A Streaming Multi-GPU Implementation of Image Simulation Algorithms for Scanning Transmission Electron Microscopy

Simulation of atomic resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. Here we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000x for PRISM and 30x for multislice are achieved relative to traditional multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic, using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic

GENFIRE: A generalized Fourier iterative reconstruction algorithm for high-resolution 3D imaging

Tomography has made a radical impact on diverse fields ranging from the study of 3D atomic arrangements in matter to the study of human health in medicine. Despite its very diverse applications, the core of tomography remains the same, that is, a mathematical method must be implemented to reconstruct the 3D structure of an object from a number of 2D projections. In many scientific applications, however, the number of projections that can be measured is limited due to geometric constraints, tolerable radiation dose and/or acquisition speed. Thus it becomes an important problem to obtain the best-possible reconstruction from a limited number of projections. Here, we present the mathematical implementation of a tomographic algorithm, termed GENeralized Fourier Iterative REconstruction (GENFIRE). By iterating between real and reciprocal space, GENFIRE searches for a global solution that is concurrently consistent with the measured data and general physical constraints. The algorithm requires minimal human intervention and also incorporates angular refinement to reduce the tilt angle error. We demonstrate that GENFIRE can produce superior results relative to several other popular tomographic reconstruction techniques by numerical simulations, and by experimentally by reconstructing the 3D structure of a porous material and a frozen-hydrated marine cyanobacterium. Equipped with a graphical user interface, GENFIRE is freely available from our website and is expected to find broad applications across different disciplines.Comment: 18 pages, 6 figure

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features in the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe$_3$O$_4$ core encased by a 25-nm-thick fluorescent silica (SiO$_2$) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles

Single-shot 3D coherent diffractive imaging of core-shell nanoparticles with elemental specificity

We report 3D coherent diffractive imaging (CDI) of Au/Pd core-shell nanoparticles with 6.1 nm spatial resolution with elemental specificity. We measured single-shot diffraction patterns of the nanoparticles using intense x-ray free electron laser pulses. By exploiting the curvature of the Ewald sphere and the symmetry of the nanoparticle, we reconstructed the 3D electron density of 34 core-shell structures from these diffraction patterns. To extract 3D structural information beyond the diffraction signal, we implemented a super-resolution technique by taking advantage of CDI’s quantitative reconstruction capabilities. We used high-resolution model fitting to determine the Au core size and the Pd shell thickness to be 65.0 ± 1.0 nm and 4.0 ± 0.5 nm, respectively. We also identified the 3D elemental distribution inside the nanoparticles with an accuracy of 3%. To further examine the model fitting procedure, we simulated noisy diffraction patterns from a Au/Pd core-shell model and a solid Au model and confirmed the validity of the method. We anticipate this super-resolution CDI method can be generally used for quantitative 3D imaging of symmetrical nanostructures with elemental specificity

LpX induces nephropathology in <i>Lcat</i>^-/- mice.

<p>(A) Effect of LpX injection on renal function. Albumin to creatinine ratios (μg/mg) in urine (UACR) were measured prior to and then every week after exogenous LpX treatment, starting on week 2. Data are expressed as mean ± SEM. P values of group differences at each time point are reported. (B) Histological analysis. Representative images of PAS-stained sections of kidneys from WT and <i>Lcat</i>^-/- mice treated or not treated with LpX. No histological alterations were present in WT mice. WT mice treated with LpX showed no changes or only mild mesangial matrix expansion. In <i>Lcat</i>^-/- mice, LpX treatment increased mesangial matrix (<i>asterisk</i>) and, occasionally, PAS-positive material in glomerular capillaries (<i>arrow</i>) was observed (Scale bars: 20 μm). (C) Representative images of PAS-stained sections of kidneys from WT (<i>upper panel</i>) and <i>Lcat</i>^-/- (<i>lower panel</i>) mice treated with LpX. Tubular cell vacuolation was present focally in <i>Lcat</i>^-/- mice. (Scale bars: 50 μm). (D) Podocyte effacement revealed by TEM (left, <i>black arrows</i>; Scale bar: 1 μm) and SEM (right, <i>white arrows</i>; Scale bar: 20 μm) in glomeruli of <i>Lcat</i>^-/- mice treated with LpX.</p

LpX plasma clearance and glomerular upake <i>in vivo.</i>

<p>LCAT deficiency markedly decreases LpX plasma clearance. WT and <i>Lcat</i>^-/- mice were injected with lissamine rhodamine B PE-tagged LpX and plasma samples were taken at the indicated times. (A) Plasma-associated fluorescence. Each data point represents the total fluorescence of pooled mouse plasma samples (mean ± S.D.; n = 3). (B) Agarose gel electrophoresis of pooled mouse plasma lipoprotein PE fluorescence (same samples as in (A)). LpX cleared from WT plasma by 240 min, whereas <i>Lcat</i>^-/- LpX remained elevated at all times. HDL-associated fluorescence was increased in WT plasma. W<i>hite line</i> indicates origin. (C) Fluorescent LpX retention in renal glomeruli is markedly increased in <i>Lcat</i>^-/- mice. Representative confocal maximum projection images of 10 μm fixed frozen kidney sections 4 hrs after injection of fluorescent-PE tagged LpX in mice chronically treated with 3 mg/wk synthetic LpX. Note the markedly increased retention of LpX in <i>Lcat</i>^-/- mice glomeruli. (D) Electron microscopic analysis of LpX in renal glomerular capillaries. Representative TEM of renal glomerular capillaries in WT (<i>left panels</i>) and <i>Lcat</i>^-/- mice (<i>right panels</i>). Endogenous multilamellar structures with features of LpX particles were occasionally present in the capillaries of (-) LpX <i>Lcat</i>^-/-, but not (-) LpX WT mice. Synthetic LpX particles resembling endogenous LpX were frequently observed in renal capillaries of both (+) LpX WT and (+) LpX <i>Lcat</i>^-/- mice. Both endogenous and exogenous synthetic LpX were often seen to be engulfed by endothelial cell processes (<i>insets</i>). Exogenous LpX in the capillary lumen bound to red blood cells in LpX-treated WT and <i>Lcat</i>^-/- mice. GBM: Glomerular Basement Membrane; PFP: Podocyte Foot Process. Scale bars = 500nm. Inset scale bars = 250 nm (WT+LpX); 100 nm (<i>Lcat</i>^-/- ± LpX).</p

Electron microscopic analysis of LpX movement through renal glomerular compartments.

<p>Circulating LpX particles (small arrows in (A, B)) bind to endothelial cell lamellipodia in (A) WT and (B) <i>Lcat</i>^-/- mouse glomerular capillaries (arrowheads), are internalized (long arrows in (A), and degraded (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s003" target="_blank">S3 Fig</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s004" target="_blank">S4 Fig</a>). LpX bound to the cell surface (B1), is partially (B2), small arrows in inset) and then completely engulfed (B3). LpX penetrates the glomerular basement membrane (GBM) in WT (C) and <i>Lcat</i>^-/- mice ((D) and, inset in (D), arrowheads), markedly disrupting its structure (C, D; asterisks). The typical intramembranous lesion as found in the peripheral GBM of human FLD is seen in the inset in D, displaying a characteristic lamellar structure within a lucent lacuna in <i>Lcat</i>^-/- mice. In (D), several lamellipodia (arrows) engulf an LpX particle in the GBM. LpX penetrates the glomerular urinary space of both WT (E, G) and <i>Lcat</i>^-/- (F, H) mice. LpX binds to podocyte cell bodies (PCBs) and foot processes (PFPs) at multiple sites (E, F: small arrows; H: arrowheads), and was internalized into PCBs (F; large arrow). Large vacuoles (G, H; large arrows) containing partially degraded LpX particles (G, H; small arrows) as well as numerous small unilamellar vesicles are often observed, consistent with cell-mediated LpX degradation. (I) In WT mice, LpX did not accumulate in the mesangial matrix and occasional foamy mesangial cells were observed. (J) Mesangial cells near the sites of LpX deposition engulf LpX particles. (K) Marked retention of LpX in <i>Lcat</i>^-/- mouse mesangial matrix. The regions near large arrows 1 & 2 in (K) are shown enlarged in K1&2. LpX binds to the mesangial cell prior to engulfment. Scale bars: A, B1, F, H, J = 200 nm; B2, D (inset), K1, K2 = 250 nm; B–E, G, I, K = 500 nm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s003" target="_blank">S3 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s004" target="_blank">S4 Fig</a>, for additional examples.</p

LpX metabolism <i>in vivo.</i>

<p>(A) Blood samples from <i>Lcat</i>^-/- and WT mice were collected prior to (“basal”) and, at 1 and 24 hrs after LpX injection. Plasma samples from WT (n = 6) and <i>Lcat</i>^-/- (n = 6) mice were pooled and lipoproteins were separated by FPLC. Phospholipid (PL), Total Cholesterol (TC), and Free Cholesterol (FC) were measured in collected fractions. Prior to LpX injection, TC, PL and FC were abundant in HDL in WT mice, whereas they were absent in <i>Lcat</i>^-/- mice, which have only small amounts of lipids in VLDL/LpX and small HDL. One hour after injection in <i>Lcat</i>^-/- mice, LpX PL and FC are clearly present in a large peak in the VLDL region, whereas in WT mice, the peak is reduced, consistent with our findings using fluorescent PE-tagged LpX (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.g001" target="_blank">Fig 1B</a>). One hour after LpX administration, a new peak in the HDL region (25 ml elution volume) appeared in WT mice; in <i>Lcat</i>^-/- mice, this peak was observed prior to LpX administration and was increased at 1 hr post-injection. At this time, the PL and FC content of the <i>Lcat</i>^-/- peak was increased compared to the <i>Lcat</i>^-/- pre-injection peak, as well as to the WT peak. (B) Characterization of particles eluted at 25 ml using native gradient gel electrophoresis 1 hr post-injection. Native gradient gel electrophoresis confirmed that lipid-containing particles were present in the 25 ml fraction in the 7–8 nm size range. (C) SDS-PAGE (16% acrylamide gel) apoA-I immunoblot of small HDL particles (25 ml elution volume) generated by LpX at I hr. ApoA-I immunostaining confirmed the presence of apoA-I in these particles, which suggests that in the presence of apoA-I, LpX-derived PL, and to a lesser extent FC, increased the pool size of small HDL particles. These findings <i>in vivo</i> are consistent with the apoA-I and LCAT-dependent remodeling of LpX that we observed in vitro (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.g001" target="_blank">Fig 1B–1D</a>). The peak is still visible 24 hours after injection in WT mice, while in <i>Lcat</i>^-/- mice, it returns to basal levels (Fig 4A).</p

LpX remodeling <i>in vitro.</i>

<p>(A) TEM analysis of synthetic LpX particles. <i>Left panel</i>: Low magnification image (Scale bar; 500 nm<i>)</i>. <i>Middle panel</i>: High magnification (Scale bar; 100 nm). <i>Right panel</i>: LpX particle size distribution. Size categories (nm): I (0–50), II (50–100), III (100–150), IV (150–200), and V (200–245). Small unilamellar vesicles as well as small, medium and, large multivesicular vesicles are seen. (B) LpX remodeling by LCAT and apoA-I in vitro. Agarose gel electrophoresis of LpX labeled with both fluorescent PE <i>(red)</i> and cholesterol (<i>blue</i>) incubated with Alexa 647-tagged apoA-I (<i>green</i>) and/or LCAT in vitro and scanned. Colocalization of LpX PE and cholesterol fluorescence is seen as <i>magenta</i> (merged image). Lane 1: ApoA-I; Lane 2: LpX; Lane 3: LpX + ApoA-I; Lanes 4–6: LpX + ApoA-I + 2, 4, or, 6 mg LCAT, respectively; Lane 7: LpX + 6 mg LCAT. (C) FPLC analysis of dual fluorescent PE- and cholesterol-tagged LpX incubated without (<i>left</i>) or with apoA-I and 6 mg LCAT <i>(right</i>). Fractions were analyzed for rhodamine (PE) fluorescence (<i>upper panels</i>) and TopFluor cholesterol fluorescence (<i>lower panels</i>). Note the additional peak (<i>arrows</i>) after incubation with apoA-I and LCAT. (D) LpX is converted to plasma HDL in vitro. Fluorescent PE-tagged LpX was incubated overnight with pooled human plasma. Agarose gels were scanned for PE fluorescence and then stained with Sudan Black. Lane 1: Fluorescent LpX. Lane 2: Pooled human plasma. Lane 3: Pooled human plasma + fluorescent LpX. <i>Arrows</i> indicate origin.</p

Kidney gene expression in <i>Lcat-/-</i> mice after LpX treatment.

<p>The expression of 84 genes involved in nephrotoxic pathways was measured. Only genes whose expression was statistically different between LpX- treated <i>vs</i> saline-treated <i>Lcat</i>^-/- mice are reported. Data are expressed as mean ± SEM. * P<0.05, ** P<0.01, paired t-test.</p