Sub-THz Characterisation of Monolayer Graphene

We explore the optical and electrical characteristics of monolayer graphene by using pulsed optoelectronic terahertz time-domain spectroscopy in the frequency range of 325-500 GHz based on fast direct measurements of phase and amplitude. We also show that these parameters can, however, be measured with higher resolution using a free space continuous wave measurement technique associated with a vector network analyzer that offers a good dynamic range. All the scattering parameters (both magnitude and phase) are measured simultaneously. The Nicholson-Ross-Weir method is implemented to extract the monolayer graphene parameters at the aforementioned frequency range

Achieving 15 nm resolution using localization with stimulated emission depletion microscopy (LocSTED)

Both the branches of high resolution nanoscopy coordinate targeted and coordinate stochastic methods discern closely spaced molecules based on the same principle switching of fluorescent molecules between bright and dark states. When stimulated emission depletion (STED) microscopy uses a coordinate targeted approach and readout by applying a spatially modulated depletion pattern to switch off the molecules, single molecule localization microscopy (SMLM) uses a stochastic approach and readout by temporally switching a subset of fluorophores at a time. This thesis introduces localization with STED microscopy (LocSTED), a correlative approach combining STED microscopy and localization microscopy. This optical scheme allows determining the position of a single emitter in space using the simplest form of a STED microscope that uses a combination of continuous waves for both excitation and depletion. Stochastic reversible blinking of fluorophores is utilized within a predefined coordinate which is nanoscopic in size. The imaging was performed in a subdiffractional extent thus limiting the scanning field to the donut minimum. The potential of the method was shown by imaging fluorescently labeled proteins attached on nanostructures fabricated by STED lithography and also by imaging DNA origami nanorulers. Photobleaching as an irreversible reaction is usually a hurdle in STED microscopy because of the higher intensity of depletion beam used. An imaging buffer containing an enzymatic oxygen scavenger system and reducing and oxidizing system (ROXS) was developed and applied. ROXS renders STED more efficient. With a simple CW STED microscope, this thesis shows that single molecules can be localized with a localization error of 5 nm. By imaging fluorescently labeled proteins attached to nanoanchors structured by STED lithography, it is shown that LocSTED microscopy can resolve molecules with a resolution of at least 15 nm. This shows that the classical resolution of a confocal microscope is improved to /35 and that of a CW STED microscope by a factor of at least 4. LocSTED microscopy thus allows estimating the total number of proteins attached on a single nanoanchor by enhancing the resolution of a simple CW STED microscope.submitted by Sujitha PuthukodanJohannes Kepler University Linz, Dissertation, 2019OeBB(VLID)454336

Localization STED (LocSTED) microscopy with 15 nm resolution

We present localization with stimulated emission depletion (LocSTED) microscopy, a combination of STED and single-molecule localization microscopy (SMLM). We use the simplest form of a STED microscope that is cost effective and synchronization free, comprising continuous wave (CW) lasers for both excitation and depletion. By utilizing the reversible blinking of fluorophores, single molecules of Alexa 555 are localized down to ~5 nm. Imaging fluorescently labeled proteins attached to nanoanchors structured by STED lithography shows that LocSTED microscopy can resolve molecules with a resolution of at least 15 nm, substantially improving the classical resolution of a CW STED microscope of about 60 nm. LocSTED microscopy also allows estimating the total number of proteins attached on a single nanoanchor

Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model.

We present the software platform 2CALM that allows for a comparative analysis of 3D localisation microscopy data representing protein distributions in two biological samples. The in-depth statistical analysis reveals differences between samples at the nanoscopic level using parameters such as cluster-density and -curvature. An automatic classification system combines multiplex and multi-level statistical approaches into one comprehensive parameter for similarity testing of the compared samples. We demonstrated the biological importance of 2CALM, comparing the protein distributions of CD41 and CD62p on activated platelets in a 3D artificial clot. Additionally, using 2CALM, we quantified the impact of the inflammatory cytokine interleukin-1β on platelet activation in clots. The platform is applicable to any other cell type and biological system and can provide new insights into biological and medical applications

CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis

Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag® with lipid membrane permeable dye, JaneliaFluor® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells

Sub-THz characterisation of monolayer graphene

We explore the optical and electrical characteristics of monolayer graphene by using pulsed optoelectronic terahertz time-domain spectroscopy in the frequency range of 325-500 GHz based on fast direct measurements of phase and amplitude. We also show that these parameters can, however, be measured with higher resolution using a free space continuous wave measurement technique associated with a vector network analyzer that offers a good dynamic range. All the scattering parameters (both magnitude and phase) are measured simultaneously. The Nicholson-Ross-Weir method is implemented to extract the monolayer graphene parameters at the aforementioned frequency range

Bio-inspired microneedle design for efficient drug/vaccine coating

Biomimetics is the interdisciplinary scientific field focused on the study and imitation of biological systems, with the aim of solving complex technological problems. In this paper, we present a new bio-inspired design for microneedles (MNs) and MN arrays, intended for rapidly coating the MNs with drug/vaccine. The biomimetic approach consists in ornamenting the lateral sides of pyramidal MNs with structures inspired by the external scent efferent systems of some European true bugs, which facilitate a directional liquid transport. To realize these MNs, two-photon polymerization (TPP) technique was used. Liquid coating capabilities of structured and non-structured MNs were compared. Moreover, both in-vivo and ex-vivo skin tests were performed to prove that MNs pierce the skin. We show that the arrays of MNs can be accurately replicated using a micro-moulding technique. We believe this design will be beneficial for the process of drug/vaccine loading onto the needles surfaces, by making it more efficient and by reducing the drug/vaccine wastage during MN coating process.(VLID)4934381Version of recor