MitoBlue as a tool to analyze the mitochondria-lysosome communication

MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations in living cells. MitoBlue is a unique tool for the study of these processes that will allow the detailed characterization of communication between mitochondria and lysosomesWe are thankful for the support given by the Spanish grants BFU2013-43513-R, SAF2016-76689-R, CTQ2015-70698-R, RTI2018-099877-B-I00, CTQ2013-49317-EXP, CTQ2013-41339-P, CTQ2016-75870-P, CTQ2015-71896-REDT (Red de Fotoquímica Biológica) and Instituto de Salud Carlos III (ISCIII; RETIC ARADYALRD16/0006/0012), Junta de Andalucía (PI0250-2016 and UMA18-FEDERJA-007), Orfeo-cinqa network CTQ2016-81797-REDC; the Xunta de Galicia (2015-CP082, ED431C 2017/19, Centro Singular de investigación de Galicia accreditation 2019–2022, ED431G 2019/03), the European Union (European Regional Development Fund - ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledged. M.E.V. also acknowledges the support provided by the Fundación AECC (IDEAS197VAZQ grant)S

Optička svojstva nanoklastera plemenitih metala unutar hibridnih sustava i njihova primjena u biosenzorici

Linear and nonlinear optical properties of functionalized forms of noble metal nanoclusters are addressed in this thesis. Noble metal nanoclusters belong to nonscalable regime in which each atom counts. They exhibit molecular like electronic transitions, due to quantum confinement effects, which are strongly influenced by structure. These systems have potential for applications in biosensing and bioimaging, based on their unique optical, electronic and structural properties, after being stabilized either by formation of hybrid systems of metal nanoclusters and biomolecules or protecting nanoclusters by ligands. Using density functional theory and its time dependent version interplay between electronic and structural properties in hybrid nanocluster-biomolecules and ligated clusters has been investigated in order to gain insight into origins of their unique optical properties. To understand optical properties of hybrid systems, silver cluster-histidine complexes, as well as nonlinear properties of ligated silver nanoclusters have been investigated theoretically, and compared with experimental results obtained by expert international collaborators. Within this thesis the concept has been worked out allowing to design systems with large two-photon cross sections which can serve for efficient imaging of tissues and cells.U ovoj tezi su opisana linearna i nelinearna optička svojstva funkcioniziranih oblika nanoklastera plemenitih metala. Nanoklasteri plemenitih metala pripadaju neskalirajućim sustavima u kojima se svaki atom broji. Zbog efekata kvantnog zatočenja, imaju elektronske prijelaze slične onima u molekulama, na koje jako utječe struktura. Zbog svojih jedinstvenih optičkih, elektronskih i strukturnih svojstava, potencijalni su kandidati za primjene u biosenzorici i biooslikavanju, nakon što se stabiliziraju formiranjem hibridnih sustava s biomolekulama ili ih se zaštiti ligandima. U tezi je istraživan je odnos elektronskih i strukturnih svojstava u hibridnim sustavima biomolekula i metalnih klastera kao i u ligandiranim klasterima kako bi se razumio uzrok njihovih jedinstvenih optički svojstava, koristeći teoriju funkcionala gustoće i njenu vremenski ovisnu varijantu. Da bi se razumjela svojstva hibridnih sustava, teorijski su istraživani hibridni sustavi srebrenih klastera i histidina te nelinearna svojstva ligandiranih srebrnih klastera, i uspoređeni s eksperimentalnim rezultatima koje su napravili međunarodni suradnici. Razvijen je koncept koji bi mogao pomoći dizajniranju sustava s jakom dvo-fotonskom apsorpcijom koji bi služili za oslikavanje tkiva i stanica

Optička svojstva nanoklastera plemenitih metala unutar hibridnih sustava i njihova primjena u biosenzorici

Linear and nonlinear optical properties of functionalized forms of noble metal nanoclusters are addressed in this thesis. Noble metal nanoclusters belong to nonscalable regime in which each atom counts. They exhibit molecular like electronic transitions, due to quantum confinement effects, which are strongly influenced by structure. These systems have potential for applications in biosensing and bioimaging, based on their unique optical, electronic and structural properties, after being stabilized either by formation of hybrid systems of metal nanoclusters and biomolecules or protecting nanoclusters by ligands. Using density functional theory and its time dependent version interplay between electronic and structural properties in hybrid nanocluster-biomolecules and ligated clusters has been investigated in order to gain insight into origins of their unique optical properties. To understand optical properties of hybrid systems, silver cluster-histidine complexes, as well as nonlinear properties of ligated silver nanoclusters have been investigated theoretically, and compared with experimental results obtained by expert international collaborators. Within this thesis the concept has been worked out allowing to design systems with large two-photon cross sections which can serve for efficient imaging of tissues and cells.U ovoj tezi su opisana linearna i nelinearna optička svojstva funkcioniziranih oblika nanoklastera plemenitih metala. Nanoklasteri plemenitih metala pripadaju neskalirajućim sustavima u kojima se svaki atom broji. Zbog efekata kvantnog zatočenja, imaju elektronske prijelaze slične onima u molekulama, na koje jako utječe struktura. Zbog svojih jedinstvenih optičkih, elektronskih i strukturnih svojstava, potencijalni su kandidati za primjene u biosenzorici i biooslikavanju, nakon što se stabiliziraju formiranjem hibridnih sustava s biomolekulama ili ih se zaštiti ligandima. U tezi je istraživan je odnos elektronskih i strukturnih svojstava u hibridnim sustavima biomolekula i metalnih klastera kao i u ligandiranim klasterima kako bi se razumio uzrok njihovih jedinstvenih optički svojstava, koristeći teoriju funkcionala gustoće i njenu vremenski ovisnu varijantu. Da bi se razumjela svojstva hibridnih sustava, teorijski su istraživani hibridni sustavi srebrenih klastera i histidina te nelinearna svojstva ligandiranih srebrnih klastera, i uspoređeni s eksperimentalnim rezultatima koje su napravili međunarodni suradnici. Razvijen je koncept koji bi mogao pomoći dizajniranju sustava s jakom dvo-fotonskom apsorpcijom koji bi služili za oslikavanje tkiva i stanica

Microscopy and Analysis

Microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been impossible to stand where we stand today in terms of understanding the structure and functions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of micro- and nanotechnologies critically depends on high-resolution imaging systems. This volume includes a series of chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Authoritative voices in their fields present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area

Development of optical methods for real-time whole-brain functional imaging of zebrafish neuronal activity

Each one of us in his life has, at least once, smelled the scent of roses, read one canto of Dante’s Commedia or listened to the sound of the sea from a shell. All of this is possible thanks to the astonishing capabilities of an organ, such as the brain, that allows us to collect and organize perceptions coming from sensory organs and to produce behavioural responses accordingly. Studying an operating brain in a non-invasive way is extremely difficult in mammals, and particularly in humans. In the last decade, a small teleost fish, zebrafish (Danio rerio), has been making its way into the field of neurosciences. The brain of a larval zebrafish is made up of 'only' 100000 neurons and it’s completely transparent, making it possible to optically access it. Here, taking advantage of the best of currently available technology, we devised optical solutions to investigate the dynamics of neuronal activity throughout the entire brain of zebrafish larvae