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Metal-Induced Energy Transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, andDictyostelium discoideumcells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and sub-cellular components across multiple timescales, from a few milliseconds to hours, with negligible phototoxic effects.

Super-resolution fluorescence imaging that surpasses the classical optical resolution limit is widely utilized for resolving the spatial organization of biological structures at molecular length scales. In one example, single-molecule localization microscopy, the lateral positions of single molecules can be determined more precisely than the diffraction limit if the camera collects individual photons separately. Using several schemes that introduce engineered optical aberrations in the imaging optics, super-resolution along the optical axis (perpendicular to the sample plane) has been achieved, and single-molecule localization microscopy has been successfully applied for the study of 3D biological structures. Nonetheless, the achievable axial localization accuracy is typically three to five times worse than the lateral localization accuracy. Only a few exceptional methods based on interferometry exist that reach nanometer 3D super-resolution, but they involve enormous technical complexity and restricted sample preparations that inhibit their widespread application. We developed metal-induced energy transfer imaging for localizing fluorophores along the axial direction with nanometer accuracy, using only a conventional fluorescence lifetime imaging microscope. In metal-induced energy transfer, experimentally measured fluorescence lifetime values increase linearly with axial distance in the range of 0-100 nm, making it possible to calculate their axial position using a theoretical model. If graphene is used instead of the metal (graphene-induced energy transfer), the same range of lifetime values occurs over a shorter axial distance (~25 nm), meaning that it is possible to get very accurate axial information at the scale of a membrane bilayer or a molecular complex in a membrane. Here, we provide a step-by-step protocol for metal- and graphene-induced energy transfer imaging in single molecules, supported lipid bilayer and live-cell membranes. Depending on the sample preparation time, the complete duration of the protocol is 1-3 d.

Over the last two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution. Similar to conventional optical microscopy, the axial resolution is by a factor three to five worse than the lateral resolution. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging which achieves an axial resolution down to nanometers. It exploits the distance dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of sub-cellular structures. Moreover, we employed spectral demixing for implementing dualcolor MIET-dSTORM that allows us to image and co-localize, in three dimensions, two different cellular structures simultaneously.

Graphene quantum dots (GQDs) are nanoparticles that consist of a nanometer-sized core of graphene with diverse chemical groups on its boundary. Due to their advantageous properties, they are considered to be a promising material for optoelectronics, bioimaging, or photovoltaics. Despite considerable efforts that have been focused on unraveling the mechanism of their photoluminescence, many fundamental details are still unclear. Here, we report on a single-particle multimodal study that provides new insight into the photoluminescence properties of emission centers of GQDs in various local chemical environments. In particular, we show that the properties that are associated with emission centers of GQDs are significantly more sensitive to the structure of the particle itself than to a nonuniform local chemical environment. A better understanding of the dependence of GQDs' emission states on the complex local chemical environment is an important step toward finding new ways of controlling the optical properties of GQDs and of optimizing their use in various applications.

Tremendous progress in nanotechnology has enabled advances in the use of luminescent nanomaterials in imaging, sensing and photonic devices. This translational process relies on controlling the photophysical properties of the building block, that is, single luminescent nanoparticles. In this Review, we highlight the importance of single-particle spectroscopy in revealing the diverse optical properties and functionalities of nanomaterials, and compare it with ensemble fluorescence spectroscopy. The information provided by this technique has guided materials science in tailoring the synthesis of nanomaterials to achieve optical uniformity and to develop novel applications. We discuss the opportunities and challenges that arise from pushing the resolution limit, integrating measurement and manipulation modalities, and establishing the relationship between the structure and functionality of single nanoparticles.

We report on proof of principle measurements of a concept for a super-resolution imaging method that is based on excitation field density-dependent lifetime modulation of semiconductor nanocrystals. The prerequisite of the technique is access to semiconductor nanocrystals with emission lifetimes that depend on the excitation intensity. Experimentally, the method requires a confocal microscope with fluorescence-lifetime measurement capability that makes it easily accessible to a broad optical imaging community. We demonstrate with single particle imaging that the method allows one to achieve a spatial resolution of the order of several tens of nanometers at moderate fluorescence excitation intensity.

We introduce a carbon-dot-based fluorescent label for F-actin with high labeling specificity that allowed us to perform three-dimensional fluorescence imaging of thick muscle tissues. Actin-binding proteins play a pivotal role in the sarcomere, regulating actin-filament morphology, stability, and dynamics. We studied the morphology of Caenorhabditis elegans nematode body-wall-muscle cells as well as changes with the mutation of various actin-binding proteins. The obtained results of the study of a newly generated by us dbn-1(vit7) C. elegans mutant muscle structure are important in order to understand the development of drebrin-like protein-caused heart failure in humans.

Graphene-induced energy transfer (GIET) was recently introduced for the precise localization of fluorescent molecules along the optical axis of a microscope. GIET is based on near-field energy transfer from an optically excited fluorophore to a single sheet of graphene. As a proof-of-concept, we demonstrated its potential by determining the distance between the two leaflets of supported lipid bilayers (SLBs) with sub-nanometer accuracy. Here, we use GIET imaging for three-dimensional reconstruction of the mitochondrial membrane architecture. We map two quasi-stationary states of the inner and outer mitochondrial membranes before and during adenosine tri-phosphate (ATP) synthesis. We trigger the ATP synthesis state in vitro by activating mitochondria with precursor molecules. Our results demonstrate that the inner membrane (IM) approaches the outer membrane (OM) while the outer membrane (OM) does not show a measurable change in average axial position upon activation. As a result, the inter-membrane space (IM-OM distance) is reduced by ∼2 nm upon activation of the mitochondria. This direct experimental observation of the subtle dynamics of mitochondrial membranes and the change in inter-membrane distance induced by ATP synthesis is relevant for our understanding of the physical functioning of mitochondria.

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and control transport of macromolecules between the two compartments. Recently, it has been shown that the axial distance between the inner nuclear membrane and the cytoplasmic side of the NPC can be measured using dual-color metal-induced energy transfer (MIET). This chapter focuses on experimental aspects of this method and discusses the details of data analysis.

The mechanisms of exciton generation and recombination in semiconductor nanocrystals are crucial to the understanding of their photophysics and for their application in nearly all fields. While many studies have been focused on type-I heterojunction nanocrystals, the photophysics of type-II nanorods, where the hole is located in the core and the electron is located in the shell of the nanorod, remain largely unexplored. In this work, by scanning single nanorods through the focal spot of radially and azimuthally polarized laser beams and by comparing the measured excitation patterns with a theoretical model, we determine the dimensionality of the excitation transition dipole of single type-II nanorods. Additionally, by recording defocused patterns of the emission of the same particles, we measure their emission transition dipoles. The combination of these techniques allows us to unambiguously deduce the dimensionality and orientation of both excitation and emission transition dipoles of single type-II semiconductor nanorods. The results show that in contrast to previously studied quantum emitters, the particles possess a 3D degenerate excitation and a fixed linear emission transition dipole.

We optimized the performance of quantum confined Stark effect QCSE based voltage nanosensors. A high throughput approach for single particle QCSE characterization was developed and utilized to screen a library of such nanosensors. Type II ZnSe CdS seeded nanorods were found to have the best performance among the different nanosensors evaluated in this work. The degree of correlation between intensity changes and spectral changes of the excitons emission under applied field was characterized. An upper limit for the temporal response of individual ZnSe CdS nanorods to voltage modulation was characterized by high throughput, high temporal resolution intensity measurements using a novel photon counting camera. The measured 3.5 us response time is limited by the voltage modulation electronics and represents about 30 times higher bandwidth than needed for recording an action potential in a neuron., Comment: 36 pages, 6 figures

This study focuses on the mechanism of fluorescence blinking of single carbon nanodots, which is one of their key but less understood properties. The results of our single-particle fluorescence study show that the mechanism of carbon nanodots blinking has remarkable similarities with that of semiconductor quantum dots. In particular, the temporal behavior of carbon nanodot blinking follows a power law both at room and at cryogenic temperatures. Our experimental data suggest that static quenching via Dexter-type electron transfer between surface groups of a nanoparticle plays a major role in the transition of carbon nanodots to off or gray states, whereas the transition back to on states is governed by an electron tunneling from the particle's core. These findings advance our understanding of the complex mechanism of carbon nanodots emission, which is one of the key steps for their application in fluorescence imaging.

The biological process of the epithelial-to-mesenchymal transition (EMT) allows epithelial cells to enhance their migratory and invasive behavior and plays a key role in embryogenesis, fibrosis, wound healing, and metastasis. Among the multiple biochemical changes from an epithelial to a mesenchymal phenotype, the alteration of cellular dynamics in cell-cell as well as cell-substrate contacts is crucial. To determine these variations over the whole time scale of the EMT, we measure the cell-substrate distance of epithelial NMuMG cells during EMT using our newly established metal-induced energy transfer (MIET) microscopy, which allows one to achieve nanometer axial resolution. We show that, in the very first hours of the transition, the cell-substrate distance increases substantially, but later in the process after reaching the mesenchymal state, this distance is reduced again to the level of untreated cells. These findings relate to a change in the number of adhesion points and will help to better understand remodeling processes associated with wound healing, embryonic development, cancer progression, or tissue regeneration.

Super-resolution microscopy has seen a tremendous development over the last two decades. It has opened new perspectives for the application of fluorescence microscopy in the life sciences. Achieving a spatial resolution beyond the diffraction limit of light allowed one to observe many biological structures that are not resolvable in conventional fluorescence microscopy. However, despite recent development of super-resolution fluorescence microscopy techniques that allowed for squeezing the lateral resolution down to tens of nanometers, the much less axial resolution remains a key limiting factor for applications where z-sectioning of a sample is needed. In this chapter, we present the recently developed fluorescence imaging method that is called metal-induced energy transfer. It combines unprecedented nanometer resolution with technical simplicity that allows life science researchers to use it with standard microscopes. We discuss basic principle of the method, its theoretical background, and its applications for imaging of various sub-cellular structures.

Imaging of complex (biological) samples in the near infrared (nIR) range of the spectrum is beneficial due to reduced light scattering, absorption, phototoxicity and autofluorescence. However, there are only few near infrared fluorescent materials known and suitable for biomedical applications. Here, we exfoliate the layered pigment CaCuSi4O10 (known as Egyptian Blue, EB) via facile tip sonication into nanosheets (EB-NS) with ultra-high nIR fluorescence stability and brightness. The size of EB-NS can be tailored by tip sonication to diameters < 20 nm and heights down to 1 nm. EB-NS fluoresce at 910 nm and the total fluorescence intensity scales with the number of Cu2+ ions that serve as luminescent centers. Furthermore, EB-NS display no bleaching and ultra-high brightness compared to other nIR fluorophores. The versatility of EB-NS is demonstrated by in vivo single-particle tracking and microrheology measurements in developing Drosophila embryos. Additionally, we show that EB-NS can be uptaken by plants and remotely detected in a low cost stand-off detection setup despite strong plant background fluorescence. In summary, EB-NS are a highly versatile, bright, photostable and biocompatible nIR fluorescent material that has the potential for a wide range of bioimaging applications both in animal and plant systems.

In recent years single-molecule localization super-resolution microscopy (SMLM) has become an indispensable tool for many fields of research. Here, for any image of a single molecule one determines its center position with much higher accuracy than the size of that image itself. A challenge of SMLM is to achieve super-resolution also along the third dimension. Recently, Metal-Induced Energy Transfer or MIET [1,2] was introduced. It exploits the energy transfer from an excited fluorophore to plasmons in a thin metal film. Similar to Forster Resonance Energy Transfer (FRET), this coupling shows a strong distance dependence, but over a range up to 150 nm and enables axial localization of fluorophores with 5-6 nm resolution at a photon budget of 1000 photons. [3,4] Here, we show that using a graphene layer the localization accuracy of MIET reaches Angstrom accuracy. At such accuracy, minute details such as nanometer-scale roughness of the sample surfaces becomes important. For proof of principle, we determined absolute distances of single molecules from a surface for samples with an a priori well-known sample geometry. We spin-coated fluorescent dye molecules (Atto655) on top of three different substrates with spacer thickness values of 10, 15, and 20 nm, defining the distance of the molecules from the graphene layer. Next, we determined the thickness of supported lipid bilayers (SLBs) by localizing fluorescent dyes attached to lipid head groups in the bottom and top leaflet of the SLB. We have demonstrated that by using graphene as the energy acceptor in MIET, the axial localization accuracy and resolution reaches sub-nanometer levels at photon budgets which are typical in conventional SMLM experiments. An interesting feature of graphene-MIET is that it provides an axial localization accuracy which now surpasses significantly that of lateral localization provided by most SMLM approaches. [1] A. I. Chizhik et al. Nat. Photonics 8, 124 (2014). [2] S. Isbaner et al. Nano Lett. 18, 2616 (2018). [3] N. Karedla et al. ChemPhysChem 15, 705 7 (2014). [4] N. Karedla et al. J. Chem. Phys. 148, 204201 (2018).

Photobleaching of fluorophores is one of the key problems in fluorescence microscopy. Overcoming the limitation of the maximum number of photons, which can be detected from a single emitter, would allow one to enhance the signal-to-noise ratio and thus the temporal and spatial resolution in fluorescence imaging. It would be a breakthrough for many applications of fluorescence spectroscopy, which are unachievable up to now. So far, the only approach for diminishing the effect of photobleaching has been to enhance the photostability of an emitter. Here, we present a fundamentally new solution for increasing the number of photons emitted by a fluorophore. We show that, by exposing a single SiO2 nanoparticle to UV illumination, one can create new luminescent centers within this particle. By analogy with nanodiamonds, SiO2 nanoparticles can possess luminescent defects in their regular SiO2 structure. However, due to the much weaker chemical bonds, it is possible to generate new defects in SiO2 nanostructures using UV light. This allows for the reactivation of the nanoparticle's fluorescence after its photobleaching.

Tremendous progress in nanotechnology has enabled advances in the use of luminescent nanomaterials in imaging, sensing and photonic devices. This translational process relies on controlling the photophysical properties of the building block, that is, single luminescent nanoparticles. In this Review, we highlight the importance of single-particle spectroscopy in revealing the diverse optical properties and functionalities of nanomaterials, and compare it with ensemble fluorescence spectroscopy. The information provided by this technique has guided materials science in tailoring the synthesis of nanomaterials to achieve optical uniformity and to develop novel applications. We discuss the opportunities and challenges that arise from pushing the resolution limit, integrating measurement and manipulation modalities, and establishing the relationship between the structure and functionality of single nanoparticles.

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

Epidermal growth factor receptor kinase mutations drive oncogenesis, but the molecular mechanism of pathological signal initiation is poorly understood. Using high-resolution microscopy methods, the authors reveal that these kinase mutations induce structural changes in the receptor ectodomain that lead to enhanced, ligand-independent dimerization., Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non–small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization.

Dual-color metal-induced energy transfer (dcMIET) imaging simultaneously resolves the axial localization of two distinct intracellular structures with nanometer accuracy. Combined with Förster resonance energy transfer, dcMIET is a powerful method to elucidate cellular architecture on the nanometer scale, also in living cells., We report a novel method, dual-color axial nanometric localization by metal-­induced energy transfer, and combine it with Förster resonance energy transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells. Our approach is based on fluorescence-lifetime-imaging microscopy and allows for precise determination of the three-dimensional architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell–matrix mechanics. In addition to resolving nanometric structural details along the z-axis, we use FRET to gain precise information on the distance between actin and vinculin at focal adhesions.

We present the results of a comprehensive photoluminescence study of defect centres in single SiO2 nanoparticles. We show that the photo-physical properties of the luminescent centres strongly resemble those of single dye molecules. However, these properties exhibit a large variability from particle to particle due to the different local chemical environment around each centre of each particle. This variability provides new insight into the complex photo-physics of single quantum emitters embedded into a random chemical environment. Moreover, a better understanding of the fundamental mechanism of the photoluminescence of defect centres in SiO2 structure is paramount for their application as white-light sources, non-toxic labels for bio-imaging, or for combining them with metallic and semiconductor nanostructures. unReviewed

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs), which are embedded in the nuclear envelope, control transport of macromolecules between the two compartments. Here, using dual-color metal-induced energy transfer (MIET), we determine the axial distance between Lap2β and Nup358 as markers for the inner nuclear membrane and the cytoplasmic side of the NPC, respectively. Using MIET imaging, we reconstruct the 3D profile of the nuclear envelope over the whole basal area, with an axial resolution of a few nanometers. This result demonstrates that optical microscopy can achieve nanometer axial resolution in biological samples and without recourse to complex interferometric approaches.

We present a new concept for measuring distance values of single molecules from a surface with nanometer accuracy using the energy transfer from the excited molecule to surface plasmons of a metal film [1]. We measure the fluorescence lifetime of individual dye molecules deposited on a dielectric spacer as a function of a spacer thickness. By using our theoretical model [2], we convert the lifetime values into the axial distance of individual molecules. Similar to Forster resonance energy transfer (FRET), this allows emitters to be localized with nanometer accuracy, but in contrast to FRET the distance range at which efficient energy transfer takes place is an order of magnitude larger. Together with orientation measurements [3], one can potentially use smMIET to localize single emitters with a nanometer precision isotropically, which will facilitate intra- and intermolecular distance measurements in biomolecules and complexes, circumventing the requirement of the knowledge of mutual orientations between two dipole emitters which severely limits the quantification of such distances from a conventional single-pair FRET (spFRET) experiment. [1] Karedla, N., Chizhik, A.I., Gregor, I., Chizhik, A.M., Schulz, O., Enderlein, J., ChemPhysChem, 15, 705-711 (2014). [2] Enderlein J., Biophyical Journal, 78, 2151-8 (2000). [3] Karedla, N., Stein, S. C., Hahnel, D., Gregor, I., Chizhik, A., and Enderlein, J., Physical Review Letters, 115, 173002 (2015).

Precise knowledge of the quantum yield is important for many fluorescence–spectroscopic techniques, for example, for Forster resonance energy transfer. However, to measure it for emitters in a complex environment and at low concentrations is far from being trivial. Using a plasmonic nanocavity, we measure the absolute quantum yield value of lipid-conjugated dyes incorporated into a supported lipid bilayer. We show that for both hydrophobic and hydrophilic molecules the quantum yield of dyes inside the lipid bilayer strongly differs from its value in aqueous solution. This finding is of particular importance for all fluorescence–spectroscopic studies involving lipid bilayers, such as protein–protein or protein–lipid interactions in membranes or direct fluorescence–spectroscopic measurements of membrane physical properties.

A means for localizing fluorescent molecules over distances of hundreds of nanometres exploits the energy transfer between a donor molecule and surface plasmons on a metal film. The technique is demonstrated by using it to profile the membranes of living cells.

While single-molecule fluorescence from emitters with high quantum efficiencies such as organic dye molecules can easily be detected by modern apparatus, many less efficient emission processes such as Raman scattering and metal luminescence require dramatic enhancement to exceed the single-particle detection limit. This enhancement can be achieved using resonant optical systems such as plasmonic particles or nanoantennas, the study of which has led to substantial progress in understanding the interaction of quantum emitters with their electromagnetic environment. This review is focused on the advances in measurement techniques and potential applications enabled by a deeper understanding of fundamental optical interaction processes occurring between single quantum systems on the nanoscale. While the affected phenomena are numerous, including molecular fluorescence and also exciton luminescence and Raman scattering, the interaction itself can often be described from a unified point of view. Starting from a single underlying model, this work elucidates the dramatic enhancement potential of plasmonic tips and nanoparticles and also the more deterministic influence of a Fabry-Pérot microresonator. With the extensive knowledge of the radiative behavior of a quantum system, insight can be gained into nonradiative factors as well, such as energy transfer phenomena or spatial and chemical configurations in single molecules.

Using our nanocavity-based method for precise determination of the absolute quantum yield values, we measure the quantum yield of each of the three different chromophores types with strongly overlapping absorption spectra in a multicolor solution, which is impossible to do by any other existing technique. By measuring the modification of the radiative rate of the chromophores at different cavity lengths, we extract the quantum yield of CdSe/ZnS semiconductor nanocrystals emitting at three different wavelengths and excited by a single wavelength in the multicolor solution. Using several detection channels, we show that only one measurement is needed for determining the quantum yield values of all the types of chromophores in the sample. The obtained results show a very good agreement with the values commonly observed using other techniques. The nanocavity-based method opens up new opportunities of determining fluorescence quantum yields in complex systems and can be used for a big variety of fluorescence studies where multichromophore samples are involved.

We present new results on the theoretical and experimental study of the excitation patterns acquired upon scanning a single molecule through the focal region of a radially polarized laser beam when the emitter is located at a glass–air interface and in bulk glass. Our calculations show that the presence of a dielectric interface, which coincides with the focal plane of a radially polarized laser beam, strongly changes the field intensity distribution within the focus, which results in the modification of the single molecule excitation pattern obtained upon scanning a molecule through the focal region. By changing the dielectric properties of the surrounding media in situ we show experimentally that the same individual molecule possessing the same orientation with respect to the laser beam axis exhibits different shapes of excitation patterns, depending on whether it is located on the dielectric interface or in medium with constant refractive index.

We study the dimensionality of the excitation transition dipole moment for single CdSe/ZnS core-shell nanocrystals using azimuthally and radially polarized laser modes. The comparison of measured and simulated single nanocrystal excitation patterns shows that single CdSe/ZnS quantum dots possess a spherically degenerated excitation transition dipole. We show that the dimensionality of the excitation transition dipole moment distribution is the same for all individual CdSe/ZnS nanocrystals, disregarding the difference in core size and irrespective of variations in the local environment. In contrast to the emission transition dipole moment, which is oriented in one plane, the excitation transition dipole moment of a single CdSe/ZnS quantum dots possesses an isotropy in three dimensions.

We present results of photoluminescence (PL) studies of single SiO 2 nanoparticles (SiO 2 NPs) and single Si nanocrystals (Si NCs). Single particle spectroscopy reveals almost identical PL spectra for both kinds of nanosized systems. The emission curves exhibit a zero-phonon line and one or two phonon bands, which can be assigned to longitudinal optical phonons in SiO 2 . Using cylindrical vector beams for imaging the fluorescence of single particles we show that they possess a linear excitation transition dipole moment (TDM). Furthermore, the single particle fluorescence patterns demonstrate upon continuous excitation dynamical effects such as blinking, bleaching, and flipping of the TDM. The latter is related to a redistribution of defect states caused by charge fluctuations in the surrounding of the embedded NP. Excitation fluorescence images visualize the intermediate state resulting from the TDM flipping.

Our paper presents the first theoretical and experimental study using single-molecule Metal-Induced Energy Transfer (smMIET) for localizing single fluorescent molecules in three dimensions. Metal-Induced Energy Transfer describes the resonant energy transfer from the excited state of a fluorescent emitter to surface plasmons in a metal nanostructure. This energy transfer is strongly distance-dependent and can be used to localize an emitter along one dimension. We have used Metal-Induced Energy Transfer in the past for localizing fluorescent emitters with nanometer accuracy along the optical axis of a microscope. The combination of smMIET with single-molecule localization based super-resolution microscopy that provides nanometer lateral localization accuracy offers the prospect of achieving isotropic nanometer localization accuracy in all three spatial dimensions. We give a thorough theoretical explanation and analysis of smMIET, describe its experimental requirements, also in its combination with lateral single-molecule localization techniques, and present first proof-of-principle experiments using dye molecules immobilized on top of a silica spacer, and of dye molecules embedded in thin polymer films.

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

Using a metal nanocavity, we measure absolute values of the photoluminescence quantum yield in a mixture of different types of chromophores (dye molecules and semiconductor nanocrystals). We show that measurements can be performed in an attoliter volume, both in liquid and solid phases, even if both types of chromophores absorb and emit light in the same spectral range. The method is based on recording photoluminescence decay curves of the chromophore mixture as a function of the cavity length. Changing the distance between the cavity mirrors modifies the local density of states of the electromagnetic field and thus, the radiative transition rate of the enclosed emitters. By extracting individual decay components, corresponding to the different types of the emitters, we determine their quantum yield values separately and simultaneously. The nanocavity-based method opens up new perspectives for studying quantum emitters in complex photophysical systems, for instance, multichromophoric thin films, fluorescent proteins, or dyes incorporated into a lipid bilayer.

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with carcinogenesis. Specific mutations common in non-small cell lung cancer (NSCLC), including EGFR-L858R and EGFR-ΔL747-P753, lead to ligand-independent phosphorylation, however the molecular mechanism by which these mutations in the EGFR kinase domain confer constitutive activity remain unknown. Here, using multiple sub-diffraction-limit imaging modalities, we reveal the altered behavior of NSCLC-associated EGFR mutants within the plasma membrane_including altered receptor dimerization, dynamics, and structure_which collectively dysregulate receptor activity. Using multi-color single particle tracking (SPT) and Hidden Markov Model analysis, EGFR mutants are shown to form stable dimers in the absence of ligand and exhibit a slower mobility that is consistent with receptor signaling. These results were confirmed using two-color, single-molecule super-resolution microscopy (dSTORM) to visualize the spatial distribution of receptors. Receptor clustering was quantified by localization-based cross-correlation analysis to show ligand-induced aggregation of EGFR as well as ligand-independent aggregation of EGFR mutants. Since the receptor ectodomain is known to play a critical role in dimerization, live cell FRET measurements between the EGFR N-terminus and the plasma membrane was used to quantify changes in ectodomain structure. We found that unliganded EGFR mutants are more readily found in the extended conformation, similar to the ligand bound wild type receptor. Therefore, mutation within the kinase domain biases the structural equilibrium of the extracellular domain toward a dimer-competent state.Collectively, these data support a model where oncogenic signaling from NSCLC-associated EGFR mutants is a result of productive dimerization between non-ligand bound receptors. Furthermore, because these mutations are found in the kinase domain, this work introduces the concept that oncogenic EGFR signaling may be controlled in part by a form of “inside-out” signaling.

Novel porphyrin-based stopcock molecules close the nanochannels of all-organic perhydrotriphenylene (PHTP) host−guest compounds as demonstrated by polarized single molecule fluorescence microscopy.

Inorganic carbon nanomaterials, also called carbon nanodots, exhibit a strong photoluminescence with unusual properties and, thus, have been the focus of intense research. Nonetheless, the origin of their photoluminescence is still unclear and the subject of scientific debates. Here, we present a single particle comprehensive study of carbon nanodot photoluminescence, which combines emission and lifetime spectroscopy, defocused emission dipole imaging, azimuthally polarized excitation dipole scanning, nanocavity-based quantum yield measurements, high resolution transmission electron microscopy, and atomic force microscopy. We find that photoluminescent carbon nanodots behave as electric dipoles, both in absorption and emission, and that their emission originates from the recombination of photogenerated charges on defect centers involving a strong coupling between the electronic transition and collective vibrations of the lattice structure.

The association state(s) and activities of the ErbB receptor family members in intact living cells differ widely depending upon expression levels and their distribution and interaction partners. There are contradictory views in the literature about the aggregation states and presumed structures of the receptors in the cell membrane. Fixation artifacts may account for apparent quantitative discrepancies. We obtained biophysical FRET/FLIM data on living cells that reveal structural features of ErbB1 (EGFR) and ErbB2 as well as the effects of EGF and various kinase inhibitors on these structures.We constructed transgenes in which an acyl carrier protein sequence was introduced between the signal peptide and the mature receptor protein sequence. ACP-ErbB1 behaves similarly to wild type ErbB1 with respect to EGF binding, activation and internalization. The ACP-ErbB2 lacks the capacity for binding ligands but can be transactivated as a heterodimer with ErbB1 or ErbB3. Enzymatic labeling of the specific serine in the ACP tag by fluorescent CoA substrates served as donors. The FRET acceptor was the novel membrane probe, NR12S, which is confined exclusively to the outer leaflet of the plasma membrane.Addition of NR12S to the cells led to a dramatic reduction in the fluorescence lifetime of the donor, indicating a close proximity of the N-terminus of the ErbB1 ectodomain to the plasma membrane, supporting the published autoinhibited structure. EGF addition caused a time-dependent increase in the donor lifetime (reduced FRET), in accordance with the extended dimeric ectodomain structure oberved by Xray-crystallography. The effects of kinase inhibitors on these states and on ensuing endocytosis were also studied. The influence(s) of ErB2 density and antibodies interfering with receptor dimerization were additional topics addressed in this study. TCSPC lifetime images were analyzed with Mathematica software developed for these studies.

This chapter presents an overview of the recently introduced concept of metal-induced energy transfer and two of its applications. We discuss the basic principle of the method and its application to the mapping of the membrane of a living cell and to the single-molecule axial localization with 2–3 nm accuracy.

We present a new concept for measuring distance values of single molecules from a surface with nanometer accuracy using the energy transfer from the excited molecule to surface plasmons of a metal film. We measure the fluorescence lifetime of individual dye molecules deposited on a dielectric spacer as a function of a spacer thickness. By using our theoretical model, we convert the lifetime values into the axial distance of individual molecules. Similar to Forster resonance energy transfer (FRET), this allows emitters to be localized with nanometer accuracy, but in contrast to FRET the distance range at which efficient energy transfer takes place is an order of magnitude larger. Our technique can be potentially used as a tool for measuring intramolecular distances of biomolecules and complexes.

We present a new method for determining absolute values of quantum yield of luminescent emitters, which is based on the modification of the radiative transition of emitters within a tunable metallic nanocavity. The method presented is easy to set up and works without any calibration. It will thus be useful for all applications where absolute and calibration-free measurements of luminescence quantum yields are needed. Moreover, it requires only a minute amount of low-concentration fluorophore solution. We give a detailed description of the theory and data evaluation of the nanocavity measurements, and report experimental results for several common dyes in aqueous solution.

The role of quantum confinement (QC) and surface-related defect centers (DCs) on the photoluminescence (PL) of individual silicon nanocrystals (Si NCs) is investigated using confocal microscopy with radially and azimuthally polarized laser beams. It is shown that the multiple-peak PL spectra of single Si NCs revealing SiO${}_{2}$ phonon side bands are associated with a linear transition dipole moment (TDM) and a short lifetime of 4 ns, indicating that the PL originates from defect centers. In a new study applied to free-standing single Si NCs obtained by Si cluster beam deposition, we could discriminate between PL centers with one- and more-dimensional TDM. Nanoparticles revealing a three-dimensional TDM are characterized by a significantly weaker and structureless Gaussian PL band at larger wavelengths and by $\ensuremath{\mu}$s lifetimes. These PL features are attributed to the radiative recombination of quantum-confined excitons generated in the Si cores. Within the same sample, we found coexistence of Si NCs exhibiting QC PL and others showing DC PL.

Using a tunable optical subwavelength microcavity, we demonstrate controlled modification of the vibronic relaxation dynamics in a single SiO(2) nanoparticle. By varying the distance between the cavity mirrors we change the electromagnetic field mode structure around a single nanoparticle and the radiative transition probability from the lowest vibronic level of the electronically excited state to the progression of phonon levels in the electronic ground state. We demonstrate redistribution of the photoluminescence spectrum between zero-phonon and phonon-assisted bands and modification of the excited state lifetime of the same individual SiO(2) particle measured at different cavity lengths. By comparing the experimental data with a theoretical model, we extract the quantum yield of a single SiO(2) nanoparticle.