Fluorescent Micro Research Articles

Micro-flow assisted synthesis of fluorescent polymer nanoparticles with tuned size and surface properties

AbstractNumerous different photonics and biomedical applications depend on the fluorescent polymer micro- and nanoparticles. Besides optical or spectroscopic properties, the performance of the polymer nanoparticles is determined by their size, size distribution, and surface charge. Moreover, in order to realize a very uniform performance, the functional polymer nanoparticles should be of high homogeneity and demand for the preparation in a minimum number of synthesis steps. Here, we present a microfluidic-assisted synthesis of different types of reproducible fluorescent polymer nanoparticles with tuned size (40 nm up to 600 nm) and surface charge (ζ potential=-52 mV up to +45 mV). Four different preparation strategies were introduced for fluorophore-functionalized nanoparticles: (a) noncovalent binding of fluorophores with high loading, (b) covalent linking of fluorophores with enhanced stability, (c) surface-anchored fluorophores by hydrophobic interactions for triple function at the same time, and (d) surface immobilization of biomolecules and fluorophore by ionic as well as secondary interactions. In this way, four different classes of nanoparticles suited for different applications were prepared with a spherical shape as a model system. Moreover, the principle has been extended to the different types of nonspherical and composite polymer nanoparticles.

Magnetic Mechanotransduction of Mesenchymal Stem Cells via a Highly Fluorescent Magnetic Micro Patterned Anti-Microbial Composite Scaffold

Magnetic Mechanotransduction of Mesenchymal Stem Cells via a Highly Fluorescent Magnetic Micro Patterned Anti-Microbial Composite Scaffold

High-brightness, high-power LED-based strobe illumination for double-frame micro particle image velocimetry

There is a need for more practical, cost-effective and adaptive flow measurement instruments. Unfortunately, most of the current equipment still has to employ bulky and expensive laser systems for flow illumination. In this work we explore the practicability of using high-brightness high-power light-emitting diodes (LED) as a strobe-light illuminator within a double-frame micro particle image velocimetry (microPIV) system. A compact, cost-effective, and controllable LED driver was designed and applied to study the optical and spectral behavior of green and blue family direct-color single chip LEDs, which have excitation wavelengths corresponding to the most widely used fluorescent micro particles. To achieve strobe illumination, LEDs were subjected to high current double-pulsed operating regimes (up to 35A at 20μs pulse duration), which are much beyond their physical damage thresholds. We have witnessed that the blue family LEDs are more suitable for the fluorescence based microPIV experiments as they exhibit more stable spectral behavior at the double-pulsed high currents compared to the green colored LEDs. Specifically, we have observed that the spectral wavelength shift of blue family LEDs due to the high current is less severe and still enough to excite particles than that of green LEDs. Also, we have studied the relation between the pulse duration (as a means of parameter controlling signal-to-noise ratio) and the motion blur effect that arises due to the prolonged pulse durations. As a result, adequate pulse durations for most widely used microPIV experimental conditions were calculated. The feasibility and robustness of the developed double-pulsed LED illumination system was validated through a series of microPIV experiments with real flows inside two different topology microchannels, where the measured velocity was in good agreement with the corresponding analytical solutions. Overall, we can conclude that the high-brightness LED devices are promising candidates to replace their laser counterparts in the microPIV systems.

A novel microfluidic switch for pH control using Chitosan based hydrogels

We report a novel pH-sensitive hydrogel based micro-valve for metered flow that has applications in a laboratory made "Intelligent valving system". The hydrogel solution was prepared through Chitosan and poly vinyl alcohol in acetic acid and crystallized using gluteraldehyde as the crosslinking agent in the form of thin wafers and it was found to be very sensitive to pH changes. The pore structure of hydrogel was investigated through Field Emission Scanning Electron Microscopy and thin wafers of the gel were physically placed inside PDMS microchannels. Flow metering in these channels was observed by controlled expansion of the hydrogel plug till complete valving was realized. This valving device was further precisely characterized with micro Particle Image Velocimetry using a solution containing fluorescent polymeric micro beads. The principle advantage of this hydrogel device is the smaller range of pH (varying between pH 3 and 7) over which the valving response is observed.

Synthesis, Photophysics, Live Cell Imaging, and Aggregation Behavior of Some Structurally Similar Alkyl Chain Containing Bromonaphthalimide Systems: Influence of Alkyl Chain Length on the Aggregation Behavior

A series of 4-bromonaphthalimide systems (BNI-Cn ; n = 4, 6, 10, 12, and 16) comprising different alkyl side chains have been synthesized and used as the building blocks to fabricate organic fluorescent micro materials. The systems have been developed basically to investigate the effect of alkyl side chains on the aggregation behavior of the systems. The aggregation behavior of these systems has been studied by spectroscopic and microscopic techniques. Microscopic investigation reveals that there is a decrease in the size of the aggregates with an increase in the linear alkyl side chain length. A change in the shape from rod-like to spherical with an increase in the length of alkyl group has also been observed during microscopic investigation. The photophysical properties of these well-characterized aggregates have been studied and compared with those in molecular form. A bathochromic shift both in absorption and in emission spectrum of the aggregates has indicated the formation of J aggregates. A confocal fluorescence microscopic investigation also reveals that the long chain systems (12 and 16 member) are cell permeable and can be used as the imaging probe in live cells.

Application of lock-in thermography for failure analysis in integrated circuits using quantitative phase shift analysis

Abstract Lock-in thermography (LIT), which is a well established technique for non-destructive evaluation, can also be used to identify and locate thermal active electrically defects like shorts and resistive opens in microelectronic devices. Defect localization on the level of the integrated circuits (IC) requires a μm resolution. But LIT can also be applied to locate buried thermal active defects within fully packaged microelectronic devices by analysing the thermal signal detected at the surface of the device. In addition to the lateral localization of the hot spot, its depth can also be determined by analysing the phase shift of the thermal signal. This is especially valued for non destructive defect localization in complex 3D integrated system in package devices (3D SiP). In comparison to competitive thermal imaging techniques, like liquid crystal imaging or fluorescent micro thermal imaging, LIT is easier to apply since it does not need any foreign thermal sensitive layer at the surface of the device. Also, the sensitivity limit of this technique within μK range is significantly better. In addition the dynamic character of LIT reduces thermal blurring, and the problem of inhomogeneous IR emissivity can be overcome by using the phase image or the 0°/−90° image. The spatial resolution limit of the used microscopic thermal imaging setup performed in the mid-wavelength range is about 5 μm, but can be improved to 1.5 μm by applying solid immersion lenses. Within the paper, the principle theory of LIT and the practical use for both, single and multiple IC devices is presented.

FRET detection of Octamer-4 on a protein nanoarray made by size-dependent self-assembly

An alternative approach for fabricating a protein array at nanoscale is suggested with a capability of characterization and/or localization of multiple components on a nanoarray. Fluorescent micro- and nanobeads each conjugated with different antibodies are assembled by size-dependent self-assembly (SDSA) onto nanometer wells that were created on a polymethyl methacrylate (PMMA) substrate by electron beam lithography (EBL). Antibody-conjugated beads of different diameters are added serially and electrostatically attached to corresponding wells through electrostatic attraction between the charged beads (confirmed by zeta potential analysis) and exposed p-doped silicon substrate underneath the PMMA layer. This SDSA method is enhanced by vibrated-wire-guide manipulation of droplets on the PMMA surface containing nanometer wells. Saturation rates of antibody-conjugated beads to the nanometer patterns are up to 97% under one component and 58-70% under two components nanoarrays. High-density arrays (up to 40,000 wells) could be fabricated, which can also be multi-component. Target detection utilizes fluorescence resonance energy transfer (FRET) from fluorescent beads to fluorescent-tagged secondary antibodies to Octamer-4 (Oct4), which eliminates the need for multiple steps of rinsing. The 100 nm green beads are covalently conjugated with anti-Oct4 to capture Oct4 peptides (39 kDa); where the secondary anti-Oct4 and F(ab)(2) fragment of anti-gIgG tagged with phycoerythrin are then added to function as an indicator of Oct4 detection. FRET signals are detected through confocal microscopes, and further confirmed by Fluorolog3 spectrofluorometer. The success rates of detecting Oct4 are 32% and 14% of the beads in right place under one and two component nanoarrays, respectively. Ratiometric FRET is used to quantify the amount of Oct4 peptides per each bead, which is estimated about 2 molecules per bead.

An experimental system for investigating the characteristics of optical sensor materials

An experimental system for investigating the characteristics of optical sensor materials, in particular, materials based on modified fluorescent micro- and nanoparticles, is described. The system can be used to obtain multicomponent gas-vapor mixtures, measure fluorescence and reflection spectra, and record color images of sensor materials with a time resolution of a few milliseconds. Experimental data obtained when investigating the responses of sensor materials to analytes are presented.

Covalent Modification of the Wool Fiber Surface: The Attachment and Durability of Model Surface Treatments

Exposure of reactive chemical functional groups through controlled surface lipid removal provides a means for covalent attachment of novel molecular entities to the wool fiber surface. Cleavage of the dominant lipid in the epicuticle, 18-methyleicosanoic acid (18-MEA), from the underlying proteinaceous layer was employed, prior to further surface modification, by means of aqueous hydroxylamine treatment. Treatments utilizing covalent bonding to the surface through thiol, carboxyl, amine, or other reactive chemical groups offer improved durability to textile processing, such as dyeing and laundering. This surface modification investigation compared and assessed the attachment and durability of various wool fiber surface modification technologies and provided proof of principle for the manipulation of surface properties via covalent attachment of treatment compounds, providing enhanced fiber performance. Following removal of the surface lipid layer, wool fibers were modified using fluorescent and hydrophobic compounds and micro and nanoparticles. The extent of modification was assessed using scanning electron, light, and fluorescence microscopy, wettability testing and X-ray photoelectron spectroscopy (XPS). Surface treatments were evaluated with regard to effectiveness and durability to dyeing and laundering. Surface modification via attachment to thiol and other nucleophilic functionalities was observed to be greatly improved after hydroxylamine treatment, demonstrating the advantage of a customized exposed and accessible surface before secondary treatment. Increased durability and effective microparticle attachment was best accomplished employing zero or short-range crosslinking mechanisms, with zero-length attachment enhanced with the use of the crosslinking promoters, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Fluorescent and hydrophobic surface modifications exhibited high durability to laundering and demonstrated the viability of customizing surface hydrophobicity.

A hydrogel based fluorescent micro array used for the characterization of liquid analytes

A fluorimetric micro spot array using non-specific recognition function is described for the analysis of liquid samples. The array was composed of binary mixtures of various fluorescence dyes which were embedded in a hydrogel matrix. The interactions between the fluorescent dyes and their molecular surrounding inside the hydrogel, influence their fluorescence wave length and intensities. The array was used for the characterization of solvent mixtures. Developed fluorescence patterns of the complete array as well as the fluorescence intensity changes of single spots were analysed. It was proven, that specific analytical information can be gained using this non-specific recognition approach. The identification of some alcoholic beverages is described as an example of the application of this method when used for quality control purposes. Analogous to the appellation “electronic nose” and “electronic tongue” the described micro spot array acts as an “optochemical tongue”.

Fluorescent organosilica micro- and nanoparticles with controllable size

This paper reports on the synthesis of uniformly dye-doped organosilica particles with narrow size distribution. The particle size can be controlled from tenths of nanometers up to several micrometers, whilst still maintaining monodispersity. Microparticles were observed to swell in various solvents up to ∼2.5 times their original volume, suggesting the presence of a gel-like internal structure. As shown by confocal microscopy, this morphological control of particle swelling has important implications for the encoding of the nano/micro particles with organic dyes, such as rhodamine B isothiocyanate. Swelling allows the dye to penetrate the organosilica matrix and produce uniformly dye-doped nano- and microparticles. Finally, we suggest a coagulation model for the particle formation which significantly differs from conventional Stöber synthesis.

Using fluorescent micro‐particle image velocimetry to interrogate the surface glycocalyx on cultured endothelial cells in collagen microchannels

Numerous studies of endothelial cells in culture have been undertaken that are intended to elucidate mechanisms of endothelial-cell function spanning a broad range of fields in cardiovascular physiology. Since the surface glycocalyx on vascular endothelial cells is likely to have important implications for endothelial-cell function, it is necessary to determine if a physiologically typical surface glycocalyx can be grown and maintained on cultured endothelial cells and required conditions are. To make this determination, we use fluorescent micro- particle image velocimetry (μ-PIV) in cylindrical collagen lined on their luminal surface with a confluent monolayer of human umbilical-cord vascular endothelial cells (HUVECs). From μ-PIV data of the velocity and positions of microspheres in an optical section through the median plane of the tube, a velocity profile of the local microfluidics in the vicinity of the HUVEC luminal surface is generated. This velocity profile was used to estimate the effective hydrodynamic thickness of glycocalyx. By modulating the composition of the perfusate solution, the μ-PIV data can be used to assess whether molecular adsorption from the perfusate is an important determinant of the hydrodynamic properties of the glycocalyx. Results will be shown for a variety of perfusate compositions before and after treatments to degrade the glycocalyx. Supported by NIH grants R01 HL76499.

Role of TEP in the microbial food web structure. I. Grazing behavior of a bacterivorous pelagic ciliate

We studied the ingestion of picoplankton-size particles by the oligotrich Strombidium sulcatum as a function of transparent exopolymeric particle (TEP) concentration. Fluorescent micro- spheres of 1 µm (FMS) were used as prey-item analogs. TEP-FMS aggregates were formed by bub- bling seawater solutions containing different TEP concentrations in the presence of FMS. After bub- bling, concentrations of TEP-FMS aggregates ranged from 0.5 to 1.7 ppm and from 11.9 to 17.9 ppm in the low and high TEP treatments, respectively. When the ciliates were exposed to similar FMS con- centrations, but different TEP concentrations, ingestion rates were higher for the low TEP treatments (14.1 FMS ciliate -1 h -1 ) than for the high TEP treatments (5.2 FMS ciliate -1 h -1 ). The number of FMS in- gested in the low TEP treatments was 2.7 times higher than in the high TEP treatments and ingestion rates were inversely correlated to TEP volume concentration. Our results suggest that the formation of TEP-FMS reduces FMS availability for micro-grazers by modifying the food size spectra and redistri- buting FMS to larger size classes, and that at a critical TEP concentration picoplankton-size prey items may become unavailable for the ciliate S. sulcatum, while concomitantly they may become available for large-particle grazers. Therefore, one role of TEP in the microbial food web would be to act as a trophic elevator, thus creating a direct lift for picoplankton-size prey items to higher trophic levels.

Micro patterning of active proteins with perforated PDMS sheets (PDMS sieve)

We propose a novel technique for patterning active proteins on a glass substrate using a perforated polydimethylsiloxane (PDMS) sheet-sieve. The sieve, which has tapering holes, is fabricated by spin-coating PDMS on a pyramidal-shaped mold. By means of this sieve, FITC (fluorescent isothiocyanate, bovine)-albumin was successfully spotted in a 5 x 5 microm(2) area in an array. The patterned spots were perfectly isolated, which eliminates the problem of non-specific binding of proteins to undesired areas. To show that proteins maintained their activity after the patterning, we used F(1)-ATPase biomolecular motors; their activity can easily be verified by observing their rotary motion after patterning. Selective patterning with three kinds of fluorescent micro beads indicated the possibility of patterning of different proteins on the same substrate by using the sieve.

Brownian modulated optical nanoprobes

Brownian modulated optical nanoprobes (Brownian MOONs) are fluorescent micro- and nanoparticles that resemble moons: one hemisphere emits a bright fluorescent signal, while an opaque metal darkens the other hemisphere. Brownian motion causes the particles to tumble and blink erratically as they rotate literally through the phases of the moon. The fluctuating probe signals are separated from optical and electronic backgrounds using principal components analysis or images analysis. Brownian MOONs enable microrheological measurements on size scales and timescales that are difficult to study with other methods. Local chemical concentrations can be measured simultaneously, using spectral characteristics of indicator dyes embedded within the MOONs.

Automated methods of zygosity determination using fluorescent micro satellite markers

Automated methods of zygosity determination using fluorescent micro satellite markers

A Simple Method for Counting Small Numbers of Cells by Flow Cytometry

We describe a flow cytometric method for the quantitation of small numbers of cells that facilitates proliferation studies in microcultures. A constant number of fluorescent latex microspheres/sample is added to single cell suspensions prepared from the cultures. By flow cytometric analysis, cells are easily distinguishable from the microspheres and can be quantitated on the basis of their light scattering and fluorescence properties is contour plots. As the number of microspheres/sample is known, the relative proportions of cells and microspheres, respectively, can be converted into absolute cell numbers. This quick method is useful for any type of studies where cell numbers less than 1 x 10(5) in a small volume are to be determined.