Activation of S6 signaling is associated with cell survival and multinucleation in hyperplastic skin after epidermal loss of AURORA-A Kinase.

The role of mitosis in the progression of precancerous skin remains poorly understood. To address this question, we deleted the mitotic Kinase Aurora-A (Aur-A) in hyperplastic mutant p53 mouse skin as an experimental tool to study the G2/M transition in precancerous keratinocytes and AUR-A's role in this process. Epidermal Aur-A deletion (Aur-AepiΔ) led to marked keratinocyte enlargement, pleomorphism, multinucleation, and  attenuated induction of cell death. This phenotype was characteristic of slippage after a stalled mitosis. We also observed altered or impaired epidermal differentiation, indicative of a partial skin barrier defect. The upregulation of mTOR/PI3K signaling was implicated as a mechanism by which keratinocytes may evade cell death after AUR-A deficiency. This was evidenced by the ectopic expression of the pathway readout, p-S6, in the basal layer of Aur-AepiΔ skin and its mitotic upregulation in isolated keratinocytes. We further tested whether our findings were extended to skin carcinoma cells. The chemical inhibition of AUR-A led to a similar mitotic delay, polyploidy/multinucleation, and attenuated cell death in skin cancer cell lines. Moreover, inhibition of mTOR/PI3K signaling ameliorated the effects caused by the deficiency of AUR-A activity but was also associated with the persistence of mitotic p-S6 detection in surviving cancer cells. These results show the induction of multinucleation/polyploidy may be a compensatory state in keratinocytes that allows for cellular survival and maintenance of partial barrier function in face of aberrant cell division or differentiation. Moreover, mTOR/PI3K signaling is active in the mitosis of hyperplastic keratinocytes expressing mutant p53 and is further enhanced by stalled mitosis, indicating a potential resistance mechanism to the use of anti-mitotic drugs in the treatment of skin cancers.

Use of a novel Förster resonance energy transfer method to identify locations of site-bound metal ions in the U2-U6 snRNA complex.

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2-U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.

Evaluation of two-beam fluorescence cross correlation spectroscopy for electrophoretic analysis of protein digests.

In this report, we evaluate experimentally and through the use of simulations and calculations the possibility of using 2-beam fluorescence cross correlation spectroscopy (2BFCCS) for the multicomponent electrophoretic analysis of peptides. The concept described has potential as a high throughput, extraordinarily sensitive means of identifying proteins and accelerating proteome studies. We present Monte Carlo simulation methods and results using them that help in understanding the capabilities and limitations of 2BFCCS for protein identification. We have calculated the expected pH dependent mobility and resultant 2BFCCS fingerprint spectra for a randomly selected subset of the peptides present upon digestion of horse myoglobin by trypsin. We demonstrate experimentally the multicomponent analysis of a mixture containing a fluorescently labeled peptide and a free fluorophore. We also demonstrate experimentally the ability to measure migration rates of dilute, single-fluorophore species over more than 2 orders of magnitude in linear velocity (between 4.2 mm s(-1) and 640 mm s(-1)).

Patterned solvent delivery and etching for the fabrication of plastic microfluidic devices.

A very simple method for micropatterning flat plastic substrates that can be used to build microfluidic devices is demonstrated. Patterned poly(dimethylsiloxane) elastomer is used as a template to control the flow path of an etching solvent through a channel design to be reproduced on the plastic substrate. The etching solvent was a acetone/ethanol mixture for poly(methyl methacrylate) substrates or a dimethylformamide/acetone mixture for polystyrene. The method is extremely fast in that duplicate plastic substrates can be patterned in just a few minutes each. We identified conditions that lead to smooth channel surfaces and characterized the rate of etching under these conditions. We determined that, for sufficiently short etching times (shallow channel depths), the etch rate is independent of the linear flow rate. This is very important since it means that the etch depth is approximately constant even in complex channel geometries where there will be a wide range of etchant flow rates at different positions in the pattern to be reproduced. We also demonstrate that the method can be used to produce channels with different depths on the same substrate as well as channels that intersect to form a continuous fluid junction. The method provides a nice alternative to existing methods to rapidly fabricate microfluidic devices in rigid plastics without the need for specialized equipment.

Fluorescence correlation spectroscopy for flow rate imaging and monitoring--optimization, limitations and artifacts.

We recently demonstrated a new method for mapping fluid velocities in 3 dimensions and with exceptionally high spatial resolution for the characterization of flow in microfluidic devices. In the method, a colloidal suspension containing fluorescent tracer particles, dye doped polymer spheres, is pumped through a microchannel and confocal microscopy combined with fluorescence correlation spectroscopy is used to measure fluid velocities. In this report, we further characterize the technique and report on optimizations that allow a 5-fold increase in speed of single point velocity measurements. This increase in measurement speed will yield a 25 fold reduction in the time needed to collect a complete velocity image. The precision of measured velocities was characterized as a function of tracer particle concentration, measurement time, and fluid velocity. In addition, we confirm the linearity of the measurement method (velocity vs. applied pressure) over a range of velocities spanning four orders of magnitude. Furthermore, we demonstrate that an artifact in velocity measurements using fluorescence correlation spectroscopy (FCS) that was interpreted by others as being caused by optical trapping forces is actually an artifact caused by detector saturation and can be avoided by careful choice of experimental conditions.

Scanning fluorescence correlation spectroscopy: a tool for probing microsecond dynamics of surface-bound fluorescent species.

In this report, a combined imaging and fluorescence correlation spectroscopy (FCS) method is described and its ability to characterize microsecond fluctuations in the fluorescence emission of a sample is demonstrated. A sample scanning laser confocal microscope is operated in the customary way while recording the time that each photon is detected with a time resolution of 50 ns using a low-cost counting board. The serial data stream of photon detection times allows access to fluorescence signal fluctuations that can be used to characterize dynamics using correlation methods. The same data stream is used to generate images of the sample. Using the technique, we demonstrate that it is possible to characterize the kinetics of transitions to and from nonemitting or "dark" states of the fluorescent dyes DiIC16 and ATTO 520. Results are similar to, but deviate slightly from, a model that has been frequently used for extracting singlet-triplet: conversion rates using conventional solution-based FCS. Like conventional FCS, the concentration, or in our case the areal density of coverage, of fluorescent species can also be obtained. Many single-molecule fluorescence experiments aim to extract kinetics from intensity trajectories; this method may be used as a rapid and convenient technique for characterization of surface-linked or thin-film samples prior to performing the more time and effort intensive single-molecule measurements. Besides the capacity to measure photophysical phenomena, the surface-sensitive FCS method could also be applied for measuring conformational changes or interaction kinetics for species immobilized on a surface. One possible scenario is measurements of the frequency and duration of association of ligand-receptor pairs where a fluorescently labeled component is freely diffusing and the other is surface immobilized. Given that microarrays of custom-designed, surface-immobilized peptides and nucleic acids are now readily available, the ability to sensitively measure association and dissociation rates of the surface-linked species with a freely diffusing species could be a useful extension to what has already become an extremely important tool for characterizing the interactions of biomolecules.

Application of fluorescence correlation spectroscopy for velocity imaging in microfluidic devices.

In this paper we present and demonstrate a technique for mapping fluid flow rates in microfluidic systems with sub-micrometer resolution using confocal microscopy in conjunction with fluorescence correlation spectroscopy (FCS). Flow velocities ranging from approximately 50 microm/s to approximately 10 cm/s can be recorded using fluorescent polymer nanospheres as fluid motion tracers. Velocity profiles and images of the flow in poly(dimethylsiloxane)-glass microchannels are presented and analyzed. Using the method, velocity images along the horizontal (top view) and vertical planes within a microdevice can be obtained. This is, to our knowledge, the first report of FCS for producing velocity maps. The high-resolution velocity maps can be used to characterize and optimize microdevice performance and to validate simulation efforts.

Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules.

We demonstrate measurements of the efficiency of competing Förster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.

Fourier analysis near-field polarimetry for measurement of local optical properties of thin films.

We present measurements of the local diattenuation and retardance of thin-film specimens by using techniques that combine near-field scanning optical microscopy (NSOM) and a novel polarization-modulation (PM) polarimetry utilizing Fourier analysis of the detected intensity signal. Generally, quantitative near-field polarimetry is hampered by the optical anisotropy of NSOM probes. For example, widely used aluminum-coated pulled-fiber aperture probes typically exhibit a diattenuation near 10%. Our analysis of aperture diattenuation demonstrates that the usual techniques for nulling a PM polarimeter result in a nonzero residual probe retardance in the presence of a diattenuating tip. However, we show that both diattenuation and retardance of the sample can be determined if the corresponding tip properties are explicitly measured and accounted for in the data. In addition, in thin films (<100 nm thick), where the sample retardance and diattenuation are often small, we show how to determine these polarimetric quantities without requiring alignment of the fast and diattenuating axes, which is a more general case than has been previously discussed. We demonstrate our techniques by using two types of polymer-film specimens: ultrahigh molecular weight block copolymers (recently noted for their photonic activity) and isotactic polystyrene spherulites. Finally, we discuss how changes in the tip diattenuation during data collection can limit the accuracy of near-field polarimetry and what steps can be taken to improve these techniques.

Spectroscopic study and evaluation of red-absorbing fluorescent dyes.

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.

Antibunching in the emission of a single tetrachromophoric dendritic system.

The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet-singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.

Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons.

A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.

High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy.

Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below approximately 10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 ns, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.