Editorial: Advances in label free tissue imaging with laser scanning microscopy techniques

Advances in Label Free Tissue Imaging with Laser Scanning Microscopy Technique

Typical photobleaching of EGFP (A) and NLS-EGFP (B) in STED <i>xt</i> acquisition.

<p>On the left of each panel, the intensity carpet is displayed; on the right it is shown the plot of the average intensity <i>vs.</i> time as determined in the dotted rectangle visible in the carpet.</p

pCF analysis of diffusing CLP-YFP.

<p>(A) Intensity and (B) pCF(3) carpets for free diffusion of CLP-YFPs. Comparison between the averaged (on 64 pixels) confocal (blue) and STED (red) pCF curves is reported for pCF(3) (C) and pCF(5) (D).</p

Calculation and characteristics of pCF.

<p>(A) From the repeated raster scan of a 64-pixel line (<i>xt</i> acquisition), an “intensity carpet” is generated in which the <i>x</i>-coordinate represents the pixel position along the line, the <i>y-</i>coordinate corresponds to the time of acquisition, and the actual intensity is expressed by a black-to-red color scale. (B) Fluorescence intensity <i>vs.</i> time along two generic columns of the carpet separated by <i>n</i> pixels. The pCF relevant to the two columns is obtained by considering a variable lag-time and then applying <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099619#pone.0099619.e015" target="_blank">equation 8</a> in main text. (C) Simulated pCF <i>vs. </i> for optically indistinguishable (blue trace, pixel distance <i>n = 2</i>) and distinguishable (green and red traces, pixel distance <i>n = 3</i> and <i>n = 5</i>) locations (columns) along the raster-scanned line.</p

Sigmoidal intensity profile along a line crossing the nuclear envelope from cytoplasm (0–1.5 mm) to as imaged in confocal (red) and STED (black) modes.

<p>Note the larger width of N/C interface detected in confocal mode (4–6 pixels) as compared to STED (2–3 pixels) mode.</p

STED-pCF analysis of NLS-GFP in cells.

<p>(A) Fluorescence intensity image of a cell expressing NLS-GFP: the nuclear accumulation is clearly visible. (B) Fluorescence intensity carpet in which the <i>x</i>-coordinate corresponds to the pixels along the scanned line (denoted as an arrow in A) and the <i>y</i>-coordinate corresponds to the time of acquisition (seconds). pCF(4) carpets obtained along the N→C (C) and C→N (D) directions.</p

Confocal-pCF analysis of NLS-GFP in cells.

<p>(A,B) Confocal-pCF(4) carpets obtained along the N→C (A) and C→N (B) directions along a line crossing the N/C interface. The two carpets are the confocal-pCF analogs of the STED-pCF carpets reported as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099619#pone-0099619-g003" target="_blank">Figure 3C,D</a> in main text. (C) Confocal (blue) and STED-pCF(4) (red) curves for pixel 28 in the scanned line across the nuclear envelope.</p

Assembly of Branched Colloidal Nanocrystals in Polymer Films Leads to Enhanced Viscous Deformation Resistance

Progress in the integration of nanocrystals with polymers has enabled the creation of materials for applications ranging from photovoltaics to biosensing. However, controlling the nanocrystal segregation and aggregation in the polymer phase remains a challenging task, especially because nanocrystals tend to form amorphous clusters inside the polymer matrix. Here, we present the ability of octapod-shaped particles to overcome their strong entropy-driven tendency to aggregate disorderly and form instead centipede-like linear arrays that are randomly oriented and fully embedded in polystyrene films upon controlled solvent evaporation. This behavior cannot be entirely described by short-range van der Waals interactions between the octapods in the polymer solution. An important role here is played by the increment of the viscosity of the medium during the evaporation of the solvent, which prevents disaggregation of the chains once they are formed. We show that increasing the octapod loading in the blends does not impact the length of the linear arrays beyond a critical length, while it favors instead chain demixing to form self-segregated regions of parallel interlocked chains. Our experiments evidence that softening of the polymer matrix by ex situ heating of the films induces a tail-to-tail coupling of the preformed chains and leads to the formation of longer linear structures of octapods, up to 2 μm long. The presence of 1D arrays of octapods in free-standing polystyrene films improves the creep response by a remarkable 37%, owing to an octapod pinning effect of the polymer matrix

Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase

Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties

Spatial-domain filter enhanced subtraction microscopy and application to mid-IR imaging

We have experimentally investigated the enhancement in spatial resolution by image subtraction in mid-infrared central solid-immersion lens (c-SIL) microscopy. The subtraction exploits a first image measured with the c-SIL point-spread function (PSF) realized with a Gaussian beam and a second image measured with the beam optically patterned by a silicon it-step phase plate, to realize a centrally hollow PSF. The intense sides lobes in both PSFs that are intrinsic to the SIL make the conventional weighted subtraction methods inadequate. A spatial-domain filter with a kernel optimized to match both experimental PSFs in their periphery was thus developed to modify the first image prior to subtraction, and this resulted in greatly improved performance, with polystyrene beads 1.4 0.1 mu m apart optically resolved with a mid-1R wavelength of 3.4 mu m in water. Spatial-domain filtering is applicable to other PSF pairs, and simulations show that it also outperforms conventional subtraction methods for the Gaussian and doughnut beams widely used in visible and near-1R microscopy. (C) 2017 Optical Society of Americ