Near-Field Fluorescence Imaging of Genetic Material: Toward the Molecular Limit

Chromosomes, DNA, and single fluorescent molecules are studied using an aperture-type near-held scanning optical microscope with tuning fork shear force feedback. Fluorescence in situ hybridization labels o­n repetitive and single copy probes o­n human metaphase chromosomes are imaged with a width of 80 nm, allowing their localization with nanometer accuracy, in direct correlation with the simultaneously obtained topography. Single fluorophores, both in polymer and covalently attached to amino- silanized glass, are imaged using two-channel fluorescence polarization detection. The molecules are selectively excited according to their dipole orientation. The orientation of the dipole moment of all molecules in o­ne image could be directly determined. Rotational dynamics o­n a 10-ms to 100-s timescale is observed. Finally, shear force imaging of double-stranded DNA with a vertical sensitivity of 0.2 nm is presented. A DNA height of 1.4 nm is measured, which indicates the nondisturbing character of the shear force mechanism

PH-responsive polysaccharide-based polyelectrolyte complexes as nanocarriers for lysosomal delivery of therapeutic proteins

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Single-molecule photobleaching probes the exciton wave function in a multichromophoric system

Ajuts: This work has been supported by the EC Program IHP- 99 (HPMF-CT-2002-01698)The exciton wave function of a trichromophoric system is investigated by means of single molecule spectroscopy at room temperature. Individual trimers exhibit superradiance and loss of vibronic structure in emission spectrum, features proving exciton delocalization. We identify two distinct photodegradation pathways for single trimers upon sequential photobleaching of the chromophores. The rate of each pathway is a measure for the contribution of the separate dyes to the collective excited state of the system, in this way probing the wave function of the delocalized exciton

Energy transfer in single molecular photonic wires

Molecular photonics is a new emerging field of research around the premise that it is possible to develop optical devices using single molecules as building blocks. Truly technological impact in the field requires focussed efforts on designing functional molecular devices as well as having access to their photonic properties on an individual basis. In this Minireview we discuss our approach towards the design and single-molecule investigation of one-dimensional multimolecular arrays intended to work as molecular photonic wires. Three different schemes have been explored: a) perylene-based dimer and trimer arrays displaying coherent exciton delocalisation at room temperature; b) DNA-based unidirectional molecular wires containing up to five different chromophores and exhibiting weak excitonic interactions between neighbouring dyes; and c) one-dimensional multichromophoric polymers based on perylene polyisocyanides showing excimerlike emission. As a whole, our single-molecule data show the importance of well-defined close packing of chromophores for obtaining optimal excitonic behaviour at room temperature. Further improvement on (bio)chemical synthesis, together with the use of single-molecule techniques, should lead in the near future to efficient and reliable photonic wires with true device functionality

Monolayer-functionalized microfluidics devices for optical sensing of acidity

This paper describes the integration of opto-chemosensors in microfluidics networks. Our technique exploits the internal surface of the network as a platform to build a sensing system by coating the surface with a self-assembled monolayer and subsequently binding a fluorescent sensing molecule to the monolayer. Fluorescent molecules were used that can switch between a fluorescent and a non-fluorescent state, depending on the acidity of the surrounding solution. Two systems were investigated. The first employs surface confinement of a Rhodamine B dye in a glass micro channel that serves as a molecular switch in organic solutions. Upon rinsing the micro channels with acidic or basic solutions it was possible to switch between the fluorescent and non- fluorescent forms reversibly. Moreover, this system could be used to monitor the mixing of two solutions of different acidity along the micro channel. To widen the scope of optical sensing in micro channels an Oregon Green dye derivative was immobilized, which functions as a sensing molecule for pH differences in aqueous solutions. In this case, a hybrid system was used consisting of a glass slide and PDMS channels. The fluorescence intensity was found to be directly correlated to the pH of the solution in contact, indicating the possibility of using such a system as a pH sensor. These systems allow real-time measurements and can be easily implemented in micro- and nanofluidics systems thus enabling analysis of extremely small sample volumes in a fast and reproducible manner