Characterization of power induced heating and damage in fiber optic probes for near-field scanning optical microscopy

This is the published version, also available here: http://dx.doi.org/10.1063/1.2740133.Tip-induced sample heating in near-field scanning optical microscopy (NSOM) is studied for fiber optic probes fabricated using the chemical etching technique. To characterize sample heating from etched NSOM probes, the spectra of a thermochromic polymer sample are measured as a function of probe output power, as was previously reported for pulled NSOM probes. The results reveal that sample heating increases rapidly to ∼55–60°C as output powers reach ∼50nW. At higher output powers, the sample heating remains approximately constant up to the maximum power studied of ∼450nW. The sample heating profiles measured for etched NSOM probes are consistent with those previously measured for NSOM probes fabricated using the pulling method. At high powers, both pulled and etched NSOM probes fail as the aluminumcoating is damaged. For probes fabricated in our laboratory we find failure occurring at input powers of 3.4±1.7 and 20.7±6.9mW for pulled and etched probes, respectively. The larger half-cone angle for etched probes (∼15° for etched and ∼6° for pulled probes) enables more light delivery and also apparently leads to a different failure mechanism. For pulled NSOM probes, high resolution images of NSOM probes as power is increased reveal the development of stress fractures in the coating at a taper diameter of ∼6μm. These stress fractures, arising from the differential heating expansion of the dielectric and the metalcoating, eventually lead to coating removal and probe failure. For etched tips, the absence of clear stress fractures and the pooled morphology of the damaged aluminumcoating following failure suggest that thermal damage may cause coating failure, although other mechanisms cannot be ruled out

Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier

The vascular endothelium is a highly dynamic structure, and the integrity of its barrier function is tightly regulated. Normally impenetrable to cells, the endothelium actively assists lymphocytes to exit the bloodstream during inflammation. The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood. Here we report that actin assembly in the EC, induced by Arp2/3 complex under control of WAVE2, is important for several steps in the process of transmigration. To begin transmigration, ECs deploy actin-based membrane protrusions that create a cup-shaped docking structure for the lymphocyte. We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2. The next step in transmigration is creation of a migratory pore, and we found that endothelial WAVE2 is needed for lymphocytes to follow a transcellular route through an EC. Later, ECs use actin-based protrusions to close the gap behind the lymphocyte, which we discovered is also driven by WAVE2. Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells

Role of cortactin homolog HS1 in transendothelial migration of natural killer cells.

Natural Killer (NK) cells perform many functions that depend on actin assembly, including adhesion, chemotaxis, lytic synapse assembly and cytolysis. HS1, the hematopoietic homolog of cortactin, binds to Arp2/3 complex and promotes actin assembly by helping to form and stabilize actin filament branches. We investigated the role of HS1 in transendothelial migration (TEM) by NK cells. Depletion of HS1 led to a decrease in the efficiency of TEM by NK cells, as measured by transwell assays with endothelial cell monolayers on porous filters. Transwell assays involve chemotaxis of NK cells across the filter, so to examine TEM more specifically, we imaged live-cell preparations and antibody-stained fixed preparations, with and without the chemoattractant SDF-1α. We found small to moderate effects of HS1 depletion on TEM, including whether the NK cells migrated via the transcellular or paracellular route. Expression of HS1 mutants indicated that phosphorylation of HS1 tyrosines at positions 222, 378 and 397 was required for rescue in the transwell assay, but HS1 mutations affecting interaction with Arp2/3 complex or SH3-domain ligands had no effect. The GEF Vav1, a ligand of HS1 phosphotyrosine, influenced NK cell transendothelial migration. HS1 and Vav1 also affected the speed of NK cells migrating across the surface of the endothelium. We conclude that HS1 has a role in transendothelial migration of NK cells and that HS1 tyrosine phosphorylation may signal through Vav1

Expression Rescue of HS1 Mutants in HS1-depleted NK Cells.

<p>A) Phosphorylation of HS1 Tyr397 in response to SDF-1α. Immunoblots probed with anti-Phospho-Tyr397 and anti-HS1. NK cells (5 x 10⁶) treated with SDF-1α (30 ng/mL) for the indicated times (min). B—D) Function of HS1 mutants in TEM by transwell assay. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box and whisker plots. Boxes: 25th to 75th percentiles; whiskers: minimum and maximum values. B) Mutations of phosphorylated tyrosine residues. Compared to control siRNA (blue), HS1 depletion by siRNA causes decreased TEM (red), and the defect is rescued by expression of wild-type HS1 (green) or siRNA-resistant wild-type HS1 (purple). Expression of siRNA-resistant forms of single-mutant HS1 Y378F (black), single-mutant HS1 Y397F (brown) or double-mutant HS1 Y378F Y397F (dark blue) does not rescue the defect, comparing their values to the value for siRNA-resistant wild-type (purple). Expression of siRNA-resistant HS1 Y222F (orange) rescues with a value that is slightly less, but not statistically significant, from that of siRNA-resistant wild type. Asterisks indicate *P>0.05, **P>0.005 (unpaired Student’s t-test, N = 6–9). C) Mutation of Arp2/3 complex binding site. Expression of siRNA-resistant HS1 with mutation of DDW residues to AAA (orange) rescues the defect, with no difference compared to siRNA-resistant wild-type HS1. N = 6 in each case. D) Mutation of SH3 domain at ligand-binding site. Expression of siRNA-resistant HS1 with the mutation W466K (orange) rescues the defect, with no difference compared to siRNA-resistant wild-type HS1. n = 6 in each case.</p

HS1 and TEM of NK cells in transwell assays.

<p>A) Diagram of transendothelial migration assay in a transwell device. B) Depletion of HS1 protein by siRNA, shown by immunoblot after 72 hrs. NK cells were treated with a pool of four siRNAs or one of the four. GAPDH is a loading control. C) Effects of HS1 knockdown on TEM. Plotted values are number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day. Box-and-whisker plots (box: 25th to 75th percentiles, whiskers: min to max, middle line: median). Asterisks indicate statistical significance (*P<0.05. Unpaired Student’s t-test, n = 5 for each condition.) D) Fluorescence micrographs of NK cells, showing expression and co-localization of expressed HS1-tdTomato (red), F-actin (green, Alexa Fluor 488 phalloidin), and total HS1, including endogenous (blue, anti-HS1 staining). E) Fluorescence micrographs of NK cells stained with anti-HS1 to show siRNA-induced depletion of HS1 and expression of siRNA-resistant HS1 protein. F) Expression of siRNA-resistant HS1 in NK cells knocked down for HS1 with siRNA, shown by immunoblot with anti-HS1. Knockdown used a combination of HS1 siRNAs 2 and 3. G) Rescue of TEM phenotype in HS1-knockdown NK cells by expression of HS1. Cells as in panels E and F. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box-and-whisker plots as in panel C. Asterisks indicate statistical significance. (* P< 0.05, *** P < 0.0005. Unpaired Student’s t-test, N = 9–12 experiments for each condition.)</p

Migration Speeds for NK Cells.

<p>Speed (μm/min) defined as net displacement (distance start to finish) divided by time for control vs HS1-depleted cells. The distributions are not Gaussian, so the values listed are median, 95% confidence interval of the median and number of data points (N). P values from two non-parametric tests of significance are listed. Data include tracks for all cells in separate experiments on three different days.</p><p>Migration Speeds for NK Cells.</p

Effect of Vav1 and HS1 plus Vav1 Depletion on Migration Speeds for NK Cells.

<p>Preparations not treated with SDF-1α. Combination of all tracks from experiments on three days. The distributions are not Gaussian, so the values listed are median, 95% confidence interval of the median and number of data points (N). P values from two non-parametric tests of significance are listed. Data include tracks for all cells in separate experiments on three different days.</p><p>Effect of Vav1 and HS1 plus Vav1 Depletion on Migration Speeds for NK Cells.</p

Role of Vav1 in TEM by NK cells.

<p>A) Immunoblots with anti-HS1 and anti-Vav1 showing depletion of HS1 and Vav1 after 72 hrs of siRNA treatment. B) Decrease in TEM in transwell assay by NK cells treated with Vav1 siRNA, compared to control siRNA. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box and whisker plots. Boxes: 25th to 75th percentiles; whiskers: minimum and maximum values. N = 6. Asterisks indicate **P<0.005 (unpaired Student’s t-test). C) Left panel: Immunoblot with anti-HS1. The left lane shows the absence of HS1 in an anti-HS1 immunoprecipitate from a whole-cell lysate of NK cells treated with siRNA targeting HS1. The right lane shows the result for cells treated with control siRNA. Middle panel: Immunoblot with anti-Vav1. The left lane shows the presence of Vav1 protein in an anti-HS1 precipitate from a lysate of NK cells treated with control siRNA. The right lane shows the presence of Vav1 in the lysate. Right panel: Similar to the middle panel, except with a lysate from NK cells depleted for HS1.</p

TEM events by NK cells on HDMVEC monolayers based on live-cell movie analysis.

<p>A) Endogenous HS1 (red) and F-actin (green) in NK cells migrating on the surface of HDMVEC monolayer observed by anti-HS1 and phalloidin fluorescence. Scale bar = 20 μm. B) DIC images from a movie (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.s006" target="_blank">S2 Movie</a>), illustrating how the passage of the NK cell through the endothelial monolayer leaves a defect. Scale bar = 10 μm. C) Speed of cell migration, based on path length. Median and 95% confidence intervals are plotted. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.t004" target="_blank">Table 4-1. D</a>) Speed of cell migration, based on net displacement. Median and 95% confidence intervals are plotted. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.t004" target="_blank">Table 4-2. E</a>) Percentage of TEM events from movie analysis with SDF-1α. The number of TEM events as a percentage of the total number of NK cells on the surface of the endothelial monolayer in the first frame. Error bars are standard error of proportion. The difference is not statistically significant by z-test (p = 0.15) or by Fisher’s exact test for a 2 x 2 contingency table (p = 0.19). Data combined from two or three experiments per day on three days. F) Percentage of TEM events from movie analysis without SDF-1α. The number of TEM events as a percentage of the total number of NK cells on the surface of the endothelial monolayer in the first frame. Error bars are standard error of proportion. The differences between control and depleted-cell values are statistically significant with p values of 0.022, 0.024 and 0.001 for HS-depleted, Vav1-depleted and HS1+Vav1-depleted NK cells, based on chi-square tests with Yates’ correction. No other differences are statistically significant. Data combined from experiments on three days.</p