Isolating strong nanoantenna-molecule interactions by ensemble-level single-molecule detection

Traditionally, the nanoscale interaction between single photon emitters and plasmonic nanostructures is studied by relying on deterministic, near-perfect, nanoscale-control, either top-down or bottom-up. However, these approaches are ultra-low throughput thus rendering systematic studies difficult and time-consuming. Here, we show a highly parallelised far-field tactic, combining multiplexed super-resolution fluorescence localization microscopy and data-driven statistical analysis, to study near-field interactions between gold nanorods and single molecules, even at bulk concentrations. We demonstrate that ensemble-level single molecule detection allows separating individual emitters according to their coupling strength with tailored resonant structures, which ultimately permits the reconstruction of super-resolved 2D interaction maps around individual nanoantennas.Peer ReviewedPostprint (author's final draft

Control of Vibronic Transition Rates by Resonant Single-Molecule-Nanoantenna Coupling

Plasmonic nanostructures dramatically alter the radiative and nonradiative properties of single molecules in their vicinity. This coupling-induced change in decay channels selectively enhances specific vibronic transitions, which can enable plasmonic control of molecular reactivity. Here, we report coupling-dependent spectral emission shaping of single Rhodamine 800 molecules in the vicinity of plasmonic gold nanorods. We show that the relative vibronic transition rates of the first two vibronic transitions of the spontaneous emission spectrum can be tuned in the weak coupling regime, by approximately 25-fold, on the single molecule level.Peer ReviewedPostprint (author's final draft

Phase control of plasmon enhanced two-photon photoluminescence in resonant gold nanoantennas

Plasmonic nanoantennas emit two-photon photoluminescence, which is much stronger than their second harmonic generation. Unfortunately, luminescence is an incoherent process and therefore generally not explored for nanoscale coherent control of the antenna response. Here, we demonstrate that, in resonant gold nanoantennas, the two-photon absorption process can be coherent, provided that the excitation pulse duration is shorter than the dephasing time of plasmon mode oscillation. Exploiting this coherent response, we show the pure spectral phase control of resonant gold nanoantennas, with effective read-out of the two-photon photoluminescence

Large-scale imaging of optical antennas and single molecules

The interaction of light and matter is of crucial importance in fundamental science as well as in high-end technology. Ultimately, this concerns the interaction between a photon and a single quantum system, e.g. the absorption or emission of a photon by a single molecule. At room temperature this interaction is very inefficient as the absorption cross-section of a molecule is small compared to the wavelength of light, which inhibits many photons from interacting and hence limits the absorption, emission and scattering of a photon. An equivalent problem, and its solution, is found in our daily lives: small electric circuits (as found e.g. in our smartphones), which radiate very poorly by themselves, are linked to (radio) antennas to radiate and transfer information efficiently. Analogously, antennas working in the visible, so-called nanoantennas, are an effective tool to link matter and light. The strength of the coupling of a single molecule with a nanoantenna depends on many factors: the overlap of the antenna resonance and the molecular absorption/emission spectrum, the molecule’s dipole orientation, the distance between molecule and nanoantenna, etc. Hence, strong interaction needs rather special conditions, which are hard to engineer. Moreover, to get a full interaction picture, a lot of single molecule encounters with different nanoantennas are needed - on one hand to make a statistically relevant statement including the many different factors and, on the other hand, to be able to observe the rare stronger interactions, that would have stayed hidden in experiments of only a few encounters. The central idea of this thesis is to statistically map and control the interactions of a very large number of single molecules with different tailored nanoantennas, to cover the landscape of interaction factors and thus extend the current knowledge of the mutual interaction. For this purpose, a home-built wide-field microscope is combined with a large array of lithographically fabricated nanoantennas, which are all probed by freely diffusing molecules. Thus in time millions of encounters are recorded in parallel. Chapter 2 introduces the necessary knowledge and methodology to understand the research work presented in chapters 3 to 5. Chapter 3 shows super-resolved nanoscale interaction maps of molecules and nanoantennas, linking the strength of interaction to the emission polarization and intensity of every encounter. Chapter 4 extends this approach by simultaneously recording the emission fluorescence and spectrum of every single molecule event, revealing strong spectral manipulation. Here, a suppression as well as an extreme enhancement of the vibrational sideband of the used molecule is observed. Additionally, the statistical mapping allows the freely diffusing molecules to encounter rare hotspots of extreme field intensities, enabling the observation of surface-enhanced Raman scattering. Finally, chapter 5 takes the first step in the direction of characterizing the interaction of molecule and nanoantenna with high sensitivity via phase measurements. Here, an interferometric wide-field microscope enables the measurement of the absolute phase of nanoparticles and demonstrates the distinction of different plasmonic and dielectric particles via their phase behavior. Furthermore, we implement a novel two-color excitation method, capable of rapidly identifying two types of nanoparticles in a single-shot image.La interacción entre la luz y la materia tiene una alta importancia en la ciencia fundamental, así como en la tecnología de punta. En última instancia, esto se refiere a la interacción entre un fotón y un sistema cuántico, por ejemplo la absorción o emisión de un fotón por una sola molécula. En condiciones ambientales y temperatura ambiente, esta interacción es muy ineficiente ya que la sección transversal de absorción de una molécula es pequeña en comparación con la longitud de onda de la luz, lo cual inhibe la interacción de muchos fotones y, por lo tanto, limita la detección de absorción, emisión y dispersión. Un problema equivalente, y su solución, se puede encontrar en nuestra vida cotidiana: pequeños circuitos eléctricos (como los que se encuentran, por ejemplo, en nuestros smartphones) están conectados a antenas (de radiofrequencia) para emitir y transferir información de manera eficiente. De manera análoga, las antenas que trabajan en el visible, así llamadas nanoantenas, son una herramienta eficaz para vincular la materia y la luz. La fuerza del acoplamiento de una sola molécula con una nanoantena depende de muchos factores: la superposición del espectro de la resonancia de la antena y de absorción/emisión molecular, la orientación del dipolo de la molécula, la distancia entre la molécula y la nanoantena, etc. Por lo tanto, la interacción fuerte necesita condiciones bastante especiales, que son difíciles de generar. Además, para obtener una imagen completa de la interacción, se necesitan muchos encuentros de una sola molécula con diferentes nanoantenas, por un lado para hacer un estudio stadísticamente relevante que incluya los multiples factores y, por otro lado, para poder observar las menos frequentes interacciones más fuertes, lo que habría permanecido oculto en experimentos de solo unos pocos encuentros. La idea central de esta tesis es mapear y controlar estadísticamente las interacciones de un gran número de moléculas individuales con diferentes nanoantenas adaptadas, para considerar todos los factores de interacción y así ampliar el conocimiento actual de la interacción mutua. Para este propósito, un microscopio hecho en casa de campo amplio se combina con una gran variedad de nanoantenas fabricadas litográficamente, que son todas probadas por moléculas que se difunden libremente. Así, con el tiempo, se registran millones de encuentros en paralelo. El capítulo 2 introduce el conocimiento y la metodología necesarios para comprender el trabajo de investigación presentado en los capítulos 3 a 5. El capítulo 3 muestra mapas de interacción nanoescalar superresueltos de moléculas y nanoantenas, vinculando la fuerza de interacción con la polarización de emisión e intensidad de cada encuentro. El Capítulo 4 amplía este enfoque al registrar simultáneamente la fluorescencia de emisión y el espectro de cada evento de una sola molécula, revelando una fuerte manipulación espectral. Aquí, se observa una supresión así como una mejora extrema de la banda lateral vibratoria de la molécula utilizada. Además, el mapeo estadístico permite que las moléculas que se difunden libremente encuentren puntos inusuales de intensidades extremas del campo eléctrico, lo que permite la observación de la espectrometría Raman de superficie mejorada. Finalmente, el capítulo 5 da el primer paso para caracterizar la interacción de la molécula y la nanoantena con alta sensibilidad a través de mediciones de fase. Aquí, un microscopio interferométrico de campo amplio permite la medición de la fase absoluta de nanopartículas y demuestra la distinción de diferentes partículas plasmónicas y dieléctricas a través de su comportamiento de fase. Además, se implementa un novedoso método de excitación de dos colores, capaz de identificar rápidamente dos tipos de nanopartículas en una imagen de un solo disparo.Postprint (published version

CD34+ circulating cells display signs of immune activation in patients with acute coronary syndrome

Abstract Bone marrow-derived endothelial progenitor cells (EPC) are released into the peripheral blood in situations of vascular repair/angiogenesis. Regulation of vascular repair and angiogenesis by EPC depends not only on the number of circulating EPC but also on their functionality. As endothelial cells can act as antigen-presenting cells in coronary artery disease (CAD), we postulated that EPC can be immune activated here as well. CD34+-EPC were isolated from peripheral blood of patients with ST-elevation myocardial infarction (STEMI, n = 12), non-STEMI/unstable angina (UA, n = 15), and stable CAD (SA, n = 18). Expression of HLA-DR, adhesion and costimulatory molecules by isolated CD34+-EPC were compared with levels in healthy controls (n = 18). There were no significant differences in VCAM-1 and CD80 expression by peripheral circulating CD34+-EPC between the four groups, yet expression of CD86 was highest in UA (p < 0.05). ICAM-1 expression was lowest in SA (p < 0.01). CD34+-EPC constitutively expressed HLA-DR across all groups. Of note, patients pretreated with HMG-CoA reductase inhibitors exhibited lower expression of VCAM-1 by CD34+-EPC throughout all patient groups; furthermore, statins significantly limited ex vivo-induced upregulation of ICAM-1 by TNF-alpha. To the best of our knowledge, this is the first study to examine the expression of immune markers in peripheral circulating CD34+-EPC ex vivo. We demonstrate that CD34+-EPC display different patterns of adhesion and costimulatory molecules in various states of CAD. Expression levels were affected by pretreatment with statins. Hence, immune activity of peripheral circulating CD34+ cells might play a pathophysiologic role in evolution of CAD

Isolating strong nanoantenna-molecule interactions by ensemble-level single-molecule detection

Traditionally, the nanoscale interaction between single photon emitters and plasmonic nanostructures is studied by relying on deterministic, near-perfect, nanoscale-control, either top-down or bottom-up. However, these approaches are ultra-low throughput thus rendering systematic studies difficult and time-consuming. Here, we show a highly parallelised far-field tactic, combining multiplexed super-resolution fluorescence localization microscopy and data-driven statistical analysis, to study near-field interactions between gold nanorods and single molecules, even at bulk concentrations. We demonstrate that ensemble-level single molecule detection allows separating individual emitters according to their coupling strength with tailored resonant structures, which ultimately permits the reconstruction of super-resolved 2D interaction maps around individual nanoantennas.Peer Reviewe

3D matrix‐embedding inhibits cycloheximide‐mediated sensitization to TNF‐alpha‐induced apoptosis of human endothelial cells

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Others and we previously demonstrated endothelial biology being intertwined with biochemical and structural composition of the subendothelial basement membrane. We now demonstrate that a three-dimensional growing environment significantly shields endothelial cells from cytokine-induced apoptosis. Detailed analysis reveals differences in intracellular signaling pathways in naive endothelial cells and cytokine-stimulated endothelial cells when cells are grown within a three-dimensional collagen-based matrix compared to cells grown on two-dimensional tissue culture plates. Main findings are significantly reduced p53 expression and level of p38-phosphorylation in three-dimensional grown endothelial cells. Despite similar concentrations of focal adhesion kinase, three-dimensional matrix-embedded endothelial cells express significantly less tyrosine-phosphorylated focal adhesion kinase. Pretreatment with antibodies against integrin αvβ3 partially reversed the protective effect of three-dimensional matrix-embedding on endothelial apoptosis. Our findings provide detailed insights into the mechanisms of endothelial apoptosis with respect to the spatial matrix environment. These results enhance our understanding of endothelial biology and may otherwise help in the design of tissue-engineered materials. Furthermore, findings on focal adhesion kinase phosphorylation might enhance our understanding of clinical studies with tyrosine kinase inhibitors. ©201