Single molecule fluorescence: from physical fascination to biological relevance

Confocal fluorescence microscopy is particularly well-known from the beautiful\ud images that have been obtained with this technique from cells. Several cellular\ud components could be nicely visualized simultaneously by staining them with\ud different fluorophores. Not only for ensemble applications but also in single\ud molecule research confocal fluorescence microscopy became a popular technique.\ud In this thesis the possibilities are shown to study a complicated biological process, which is Nucleotide Excision Repair (NER), on the single molecule level with confocal fluorescence microscopy. In chapter one an introduction is given in single molecule fluorescence, properties of fluorescent labels and immobilization methods. Also the NER process is described and the configuration of the confocal fluorescence microscope used

In vivo endoscopic autofluorescence microspectro-imaging of bronchi and alveoli

Fibered confocal fluorescence microscopy (FCFM) is a new technique that can be used during a bronchoscopy to analyze the nature of the human bronchial and alveolar mucosa fluorescence microstructure. An endoscopic fibered confocal fluorescence microscopy system with spectroscopic analysis capability was developed allowing real-time, simultaneous images and emission spectra acquisition at 488 nm excitation using a flexible miniprobe that could be introduced into small airways. This flexible 1.4 mm miniprobe can be introduced into the working channel of a flexible endoscope and gently advanced through the bronchial tree to the alveoli. FCFM in conjunction with bronchoscopy is able to image the in vivo autofluorescence structure of the bronchial mucosae but also the alveolar respiratory network outside of the usual field of view. Microscopic and spectral analysis showed that the signal mainly originates from the elastin component of the bronchial subepithelial layer. In non smokers, the system images the elastin backbone of the aveoli. In active smokers, a strong autofluorescence signal appears from alveolar macrophages. The FCFM technique appears promising for in vivo exploration of the bronchial and alveolar extracellular matrix

Electrochemically Induced pH Change: Time-Resolved Confocal Fluorescence Microscopy Measurements and Comparison with Numerical Model

Confocal fluorescence microscopy is a proven technique, which can image near-electrode pH changes. For a complete understanding of electrode processes, time-resolved measurements are required, which have not yet been provided. Here we present the first measurements of time-resolved pH profiles with confocal fluorescence microscopy. The experimental results compare favorably with a one-dimensional reaction-diffusion model; this holds up to the point where the measurements reveal three-dimensionality in the pH distribution. Specific factors affecting the pH measurement such as attenuation of light and the role of dye migration are also discussed in detail. The method is further applied to reveal the buffer effects observed in sulfate-containing electrolytes. The work presented here is paving the way toward the use of confocal fluorescence microscopy in the measurement of 3D time-resolved pH changes in numerous electrochemical settings, for example in the vicinity of bubbles.Comment: 10 pages, 7 figures, Supplementary informatio

Surface Modification of Melt Extruded Poly(ε-caprolactone) Nanofibers: Toward a New Scalable Biomaterial Scaffold.

A photochemical modification of melt-extruded polymeric nanofibers is described. A bioorthogonal functional group is used to decorate fibers made exclusively from commodity polymers, covalently attach fluorophores and peptides, and direct cell growth. Our process begins by using a layered coextrusion method, where poly(ε-caprolactone) (PCL) nanofibers are incorporated within a macroscopic poly(ethylene oxide) (PEO) tape through a series of die multipliers within the extrusion line. The PEO layer is then removed with a water wash to yield rectangular PCL nanofibers with controlled cross-sectional dimensions. The fibers can be subsequently modified using photochemistry to yield a "clickable" handle for performing the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction on their surface. We have attached fluorophores, which exhibit dense surface coverage when using ligand-accelerated CuAAC reaction conditions. In addition, an RGD peptide motif was coupled to the surface of the fibers. Subsequent cell-based studies have shown that the RGD peptide is biologically accessible at the surface, leading to increased cellular adhesion and spreading versus PCL control surfaces. This functionalized coextruded fiber has the advantages of modularity and scalability, opening a potentially new avenue for biomaterials fabrication

Novel nontoxic mitochondrial probe for confocal fluorescence microscopy.

We propose a 2,5-Bis[1-(4-N-methylpyridinium)ethen-2-yl)]-N-methylpyrrole ditriflate (PEPEP) as a novel nontoxic, nonpotentiometric mitochondrial probe for confocal fluorescence microscopy. PEPEP is a representative chromophore of a large family of heterocyclic fluorescent dyes that show fluorescence emission in aqueous media and great DNA affinity. We check its cytotoxicity and intracellular localization in mammalian and yeast cell cultures. We demonstrate that PEPEP is a very efficient dye for fluorescence confocal microscopy and a valuable alternative to the most frequently used mitochondrial stains. © 2006 Society of Photo-Optical Instrumentation Engineers

Red-Emitting Tetracoordinate Organoboron Chelates: Synthesis, Photophysical Properties, and Fluorescence Microscopy

Seven tetracoordinate organoboron fluorophores with heterobiaryl N,O- or N,N-chelate ligands were prepared and photophysically characterized (in toluene). The electronic variation of the heteroaromatic moiety provided a means for the fine-tuning of the UV/vis absorption and emission spectra. In the most interesting cases, the spectra were red-shifted to maximum absorbance at wavelengths longer than 500 nm and emission maxima between 620 and 660 nm. The pronounced intramolecular charge-transfer character of the dyes yielded large Stokes shifts (3500-5100 cm), while maintaining appreciable fluorescence quantum yields of up to 0.2 for emission maxima longer than 600 nm. The lipophilic character of the dyes enabled their application as stains of vesicle substructures in confocal fluorescence microscopy imaging.Ministerio de Economía y Competitividad CTQ2014-54729 - C2 - 1 - P for U.P., CTQ2013 - 48164 - C2 - 1 - P , CTQ2013 - 48164 - C2 - 2 - P for A.R., CTQ2013 - 41339 - P, CTQ2015 - 71896 - REDT for E.P.I., Ramón y Cajal contract RYC - 2013 - 12585 for A.R.FEDER FundJunta de Andalucía 2012/FQM - 2140 for U.P., 2009/FQM - 4537 and 2012/FQM - 1078 for A.R

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes

Gold nanoparticle coated silicon nitride chips for intracellular surface-enhanced raman spectroscopy

Using surface-enhanced Raman spectroscopy on gold-nanoparticle-decorated silicon nitride chips, we monitor the release of dextran-rhodamin molecules from capsules inside living cells. This demonstrates the feasibility of using photonic chips for intracellular sensing at visible wavelengths