Diffusion of Alexa Fluor 488-Conjugated Dendrimers in Rat Aortic Tissue

In this study, the distribution of labeled dendrimers in native and aneurysmal rat aortic tissue was examined. Adult male rats underwent infrarenal aorta perfusion with generation 5 (G5) acetylated Alexa Fluor 488-conjugated dendrimers for varying lengths of time. In a second set of experiments, rats underwent aortic elastase perfusion followed by aortic dendrimer perfusion 7 days later. Aortic diameters were measured prior to and postelastase perfusion, and again on the day of harvest. Aortas were harvested 0, 12, or 24 h postperfusion, fixed, and mounted. Native aortas were harvested and viewed as negative controls. Aortic cross-sections were viewed and imaged using confocal microscopy. Dendrimers were quantified (counts high-powered field). Results were evaluated by repeated measures ANOVA and Student's t -test. We found that in native aortas, dendrimers penetrated the aortic wall in all groups. For all perfusion times, fewer dendrimers were present as time between dendrimer perfusion and aortic harvest increased. Longer perfusion times resulted in increased diffusion of dendrimers throughout the aortic wall. By 24 h, the majority of the dendrimers were through the wall. Dendrimers in aneurysmal aortas, on day 0 postdendrimer perfusion, diffused farther into the aortic wall than controls. In conclusion, this study documents labeled dendrimers delivered intra-arterially to native rat aortas in vivo , and the temporal diffusion of these molecules within the aortic wall. Increasing perfusion time and length of time prior to harvest resulted in continued dendrimer diffusion into the aortic wall. These preliminary data provide a novel mechanism whereby local inhibitory therapy may be delivered locally to aortic tissue.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72448/1/annals.1383.004.pd

Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

Background: The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. Methodology/Principal Findings: A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells

Nanoscale control of Ag nanostructures for plasmonic fluorescence enhancement of near-infrared dyes

Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based detection techniques. Metal induced fluorescence enhancement offers the possibility of increasing the sensitivity of protein detection in clinical applications. We report the use of tunable plasmonic silver nanostructures for the fluorescence enhancement of a near-infrared (NIR) dye (Alexa Fluor 790). Extensive fluorescence enhancement of ∼2 orders of magnitude is obtained by the nanoscale control of the Ag nanostructure dimensions and interparticle distance. These Ag nanostructures also enhanced fluorescence from a dye with very high quantum yield (7.8 fold for Alexa Fluor 488, quantum efficiency (Qy) = 0.92). A combination of greatly enhanced excitation and an increased radiative decay rate, leading to an associated enhancement of the quantum efficiency leads to the large enhancement. These results show the potential of Ag nanostructures as metal induced fluorescence enhancement (MIFE) substrates for dyes in the NIR “biological window” as well as the visible region. Ag nanostructured arrays fabricated by colloidal lithography thus show great potential for NIR dye-based biosensing applications

Controlled reduction of photobleaching in DNA origami gold nanoparticle hybrids

The amount of information obtainable from a fluorescence-based measurement is limited by photobleaching: Irreversible photochemical reactions either render the molecules nonfluorescent or shift their absorption and/or emission spectra outside the working range. Photobleaching is evidenced as a decrease of fluorescence intensity with time, or in the case of single molecule measurements, as an abrupt, single-step interruption of the fluorescence emission that determines the end of the experiment. Reducing photobleaching is central for improving fluorescence (functional) imaging, single molecule tracking, and fluorescence-based biosensors and assays. In this single molecule study, we use DNA self-assembly to produce hybrid nanostructures containing individual fluorophores and gold nanoparticles at a controlled separation distance of 8.5 nm. By changing the nanoparticles? size we are able to systematically increase the mean number of photons emitted by the fluorophores before photobleaching.Fil: Pellegrotti, Jesica Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Acuña, Guillermo. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Puchkova, Anastasiya. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Holzmeister, Phil. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Gietl, Andreas. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Lalkens, Birka. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Tinnefeld, Philip. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemani

Working with Commercially Available Quantum Dots for Immunofluorescence on Tissue Sections

Quantum dots are semiconductor fluorescent nanocrystals that exhibit excellent characteristics compared with more commonly used organic fluorescent dyes. For many years quantum dot conjugated products have been available in multiple forms for fluorescence imaging of tissue sections under the trademark name Qdot®. They have much increased brightness, narrow emission spectrum, large Stokes shift and photostability compared with conventional organic fluorescent dyes, which together make them the fluorophores of choice for demanding requirements. Vivid Qdots are recent replacements for original Qdots, modified to improve brightness, however this has affected the fluorescence stability in commonly used conditions for immunohistochemistry. We present here our investigation of the stability of original and Vivid Qdots in solution and in immunohistochemistry, highlight the potential pitfalls and propose a protocol for stable and reliable multiplex staining with current commercially available original and Vivid Qdots

Structural Heterogeneity and Quantitative FRET Efficiency Distributions of Polyprolines through a Hybrid Atomistic Simulation and Monte Carlo Approach

Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies