is necessary for chirality. Our results demonstrate that through a handful of surface molecules cells can fundamentally reorganize their migration patterns, which might affect intra- and interspecific competitions through colony morphology or other mechanisms.

Manipulation and Motion of Organelles and Single Molecules in Living Cells.

The biomolecule is among the most important building blocks of biological systems, and a full understanding of its function forms the scaffold for describing the mechanisms of higher order structures as organelles and cells. Force is a fundamental regulatory mechanism of biomolecular interactions driving many cellular processes. The forces on a molecular scale are exactly in the range that can be manipulated and probed with single molecule force spectroscopy. The natural environment of a biomolecule is inside a living cell, hence, this is the most relevant environment for probing their function. In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theoretical tools for analyzing single molecule data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly used force spectroscopy techniques, namely optical tweezers, magnetic tweezers, and atomic force microscopy, are described in detail, and their strength and limitations related to in vivo experiments are discussed. Finally, recent exciting discoveries within the field of in vivo manipulation and dynamics of single molecule and organelles are reviewed.

Single Particle and PET-based Platform for Identifying Optimal Plasmonic Nano-Heaters for Photothermal Cancer Therapy.

Plasmonic nanoparticle-based photothermal cancer therapy is a promising new tool to inflict localized and irreversible damage to tumor tissue by hyperthermia, without harming surrounding healthy tissue. We developed a single particle and positron emission tomography (PET)-based platform to quantitatively correlate the heat generation of plasmonic nanoparticles with their potential as cancer killing agents. In vitro, the heat generation and absorption cross-section of single irradiated nanoparticles were quantified using a temperature sensitive lipid-based assay and compared to their theoretically predicted photo-absorption. In vivo, the heat generation of irradiated nanoparticles was evaluated in human tumor xenografts in mice using 2-deoxy-2-[F-18]fluoro-D-glucose ((18)F-FDG) PET imaging. To validate the use of this platform, we quantified the photothermal efficiency of near infrared resonant silica-gold nanoshells (AuNSs) and benchmarked this against the heating of colloidal spherical, solid gold nanoparticles (AuNPs). As expected, both in vitro and in vivo the heat generation of the resonant AuNSs performed superior compared to the non-resonant AuNPs. Furthermore, the results showed that PET imaging could be reliably used to monitor early treatment response of photothermal treatment. This multidisciplinary approach provides a much needed platform to benchmark the emerging plethora of novel plasmonic nanoparticles for their potential for photothermal cancer therapy.

Dynamic buckling of actin within filopodia.

Filopodia are active tubular structures protruding from the cell surface which allow the cell to sense and interact with the surrounding environment through repetitive elongation-retraction cycles. The mechanical behavior of filopodia has been studied by measuring the traction forces exerted on external substrates.(1) These studies have revealed that internal actin flow can transduce a force across the cell surface through transmembrane linkers like integrins. In addition to the elongation-retraction behavior filopodia also exhibit a buckling and rotational behavior. Filopodial buckling in conjunction with rotation enables the cell to explore a much larger 3-dimensional space and allows for more complex, and possibly stronger, interactions with the external environment.(2) Here we focus on how bending of the filopodial actin dynamically correlates with pulling on an optically trapped microsphere which acts like an external substrate attached to the filopodial tip. There is a clear correlation between presence of actin near the tip and exertion of a traction force, thus demonstrating that the traction force is transduced along the actin shaft inside the filopodium. By extending a filopodium and holding it while measuring the cellular response, we also monitor and analyze the waiting times for the first buckle observed in the fluorescently labeled actin shaft.