Combined transmission, dark field and fluorescence microscopy for intact, 3D tissue analysis of biopsies.

SIGNIFICANCE: Currently, tissue biopsies are sectioned into 3- to 5-µm-thick slices that are used for conventional pathology analysis. Previous work by confocal microscopy and light-sheet microscopy has shown that analyzing biopsies intact in three-dimensions (3D) is possible and may lead to a better understanding of cancer growth patterns. Although accurate, these methods require fluorescent staining of the tissue, in addition to tissue clearing. If the 3D biopsy analysis could be done sufficiently swiftly, this approach may be used for on-site assessment of the adequacy of a biopsy taken. AIM: We aim to show that, by transmission microscopy of optically cleared tissue punches, the tissue architecture can be determined without the need for fluorescent staining. APPROACH: Transmission microscopy is used by combining bright field microscopy with dark field and epifluorescent microscopy to compare samples that have also been analyzed by fluorescent confocal microscopy. RESULTS: With increasing distance to the focal plane, the higher-frequency part of the spatial frequency spectrum of transmitted light is attenuated increasingly. This property is exploited for tissue segmentation, detecting whether tissue is present at a certain position in the focal plane image. Using this approach, we show that a 3D rendering of the internal cavity or tubules structure of punch biopsies, which are up to 1-mm thick, can be acquired in ≈1 min scan time per imaging modality. The images of the overall tissue architecture that are obtained are similar to those from the confocal microscopy benchmark, without requiring fluorescent staining. CONCLUSIONS: Images of the overall tissue architecture can be obtained from transmission microcopy; they are similar to those from the confocal microscopy benchmark without requiring fluorescent staining. Tissue clearing is still needed. The total scan time of the present method is significantly shorter at a fraction of the device costs.

Dynamic Properties of Supercooled Chlorinated Biphenyls.

A study of the dynamics of a series of biphenyl compounds having varying chlorine levels was carried out. Increasing the chlorine content increases the glass transition temperature and makes the dynamics substantially more sensitive to density changes. Nonetheless, in the vicinity of their respective glass transitions, the different liquids display very similar extents of dynamic correlation and dynamic heterogeneity. The slight narrowing of the relaxation peak with increasing chlorine follows the general trend of the effect of increasing molecular polarity. This relationship between the peak breadth and dipole moment was reproduced in molecular dynamics simulations of a simplified model of the Aroclor molecule.

Structurally Related Scaling Behavior in Ionic Systems.

We examine the density scaling properties of two ionic materials, a classic aprotic low molecular weight ionic liquid, 1-butyl-3-methylimidazolium bis(perfluoroethylsulfonyl)imide ([BMIm][BETI]), and a polymeric ionic liquid, poly(3-methyl-1,2,3-triazolium bis(trifluoromethylsulfonyl)imide) (TPIL). Density scaling is known to apply rigorously to simple liquids lacking specific intermolecular associations such as hydrogen bonds. Previous work has found that ionic liquids conform to density scaling over limited ranges of temperature and pressure. In this work, we find that the dc-conductivity of [BMIm][BETI] accurately scales for density changes of 17%; however, there is a departure from scaling for TPIL for even more modest variations of temperature and pressure. The entropy of both ionic samples conforms to density scaling only if the scaling exponent is allowed to vary linearly with the magnitude of the entropy.

Pressure densified 1,3,5-tri(1-naphthyl)benzene glass. I. Volume recovery and physical aging.

The effects of pressure densification on 1,3,5-tri(1-naphthyl)benzene (TNB) are assessed from volumetric and calorimetric measurements. The pressure densified glass (PDG) has higher density than conventional glass (CG), but unlike ultrastable TNB glass prepared using vapor deposition which also has elevated density, TNB PDG exhibits higher enthalpy and lower thermal stability than when formed at ambient pressure. PDG also exhibits anomalous physical aging. Rather than evolving monotonically toward the equilibrium density, there is an overshoot to a lower density state. Only when the density of the PDG becomes equivalent to the corresponding CG does the density begin a slow approach toward equilibrium.

The complex behavior of the "simplest" liquid: Breakdown of density scaling in tetramethyl tetraphenyl trisiloxane.

Dielectric relaxation measurements, in combination with density determinations, on tetramethyl tetraphenyl trisiloxane (DC704) over an unusually broad range of temperatures and pressures revealed a state-point dependency in its density scaling exponent. This is the first unambiguous experimental demonstration of a breakdown of density scaling in a nonassociated glass-forming material, and unanticipated for DC704, among the "simplest" of liquids, having a constant breadth of the relaxation dispersion and a Prigogine-Defay ratio near unity characteristic of approximate single-parameter systems. We speculate that the anomalous behavior has origins in the large value of its scaling exponent and relative flexibility of the chemical structure.

Intermolecular distance and density scaling of dynamics in molecular liquids.

A broad variety of liquids conform to density scaling: relaxation times can be expressed as a function of the ratio of temperature to density, the latter raised to a material constant γ. For atomic liquids interacting only through simple pair potentials, the exponent γ is very nearly equal to n/3, where n is the steepness of the intermolecular potential, while for molecular liquids having rigid bonds and built using the same interatomic potential, γ > n/3. We find that for this class of molecular liquids, γ = n/δ, where the parameter δ relates the intermolecular distance to the density along an isomorph (the line of approximately constant dynamics and structure). δ depends only on the molecular structure and not the interatomic potential.

Segmental and secondary dynamics of nanoparticle-grafted oligomers.

The local segmental and secondary dynamics of tetramethylene oxide oligomer grafted to silica nanoparticles (NPs) were investigated as a function of grafting density and molecular weight. Grafting slows the segmental (α) dynamics, but gives rise to faster secondary (ß) motions. Interestingly, the magnitude of these effects decreases with the extent of grafting (i.e., surface coverage), as well as with oligomer molecular weight. The disparity in dynamical effects reflects the decoupling of the segmental and more local ß dynamics, the former is associated with stronger dynamic correlations that extend over a greater spatial range. This results in greater sensitivity to interactions, including tethering of the chains to the NP surface.

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

Nonlinear dielectric spectroscopy of propylene carbonate derivatives.

Nonlinear dielectric measurements were carried out on two strongly polar liquids, 4-vinyl-1,3-dioxolan-2-one (VPC) and 4-ethyl-1,3-dioxolan-2-one (EPC), having chemical structures differing from propylene carbonate (PC) only by the presence of a pendant group. Despite their polarity, the compounds are all non-associated, "simple" liquids. From the linear component of the dielectric response, the α relaxation peak breadth was found to be invariant at a fixed value of the relaxation time, τα. From spectra from the nonlinear component, the number of dynamically correlated molecules was determined; it was also constant at fixed τα. Thus, two manifestations of dynamic heterogeneity depend only on the time constant for structural reorientation. More broadly, the cooperativity of molecular motions for non-associated glass-forming materials is connected to (i.e., reciprocally governs) the time scale. The equation of state for the two liquids was also obtained from density measurements made over a broad range of pressures and temperatures. Using these data, it was determined that the relaxation times of both liquids conform to density scaling. The effect of density, relative to thermal effects, on the α relaxation increases going from PC < VPC < EPC.

Communication: Effect of density on the physical aging of pressure-densified polymethylmethacrylate.

The rate of physical aging of glassy polymethylmethacrylate (PMMA), followed from the change in the secondary relaxation with aging, is found to be independent of the density, the latter controlled by the pressure during glass formation. Thus, the aging behavior of the secondary relaxation is the same whether the glass is more compacted or less dense than the corresponding equilibrium liquid. This equivalence in aging of glasses formed under different pressures indicates that local packing is the dominant variable governing the glassy dynamics. The fact that pressure densification yields different glass structures is at odds with a model for non-associated materials having dynamic properties exhibited by PMMA, such as density scaling of the relaxation time and isochronal superposition of the relaxation dispersion.

A test for the existence of isomorphs in glass-forming materials.

We describe a method to determine whether a material has isomorphs in its thermodynamic phase diagram. Isomorphs are state points for which various properties are invariant in reduced units. Such materials are commonly identified from strong correlation between thermal fluctuations of the potential energy, U, and the virial W, but this identification is not generally applicable to real materials. We show from molecular dynamic simulations of atomic, molecular, and polymeric materials that systems with strong U-W correlation cannot be pressure densified, that is, the density obtained on cooling to the glassy state and releasing the pressure is independent of the pressure applied during cooling.

Dynamics of poly(vinyl methyl ketone) thin films studied by local dielectric spectroscopy.

Local dielectric spectroscopy, which entails measuring the change in resonance frequency of the conducting tip of an atomic force microscope to determine the complex permittivity of a sample with high spatial (lateral) resolution, was employed to characterize the dynamics of thin films of poly(vinyl methyl ketone) (PVMK) having different substrate and top surface layers. A free surface yields the usual speeding up of the segmental dynamics, corresponding to a glass transition suppression of 6.5° for 18 nm film thickness. This result is unaffected by the presence of a glassy, compatible polymer, poly-4-vinyl phenol (PVPh), between the metal substrate and the PVMK. However, covering the top surface with a thin layer of the PVPh suppresses the dynamics. The speeding up of PVMK segmental motions observed for a free surface is absent due to interfacial interactions of the PVMK with the glass layer, an effect not seen when the top layer is an incompatible polymer.

Role of structure in the α and ß dynamics of a simple glass-forming liquid.

The elusive connection between dynamics and local structure in supercooled liquids is an important piece of the puzzle in the unsolved problem of the glass transition. The Johari-Goldstein ß relaxation, ubiquitous in glass-forming liquids, exhibits mean properties that are strongly correlated to the long-time α dynamics. However, the former comprises simpler, more localized motion, and thus has perhaps a more straightforward connection to structure. Molecular dynamics simulations were carried out on a two-dimensional, rigid diatomic molecule (the simplest structure exhibiting a distinct ß process) to assess the role of the local liquid structure on both the Johari-Goldstein ß and the α relaxation. Although the average properties for these two relaxations are correlated, there is no connection between the ß and α properties of a given (single) molecule. The propensity for motion at long times is independent of the rate or strength of a molecule's ß relaxation. The mobility of a molecule averaged over many initial energies, a measure of the influence of structure, was found to be heterogeneous, with clustering at both the ß and α time scales. This heterogeneity is less extended spatially for the ß than for the α dynamics, as expected; however, the local structure is the more dominant control parameter for the ß process. In the glassy state, the arrangement of neighboring molecules determines entirely the relaxation properties, with no discernible effect from the particle momenta.

The "anomalous" dynamics of decahyroisoquinoline revisited.

Decahydroisoquinoline (DHIQ) appears to be a unique material-the only non-associated, simple liquid with dynamics deviating from density scaling. To examine whether this anomaly is real, the density, ρ, of DHIQ was measured at temperatures, T, as low as 214 K and pressures up to ∼1.2 GPa. This enabled the equation of state (EoS) to be determined, without extrapolation, over the range of thermodynamic conditions for which the relaxation times had been reported. Using this less ambiguous EoS, we find that within the precision of the available relaxation times, the latter are a function of T/ρ(3.9), contrary to previous reports. Thus, the behavior of DHIQ is unexceptional; similar to every non-associated liquid tested to date, its dynamics comply with density scaling.

The effect of nanoclay on the rheology and dynamics of polychlorinated biphenyl.

The thermal, rheological, and mechanical and dielectric relaxation properties of exfoliated dispersions of montmorillonite clay in a molecular liquid, polychlorobiphenyl (PCB), were studied. The viscosity enhancement at low concentrations of clay (≤5%) exceeded by a factor of 50 the increase obtainable with conventional fillers. However, the effect of the nanoclay on the local dynamics, including the glass transition temperature, was quite small. All materials herein conformed to density-scaling of the reorientation relaxation time of the PCB for a common value of the scaling exponent. A new relaxation process was observed in the mixtures, associated with PCB molecules in proximity to the clay surface. This process has an anomalously high dielectric strength, suggesting a means to exploit nanoparticles to achieve large electrical energy absorption. This lower frequency dispersion has a weaker dependence on pressure and density, consistent with dynamics constrained by interactions with the particle surface.

Rotational dynamics of simple asymmetric molecules.

Molecular dynamic simulations were carried out on rigid diatomic molecules, which exhibit both α (structural) and ß (secondary) dynamics. The relaxation scenarios range from onset behavior, in which a distinct α process emerges on cooling, to merging behavior, associated with two relaxation peaks that converge at higher temperature. These properties, as well as the manifestation of the ß peak as an excess wing, depend not only on thermodynamic conditions, but also on both the symmetry of the molecule and the correlation function (odd or even) used to analyze its dynamics. These observations help to reconcile divergent results obtained from different experiments. For example, the ß process is more intense and the α-relaxation peak is narrower in dielectric relaxation spectra than in dynamic light scattering or NMR measurements. In the simulations herein, this follows from the weaker contribution of the secondary relaxation to even-order correlation functions, related to the magnitude of the relevant angular jumps.

Dynamic correlation length scales under isochronal conditions.

The origin of the dramatic changes in the behavior of liquids as they approach their vitreous state-increases of many orders of magnitude in dynamic time scales and transport properties-is a major unsolved problem in condensed matter. These changes are accompanied by greater dynamic heterogeneity, which refers to both spatial variation and spatial correlation of molecular mobilities. The question is whether the changing dynamics are coupled to this heterogeneity; that is, does the latter cause the former? To address this, we carried out the first nonlinear dielectric experiments at elevated hydrostatic pressures on two liquids, to measure the third-order harmonic component of their susceptibilities. We extract from this the number of dynamically correlated molecules for various state points and find that the dynamic correlation volume for non-associated liquids depends primarily on the relaxation time, sensibly independent of temperature and pressure. We support this result by molecular dynamic simulations showing that the maximum in the four-point dynamic susceptibility of density fluctuations is essentially invariant along isochrones for molecules that do not form hydrogen bonds. Our findings are consistent with dynamic cooperativity serving as the principal control parameter for the slowing down of molecular motions in supercooled materials.

Effect of Interface Interaction on the Segmental Dynamics of Poly(vinyl acetate) Investigated by Local Dielectric Spectroscopy.

The segmental dynamics of poly(vinyl acetate) (PVAc) thin films were measured in the presence of an aluminum interface and in contact with an incompatible polymer, poly(4-vinylpyridine). The local dielectric relaxation was found to be faster in thin films than in the bulk; however, no differences were observed for the various interfaces, including a PVAc/air interface. These results show that capping of thin films, even with a rigid material, does not necessarily affect the dynamics, the speeding up herein for capped PVAc was equivalent to that for the air interface. The insensitivity of the dynamics to the nature of the interface affords a means to engineer thin films while maintaining desired mechanical properties. Our findings for PVAc also may explain the discordant results that have been reported in general for the effect of air versus rigid interfaces on the local segmental relaxation of thin films.

Dynamic correlations and heterogeneity in the primary and secondary relaxations of a model molecular liquid.

Molecular dynamics simulations were carried out on a series of Lennard-Jones binary mixtures of rigid, asymmetric, dumbbell-shaped molecules. Below an onset temperature, the rotational and translational dynamics split into the slow structural α relaxation and a higher-frequency Johari-Goldstein ß relaxation. Both processes are dynamically heterogeneous, having broad distributions of relaxation times. However, only the α relaxation shows strong dynamic correlations; correlations at the ß time scale are weak, in particular for molecules having shorter bonds. Despite the close connection between the two processes, we find no correlation between the α and ß relaxation times of individual molecules; that is, a molecule exhibiting slow ß motion does not necessarily undergo slow α dynamics and likewise for fast molecules. However, the single-molecule α relaxation times do correlate with both the α and ß relaxation strengths.