Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins.

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, and Dictyostelium discoideum cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and subcellular components across multiple time scales, from a few milliseconds to hours, with negligible phototoxic effects.

Excitation Intensity-Dependent Quantum Yield of Semiconductor Nanocrystals.

One of the key phenomena that determine the fluorescence of nanocrystals is the nonradiative Auger-Meitner recombination of excitons. This nonradiative rate affects the nanocrystals' fluorescence intensity, excited state lifetime, and quantum yield. Whereas most of the above properties can be directly measured, the quantum yield is the most difficult to assess. Here we place semiconductor nanocrystals inside a tunable plasmonic nanocavity with subwavelength spacing and modulate their radiative de-excitation rate by changing the cavity size. This allows us to determine absolute values of their fluorescence quantum yield under specific excitation conditions. Moreover, as expected considering the enhanced Auger-Meitner rate for higher multiple excited states, increasing the excitation rate reduces the quantum yield of the nanocrystals.

Isotropic three-dimensional dual-color super-resolution microscopy with metal-induced energy transfer.

Over the past 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, which is by a factor of 2 to 3 worse. 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 subcellular structures. Moreover, we used spectral demixing for implementing dual-color MIET-dSTORM that allows us to image and colocalize, in three dimensions, two different cellular structures simultaneously.

Excited state lifetime modulation in semiconductor nanocrystals for super-resolution imaging.

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.

Mapping Activity-Dependent Quasi-stationary States of Mitochondrial Membranes with Graphene-Induced Energy Transfer Imaging.

Graphene-induced energy transfer (GIET) was recently introduced for sub-nanometric axial localization of fluorescent molecules. GIET relies on near-field energy transfer from an optically excited fluorophore to a single sheet of graphene. Recently, we demonstrated its potential by determining the distance between two leaflets of supported lipid bilayers. Here, we use GIET imaging for mapping quasi-stationary states of the inner and outer mitochondrial membranes before and during adenosine triphosphate (ATP) synthesis. We trigger the ATP synthesis state in vitro by activating mitochondria with precursor molecules. Our results demonstrate that the inner membrane approaches the outer membrane, while the outer membrane does not show any measurable change in average axial position upon activation. The inter-membrane space is reduced by ∼2 nm. This direct experimental observation of the subtle dynamics of mitochondrial membranes and the change in intermembrane distance upon activation is relevant for our understanding of mitochondrial function.

Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution.

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.

Absolute quantum yield measurements of fluorescent proteins using a plasmonic nanocavity.

One of the key photophysical properties of fluorescent proteins that is most difficult to measure is the quantum yield. It describes how efficiently a fluorophore converts absorbed light into fluorescence. Its measurement using conventional methods become particularly problematic when it is unknown how many of the proposedly fluorescent molecules of a sample are indeed fluorescent (for example due to incomplete maturation, or the presence of photophysical dark states). Here, we use a plasmonic nanocavity-based method to measure absolute quantum yield values of commonly used fluorescent proteins. The method is calibration-free, does not require knowledge about maturation or potential dark states, and works on minute amounts of sample. The insensitivity of the nanocavity-based method to the presence of non-luminescent species allowed us to measure precisely the quantum yield of photo-switchable proteins in their on-state and to analyze the origin of the residual fluorescence of protein ensembles switched to the dark state.

Emission States Variation of Single Graphene Quantum Dots.

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.

Dual-Color Metal-Induced Energy Transfer (MIET) Imaging for Three-Dimensional Reconstruction of Nuclear Envelope Architecture.

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.

Single-particle spectroscopy for functional nanomaterials.

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.

Excitation and Emission Transition Dipoles of Type-II Semiconductor Nanorods.

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.

Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids.

Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein's hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.

Three-dimensional single-molecule localization with nanometer accuracy using Metal-Induced Energy Transfer (MIET) imaging.

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.

Dual-color metal-induced and Förster resonance energy transfer for cell nanoscopy.

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.

Charge-Driven Fluorescence Blinking in Carbon Nanodots.

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.

Three-Dimensional Reconstruction of Nuclear Envelope Architecture Using Dual-Color Metal-Induced Energy Transfer Imaging.

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 Lap2ß 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.

Cell-Substrate Dynamics of the Epithelial-to-Mesenchymal Transition.

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.

Quantum Yield Measurements of Fluorophores in Lipid Bilayers Using a Plasmonic Nanocavity.

Precise knowledge of the quantum yield is important for many fluorescence-spectroscopic techniques, for example, for Förster 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.

Photoactivation of Luminescent Centers in Single SiO2 Nanoparticles.

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.

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

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.