Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

FLUORINATED EMITTER MOLECULES FOR TRIPLET-TRIPLET ANNIHILATION UP-CONVERSION MEDIA

This thesis details the synthesis, characterisation and photophysical properties of various fluorinated emitters for triplet-triplet annhilation up-conversion systems. The theory and mechanism of TTAUC is evaluated and the molecular design of the annihilating emitter molecule reviewed to allow improvement of the external up-conversion quantum yield and overall energy efficiency of the process. Three main series of chromaphores were investigated, based on diphenylanthracenes, bisphenylperylenes, and 3,5,8-triphenylBODIPYs. These were synthesised by metal catalysed aryl-aryl coupling (Suzuki-Miyaura) or nucleophilic substitution reactions and, when paired with appropriate sensitizing molecules, allowed the up-conversion of green to blue; red to green; and near IR/red to orange respectively. The effect of increasing fluorination on the ease of synthesis and photophysical properties of these emitter systems was studied with a view to their application in up-conversion systems. Fluorinated emitter molecules were shown to be highly resistant to degradation by UV light compared to their non-fluorinated analogues. The up-conversion ability of these systems was evaluated and novel fluorinated BODIPY based dyes were produced that have high fluorescence quantum yields of over 90%. Finally the up-conversion of up-converting nanoparticles incorporating fluorinated emitters was evaluated

Rare earth based nanostructured materials: Synthesis, functionalization, properties and bioimaging and biosensing applications

Rare earth based nanostructures constitute a type of functional materials widely used and studied in the recent literature. The purpose of this review is to provide a general and comprehensive overview of the current state of the art, with special focus on the commonly employed synthesis methods and functionalization strategies of rare earth based nanoparticles and on their different bioimaging and biosensing applications. The luminescent (including downconversion, upconversion and permanent luminescence) and magnetic properties of rare earth based nanoparticles, as well as their ability to absorb X-rays, will also be explained and connected with their luminescent, magnetic resonance and X-ray computed tomography bioimaging applications, respectively. This review is not only restricted to nanoparticles, and recent advances reported for in other nanostructures containing rare earths, such as metal organic frameworks and lanthanide complexes conjugated with biological structures, will also be commented on.European Union 267226Ministerio de Economía y Competitividad MAT2014-54852-

Luminescent Transition Metal Complexes: Optical Characterization, Integration into Polymeric Nanoparticles and Sensing Applications

Photoluminescence is a fascinating phenomenon which has a huge impact on our daily life. Many important applications are based on this principle, such as imaging diagnostics, bioanalytic, photocatalysis, solar cells, or optoelectronic devices. The emerging demands for versatile photoluminescent materials have encouraged generations of scientists to develop different types of luminophores, ranging from molecular dyes to luminescent nanomaterials. Among them, luminescent transition metal complexes (TMCs), which consist of one (or more) metal center and several organic or inorganic ligands, are drawing increasing interest due to their unique photophysical and photochemical properties, such as large Stokes shift, long-lived triplet excited state, sharp emission band (f-block metal complexes), and multiple stale oxidation states (d-block metal complexes). These distinct optical properties are not only of great research interest, but also have led to commercial applications, such as imaging agents, optical sensors, light-harvesting materials, optical barcoding, and displays. The demands and desires of optoelectronic devices and higher requirements in bioanalytic make the development of new TMCs necessary, which needs to be examined in detail not only in terms of their chemical but much more importantly their photophysical properties. In this work, a series of new types of luminescent TMCs are involved, including Cr(III)-, Pt(II)-, and Pd(II) complexes. Based on their optical studies, a series of proof-of-concept applications were designed by introducing these metal complexes to different nanomatrix, such as polymeric nanoparticles and metal-organic frameworks (MOFs), resulting various luminescent nanosensors or energy-conversion materials. The major part of this work is based on the [Cr(ddpd)2]3+ complex (ddpd = N, N′‐dimethyl‐N, N´-dipyridine‐2‐ylpyridine‐2,6‐diamine) and his derivatives. Fundamental photophysical studies of these Cr(III) complexes showed that their photoluminescence properties can be significantly enhanced by ligand and solvent deuteration. Moreover, a choice of bulky counter anions can provide an enhancement in the photoluminescence properties as well as the oxygen sensitivity. In addition, based on the photophysical understanding of the [Cr(ddpd)2]3+ complex, a proof-of-concept study of photon upconversion in molecular chromium ytterbium salts was completed. Upon an excitation of the Yb3+ sensitizers at 976 nm, these solid-state salts produced upconverted luminescence of the Cr3+ activator at 780 nm at room temperature. Another proof-of-concept study based on the [Cr(ddpd)2]3+ complex was investigated by designing and developing multianalyte nanosensors for simultaneously measuring temperature (“T”), oxygen (“O”), and pH (“P”) in aqueous phase under one excitation wavelength. Apart from the [Cr(ddpd)2]3+ complexes, four novel Pt(II)- and Pd(II)-complexes bearing tetradentate ligands were also studied regarding their photophysical properties in solutions and in polystyrene nanoparticles (PS-NPs). In PS-NPs, the aggregation-induced Metal-Metal-to-Ligand Charge-Transfer (3MMLCT) state of the fluorinated Pt(II) complex is red-shifted compared to the monomeric emission and performs insensitive to oxygen, allowing the particles as self-referenced oxygen nanosensor in both the luminescence intensity and lifetime domains. Additionally, a triplet–triplet annihilation upconversion (TTA-UC) system was developed based on a crystalline MOF. A Pd(II) porphyrin complex acted as the sensitizer immobilized in the MOF walls, while a 9,10-diphenylanthracene annihilator was filled in the channels. Upon green light excitation at 532 nm, the resulting MOF crystalline showed an upconverted blue emission with delayed lifetime from 4 ns to 373 µs and a triplet–triplet energy transfer efficiency of 82%

Spectral management for quantum solar energy harvesting: changing the colour of the sun

The study and deployment of solar energy conversion systems are justified on many grounds: environmental, economic, geopolitical, and societal. Collectively, these justifications provide a dynamic and compelling backdrop for the continuing narrative of solar energy. The energy conversion efficiency of a solar cell is set by the design of the cell and by the properties of the incident sunlight. Thus in addition to works aimed at improving solar cells directly, are those directed towards shaping the solar spectrum incident on the cell, prior to sunlight absorption. So-called spectral management is distinct from, but closely related to, solar cells. Two such techniques are documented here. The first, luminescent concentration, downshifts energy and concentrates photon flux within a luminophore-doped waveguide. Problems associated with luminescence concentrators are reported, motivating a novel arrangement of the light absorbing centers aimed at ameliorating lossy emission by induced photon anisotropy. We present the first experimentally-realised implementation of the design. The second portion of work concerns triplet-triplet annihilation upconversion (TTA-UC), a means by which sub-band gap photon losses in solar cells can be reduced. We present schemes for tethering TTA-UC absorbers to nanostructured solids in a bid to increase chromophore concentration and UC efficiency. Kinetic studies of these materials are presented. Results show the formation of heterogeneous structures dependent on the chromophore, binding mechanism and scaffold. Solar cell enhancement experiments were used to show the enhancement of a H-passivated a-Si solar cell by a solid-tethered upconverter, producing modest gains in short-circuit current. The action spectrum, a novel photoluminescence technique for measuring TTA-UC efficiency, was measured for two materials, and the results corroborated using rate measurements. The action spectrum is a promising new upconversion characterisation method

Triplet-triplet annihilation mediated photon upconversion solar energy systems

Solar energy harvesting is among the best solutions for a global transition toward carbon-neutral energy technologies. The existing solar energy harvesting technologies like photovoltaics (PV) and emerging molecular concepts such as solar fuels and molecular solar thermal energy storage (MOST) are rapidly developing. However, to realize their full potential, fundamental solar energy loss channels like photon transmission, recombination, and thermalization need to be addressed. Triplet-triplet annihilation mediated photon upconversion (TTA-UC) is emerging as a way to overcome losses due to the transmission of photons below the PV/chromophore band gap. However, there are several challenges related to the integration of efficient solid-state TTA-UC systems into efficient devices such as: wide band absorption, materials sustainability, and device architecture. In this article, we review existing work, identify and discuss challenges as well as present our perspective toward possible future directions

New photo-luminescent inorganic materials: high-tec application in chemical sensing and labeling

This thesis describes the potential of various kinds of luminescent nanoparticles with respect to chemical sensing and biosensing. First, fluorescent silica nanoparticles (SiNPs) were prepared by covalent attachment of fluorophores to the amino-modified surface of SiNPs with a typical diameter of 15 nm. The SiNPs were used in novel kinds of Förster resonance energy transfer (FRET)-based affinity assays at the interface between nanoparticle and sample solution. Various labels were employed to obtain a complete set of colored SiNPs, with excitation maxima ranging from 337 to 659 nm and emission maxima ranging from 436 nm to the near infrared (710 nm). The nanoparticles were characterized in terms of size and composition using transmission electron microscopy, thermogravimetry, elemental analysis, and dynamic light scattering. The surface of the fluorescent SiNPs was biotinylated, and binding of labeled avidin to the surface was studied via FRET in two model cases. Secondly, the upconverting luminescent nanoparticles (UCLNPs) consist of hexagonal NaYF4 nanocrystals doped with trivalent rare earth ions were synthesized by both the oleic acid (solvothermal) method and the ethylenediaminetetraacetic acid (coprecipitation) method. The nanoparticles were codoped using Yb3+ as the sensitizer ion, Er3+, Tm3+, or Ho3+ respectively as the emitting activator ions. An affinity system was demonstrated based on the interaction of two types of nanoparticles. The first type consists of UCLNPs of the type NaYF4:Yb,Er absorbing light in the infrared and showing green luminescence at 521 and 543 nm and red luminescence at 657 nm. The second type consists of gold nanoparticles (Au-NPs) with a size of about 50 nm, which absorb the green luminescence of the UCLNPs, but do not influence their red luminescence. A model system for a self referenced affinity system were established by labeling the UCLNPs with avidin and the AuNPs with biotin. In the presence of avidin-modified UCLNPs, the biotinylated Au-NPs can be detected in the range from 12 to 250 µg•mL-1 by rationing the intensity of the red (analyte-independent) emission band to that of the green (analyte-dependent) emission band. All nanoparticles were characterized in terms of size and composition using transmission electron microscopy, thermo-gravimetry, and FTIR spectroscopy. Thirdly, different types of nanoparticles (made from silica, polystyrene and UCLNPs) carrying longwave absorbing and emitting fluorescent labels were prepared by conjugating reactive dyes to the surface of amino-modified particles. The dyes have a reactive chloro group capable of reacting with amino groups and thereby undergoing a change in color, typically from green to blue (the so-called chameleon effect). The latter show the effect of upconversion in that near-infrared laser light is converted into visible luminescence. They also show the unusual property of displaying dual emission, depending on whether their luminescence is photoexcited with visible light or near-infrared light. The amino groups on the surface of nanoparticles were detected via the chameleon effect of the applied amino-reactive dyes. Fourth, the quenching effect of heavy metal ions and halide ions on the luminescence of UCLNPs in aqueous solution was studied. The effect was investigated for the ions Cu(II), Hg(II), Pb(II), Cd(II), Co(II), Ag(I), Fe(III), Zn(II), bromide and iodide, and was found to be particularly strong for Hg(II). Stern-Volmer plots were virtually linear up to 10 – 25 mM concentrations of the quencher, but deviate from linearity at higher quencher concentrations where static quenching caused an additional effect. The UCLNPs display two main emission bands (blue, green, red or near-infrared), and the quenching efficiencies for these found to be different. The effect seems to be generally associated with UCLNPs because it was observed for all particles doped with trivalent lanthanide ions including Yb(III), Er(III), Ho(III), and Tm(III)