Fabrication and characterization of spiral phase masks for super-resolution

Confocal microscopy, with both high lateral and axial resolution, has enabled the observation of the inner workings of cells and tissues with great detail. Strong scattering and absorption of light, though, strongly limit the depth at which samples can be imaged, and resolution is limited to a wavelength-wide area in the focal plane by diffraction. Sample penetration of hundreds of µm can be reached by nonlinear microscopy, based on the interaction between the tissue and multiple infrared photons, that undergo much less scattering and absorption. Resolutions of just tens of nanometers can also be reached with STED microscopy, an upgrade to the confocal architecture. Two-photon excitation STED (TPE-STED) microscopes have been invented in the last decade to combine the two properties, with resolution gains up to 4-5 times the diffraction-limited systems, at depths of tens of microns. Still, scattering and absorption of the depletion beam limit the observation of super-resolved features in the 100 µm regions. The aim of this thesis was the development of the first TPE-STED microscope working with excitation wavelengths in the [1000−1500] nm range and depletion wavelengths near 800 nm, capable of surpassing the depth limit of current STED microscopes. Suitable fluorophores must be used in this regime, so we tested the depletion performance of ATTO 594, ATTO 647N and mGarnet2. In parallel, we used the dual-beam nature of the platform to provide simultaneous nonlinear imaging with both degenerate and nondegenerate absorption of photons at the different wavelengths. A consistent part of the thesis work was also centered on the development of a protocol of fabrication and characterization optical elements for the manipulation of the STED beam. This was done in order to be able to freely couple every selected fluorophore with its most efficient depletion wavelength, without the need for long waiting times of commercial applications

Multiphoton Label-Free ex-vivo imaging using a custom-built dual-wavelength microscope with chromatic aberrations compensation

Label-Free Multiphoton Microscopy is a very powerful optical microscopy that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of a Multiphoton approach, like longer penetration depths and intrinsic optical sectioning, while opening the possibility of serial examinations with different kinds of techniques. Among the many variations of Label-Free MPM, Higher Harmonic Generation (HHG) is one of the most intriguing due to its generally low photo-toxicity, which enables the examination of specimens particularly susceptible to photo-damages. HHG and common Two-Photon Microscopy (TPM) are well-established techniques, routinely used in several research fields. However, they require a significant amount of fine-tuning in order to be fully exploited and, usually, the optimized conditions greatly differ, making them quite difficult to perform in parallel without any compromise on the extractable information. Here we present our custom-built Multiphoton microscope capable of performing simultaneously TPM and HHG without any kind of compromise on the results thanks to two, separate, individually optimized laser sources with full chromatic aberration compensation. We also apply our setup to the examination of a plethora of ex vivo samples in order to prove the significant advantages of our approach

Fabrication and characterization of spiral phase masks for super-resolution

Confocal microscopy, with both high lateral and axial resolution, has enabled the observation of the inner workings of cells and tissues with great detail. Strong scattering and absorption of light, though, strongly limit the depth at which samples can be imaged, and resolution is limited to a wavelength-wide area in the focal plane by diffraction. Sample penetration of hundreds of µm can be reached by nonlinear microscopy, based on the interaction between the tissue and multiple infrared photons, that undergo much less scattering and absorption. Resolutions of just tens of nanometers can also be reached with STED microscopy, an upgrade to the confocal architecture. Two-photon excitation STED (TPE-STED) microscopes have been invented in the last decade to combine the two properties, with resolution gains up to 4-5 times the diffraction-limited systems, at depths of tens of microns. Still, scattering and absorption of the depletion beam limit the observation of super-resolved features in the 100 µm regions. The aim of this thesis was the development of the first TPE-STED microscope working with excitation wavelengths in the [1000−1500] nm range and depletion wavelengths near 800 nm, capable of surpassing the depth limit of current STED microscopes. Suitable fluorophores must be used in this regime, so we tested the depletion performance of ATTO 594, ATTO 647N and mGarnet2. In parallel, we used the dual-beam nature of the platform to provide simultaneous nonlinear imaging with both degenerate and nondegenerate absorption of photons at the different wavelengths. A consistent part of the thesis work was also centered on the development of a protocol of fabrication and characterization optical elements for the manipulation of the STED beam. This was done in order to be able to freely couple every selected fluorophore with its most efficient depletion wavelength, without the need for long waiting times of commercial applications.La microscopia confocale, con la sua alta risoluzione laterale e assiale, ha permesso l'osservazione delle dinamiche interne delle cellule e dei tessuti in modo dettagliato. La profondità alla quale è possibile osservare i campioni, però, è fortemente limitata dalla diffusione e dall'assorbimento subiti dalla luce, inoltre la risoluzione sul piano focale è limitata ad un'area di grandezza paragonabile alla lunghezza d'onda utilizzata, a causa della diffrazione. È possibile raggiungere profondità di centinaia di µm usando la microscopia nonlineare, basata sull'interazione tra il tessuto e più fotoni infrarossi, che subiscono molto di meno gli effetti della diffusione e dell'assorbimento nel tessuto. Risoluzioni di poche decine di nanometri possono inoltre essere ottenute grazie alla microscopia STED, un miglioramento della modalità confocale. Nell'ultimo decennio, sono stati sviluppati microscopi STED con eccitazione a due fotoni (TPE-STED), in modo da combinare queste due proprietà, con risoluzioni che a profondità di decine di micron arrivano fino a valori 4-5 volte migliori dei sistemi limitati dalla diffrazione. Ciononostante, la diffusione e l'assorbimento del fascio di deplezione limitano la profondità alla quale poter ancora osservare dettagli super-risolti a non più di un centinaio di micron. Lo scopo di questa tesi è stato lo sviluppo del primo microscopio TPE-STED con eccitazione nel range [1000-1500] nm e lunghezze d'onda di deplezione vicine agli 800 nm, in modo da poter sorpassare il limite di profondità degli attuali microscopi STED. In questo regime, sono necessari fluorofori adatti, e per questo abbiamo testato la performance delle molecole ATTO 594, ATTO 647N e mGarnet2. In parallelo, abbiamo usato il sistema a due fasci della piattaforma per fornire simultaneamente imaging nonlineare con assorbimento degenere e nondegenere di fotoni a diverse lunghezze d'onda. Una parte consistente del lavoro di tesi è stato anche concentrato sullo sviluppo di un protocollo di fabbricazione e caratterizzazione di elementi ottici per la manipolazione del fascio STED. Lo sforzo è stato compiuto con l'intenzione di poter abbinare liberamente ogni fluoroforo selezionato con la lunghezza di deplezione più efficiente, senza dover attendere i lunghi tempi necessari per la richiesta di soluzioni commerciali

A perspective on NETosis in diabetes and cardiometabolic disorders

Aims: To review the significance of a new type of neutrophil cell death (NETosis) in diabetes and cardiometabolic diseases. Data synthesis: Diabetes and the metabolic syndrome are characterized by activation of the innate immune system. In this framework, neutrophils are front line defences against infections, but can also turn deleterious if abnormally stimulated. NETosis refers to a type of cell death whereby neutrophils release nuclear material and granule enzymes that together form the NETs (neutrophil extracellular traps). As NETs entrap bacteria, NETosis is instrumental to the clearance of microorganisms, but an exaggerated NETosis response can also lead to tissue damage in several pathological conditions. In diabetes, the finely tuned balance of NETosis required to protect the human body from microorganisms yet avoiding self-damage seems to be lost. In fact, in vitro induction of NETosis and circulating concentrations of NET-associated proteins appear to be enhanced in diabetic patients. Furthermore, NETs contribute to endothelial damage, thrombosis, and ischemia/reperfusion injury, making it a novel player in the pathobiology of cardiovascular disease. Though the cellular events taking place during NETosis have been described and directly visualized, its molecular machinery is still incompletely understood. Protein kinase C (PKC) and NADPH oxidase (NOX) are two important targets to counter NETosis in the setting of diabetes. Conclusions: NETosis appears to be part of an abnormal response to damage in diabetes that, in turn, can promote or aggravate end-organ complications. We suggest that this will be a hot topic of investigation in diabetology in the near futur

Aqueye+: a wavefront sensorless adaptive optics system for narrow field coronagraphy

We have designed Aqueye+, an instrument for the Copernicus 182 cm Asiago Telescope, with two channels, one devoted to ultrafast photometry based on four single photon avalanche photodiodes, the second dedicated to stellar coronagraphy based on innovative optical vortex coronagraph system. The OVC requires a very good image quality, therefore an adaptive optic system AO was designed for the instrument. The peculiarity of this AO system is that there is no wavefront sensors, but the feedback for the deformable mirror is instead given by the photometric channel of Aqueye+

The risks associated with percutaneous native kidney biopsies: a prospective study

The known risks and benefits of native kidney biopsies are mainly based on the findings of retrospective studies. The aim of this multicentre prospective study was to evaluate the safety of percutaneous renal biopsies and quantify biopsy-related complication rates in Italy