scholarly journals Virus-Dependent Immune Conditioning of Tissue Microenvironments

2021 ◽  
Author(s):  
Sizun Jiang ◽  
Chi Ngai Chan ◽  
Xavier Rovira-Clave ◽  
Han Chen ◽  
Yunhao Bai ◽  
...  
Keyword(s):  
Ion Beam ◽  
Tumor Biology ◽  
Functional Markers ◽  
Lymphoid Tissues ◽  
Protein Levels ◽  
Preventative Measures ◽  
Immunodeficiency Virus ◽  
Cellular Phenotypes ◽  
Viral Dna Integration

A thorough understanding of complex spatial host-disease interactions in situ is necessary in order to develop effective preventative measures and therapeutic strategies. Here, we developed Protein And Nucleic acid IN situ Imaging (PANINI) and coupled it with Multiplexed Ion Beam Imaging (MIBI) to sensitively and simultaneously quantify DNA, RNA, and protein levels within the microenvironments of tissue compartments. The PANINI-MIBI approach was used to measure over 30 parameters simultaneously across large sections of archival lymphoid tissues from non-human primates that were healthy or infected with simian immunodeficiency virus (SIV), a model that accurately recapitulates human immunodeficiency virus infection (HIV). This enabled multiplexed dissection of cellular phenotypes, functional markers, viral DNA integration events, and viral RNA transcripts as resulting from viral infection. The results demonstrated immune coordination from an unexpected upregulation of IL10 in B cells in response to SIV infection that correlated with macrophage M2 polarization, thus conditioning a potential immunosuppressive environment that allows for viral production. This multiplexed imaging strategy also allowed characterization of the coordinated microenvironment around latently or actively infected cells to provide mechanistic insights into the process of viral latency. The spatial multi-modal framework presented here is applicable to deciphering tissue responses in other infectious diseases and tumor biology.

scholarly journals MAUI (MBI Analysis User Interface)—An image processing pipeline for Multiplexed Mass Based Imaging

2021 ◽  
Vol 17 (4) ◽  
pp. e1008887
Author(s):  
Alex Baranski ◽  
Idan Milo ◽  
Shirley Greenbaum ◽  
John-Paul Oliveria ◽  
Dunja Mrdjen ◽  
...  
Keyword(s):  
User Interface ◽  
User Interfaces ◽  
Ion Beam ◽  
Parameter Tuning ◽  
Crosstalk Noise ◽  
Domain Experts ◽  
Imaging Artifacts ◽  
Cellular Phenotypes ◽  
Insight Into

Mass Based Imaging (MBI) technologies such as Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and Imaging Mass Cytometry (IMC) allow for the simultaneous measurement of the expression levels of 40 or more proteins in biological tissue, providing insight into cellular phenotypes and organization in situ. Imaging artifacts, resulting from the sample, assay or instrumentation complicate downstream analyses and require correction by domain experts. Here, we present MBI Analysis User Interface (MAUI), a series of graphical user interfaces that facilitate this data pre-processing, including the removal of channel crosstalk, noise and antibody aggregates. Our software streamlines these steps and accelerates processing by enabling real-time and interactive parameter tuning across multiple images.

scholarly journals Rapid and Irreversible CD4+ T-Cell Depletion Induced by the Highly Pathogenic Simian/Human Immunodeficiency Virus SHIVDH12R Is Systemic and Synchronous

2002 ◽  
Vol 76 (1) ◽  
pp. 379-391 ◽  
Author(s):  
Tatsuhiko Igarashi ◽  
Charles R. Brown ◽  
Russell A. Byrum ◽  
Yoshiaki Nishimura ◽  
Yasuyuki Endo ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Virus Infection ◽  
Lymph Nodes ◽  
T Lymphocyte ◽  
Lymphoid Tissues ◽  
Highly Pathogenic ◽  
Dna And Rna ◽  
Immunodeficiency Virus

ABSTRACT Highly pathogenic simian/human immunodeficiency virus chimeric viruses are known to induce a rapid, irreversible depletion of CD4+ T lymphocytes in the peripheral blood of acutely infected macaque monkeys. To more fully assess the systemic effects of this primary virus infection, specimens were collected serially between days 3 and 21 postinfection from variety of lymphoid tissues (lymph nodes, thymus, and spleen) and gastrointestinal tract and examined by DNA and RNA PCR, in situ hybridization, and immunohistochemical assays. In addition, the lymphoid tissues were evaluated by fluorescence-activated cell sorting. Virus infection was initially detected by DNA PCR on day 3 postinfection in lymph node samples and peaked on day 10 in the T-lymphocyte-rich areas of this tissue. CD4+ T-cell levels remained stable through day 10 in several lymphoid tissue specimens examined but fell precipitously between days 10 and 21. In situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed the accumulation of apoptotic cells during the second week of infection in both lymph nodes and thymus, which colocalized, to a large extent, to sites of both virus replication and CD4+ T-lymphocyte loss.

scholarly journals Wide Range of Viral Load in Healthy African Green Monkeys Naturally Infected with Simian Immunodeficiency Virus

2000 ◽  
Vol 74 (24) ◽  
pp. 11744-11753 ◽  
Author(s):  
Simoy Goldstein ◽  
Ilnour Ourmanov ◽  
Charles R. Brown ◽  
Brigitte E. Beer ◽  
William R. Elkins ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Lymphoid Tissues ◽  
Plasma Viremia ◽  
Dna And Rna ◽  
Chlorocebus Pygerythrus ◽  
Natural Host Species ◽  
Wide Range ◽  
Immunodeficiency Virus ◽  
Green Monkeys

ABSTRACT The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies (Chlorocebus pygerythrus) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (<1,000 to 800,000 copies per ml), and the levels appeared to be stable over long periods of time. The relative numbers of SIV-expressing cells in tissues of two monkeys correlated with the extent of plasma viremia. SIV expression was observed in lymphoid tissues and was not associated with immunopathology. Virus-expressing cells were observed in the lamina propria and lymphoid tissue of the gastrointestinal tract, as well as within alveolar macrophages in the lung tissue of one AGM. The range of plasma viremia in naturally infected AGM was greater than that reported in naturally infected sooty mangabeys. However, the degree of viremia in some AGM was similar to that observed during progression to AIDS in human immunodeficiency virus-infected individuals. Therefore, containment of viremia is an unlikely explanation for the lack of pathogenicity of SIVagm in its natural host species, AGM.

scholarly journals In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo

2001 ◽  
Vol 82 (9) ◽  
pp. 2225-2234 ◽  
Author(s):  
Carmen Cantó-Nogués ◽  
Sue Jones ◽  
Rebecca Sangster ◽  
Peter Silvera ◽  
Robin Hull ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Simian Immunodeficiency Virus ◽  
Lymphoid Tissues ◽  
Mesenteric Lymph Nodes ◽  
Mesenteric Lymph ◽  
Immunodeficiency Virus ◽  
Early Replication ◽  
Dna Pcr

The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID50 of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.i.), detection of the virus was sporadic. By day 7 p.i., however, significant SIV loads were detected in the blood and lymphoid tissues by DNA PCR and virus co-cultivation. Large numbers of cells expressing SIV RNA were detected in mesenteric lymph nodes by ISH and significantly fewer (P<0·05) in the spleen. Significant numbers of ISH-positive cells were also observed in sections of ileum. By day 14 p.i., the distribution of SIV was more even in all lymphoid tissues analysed. By day 28, most of the tissues were negative by ISH, but all remained positive by virus isolation and DNA PCR. Immunolabelling of sections of mesenteric lymph node with monoclonal antibodies specific for SIV envelope and Nef largely confirmed the observations from ISH. These results indicate that, even following intravenous challenge, a major site of the initial replication of SIV is gut-associated lymphoid tissue. Vaccines that induce protection at this site may therefore be superior, even against parenteral challenge.

Direct Observations of the Epitaxial Growth of Sputtered Ag on Si

1973 ◽  
Vol 31 ◽  
pp. 122-123
Author(s):  
J. S. Maa ◽  
Thos. E. Hutchinson
Keyword(s):  
Epitaxial Growth ◽  
Film Growth ◽  
Ion Beam ◽  
Ion Beam Sputtering ◽  
Microscopy Technique ◽  
Direct Observations ◽  
In Situ Electron Microscopy ◽  
Beam Sputtering ◽  
The Subject

The growth of Ag films deposited on various substrate materials such as MoS2, mica, graphite, and MgO has been investigated extensively using the in situ electron microscopy technique. The three stages of film growth, namely, the nucleation, growth of islands followed by liquid-like coalescence have been observed in both the vacuum vapor deposited and ion beam sputtered thin films. The mechanisms of nucleation and growth of silver films formed by ion beam sputtering on the (111) plane of silicon comprise the subject of this paper. A novel mode of epitaxial growth is observed to that seen previously.The experimental arrangement for the present study is the same as previous experiments, and the preparation procedure for obtaining thin silicon substrate is presented in a separate paper.

Nucleation and Early Growth of Sputtered thin Films

1970 ◽  
Vol 28 ◽  
pp. 516-517
Author(s):  
Dudley M. Sherman ◽  
Thos. E. Hutchinson
Keyword(s):  
Thin Films ◽  
Electron Beam ◽  
Ion Beam ◽  
Ion Source ◽  
Water Cooling ◽  
Stages Of Growth ◽  
Lens Assembly ◽  
Microscope Technique ◽  
Ion Beam Sputter Deposition

The in situ electron microscope technique has been shown to be a powerful method for investigating the nucleation and growth of thin films formed by vacuum vapor deposition. The nucleation and early stages of growth of metal deposits formed by ion beam sputter-deposition are now being studied by the in situ technique.A duoplasmatron ion source and lens assembly has been attached to one side of the universal chamber of an RCA EMU-4 microscope and a sputtering target inserted into the chamber from the opposite side. The material to be deposited, in disc form, is bonded to the end of an electrically isolated copper rod that has provisions for target water cooling. The ion beam is normal to the microscope electron beam and the target is placed adjacent to the electron beam above the specimen hot stage, as shown in Figure 1.

Electron and ion irradiation-induced dissolution of mechanical twins in the intermetalic U3Si: An in situ HVEM study

1989 ◽  
Vol 47 ◽  
pp. 652-653
Author(s):  
Charles W. Allen ◽  
Robert C. Birtcher
Keyword(s):  
Elevated Temperatures ◽  
Ion Irradiation ◽  
Ion Beam ◽  
National Laboratory ◽  
Argonne National Laboratory ◽  
Heavy Ion ◽  
High Energy ◽  
Mechanical Twinning ◽  
In Situ Experiments

The uranium silicides, including U3Si, are under study as candidate low enrichment nuclear fuels. Ion beam simulations of the in-reactor behavior of such materials are performed because a similar damage structure can be produced in hours by energetic heavy ions which requires years in actual reactor tests. This contribution treats one aspect of the microstructural behavior of U3Si under high energy electron irradiation and low dose energetic heavy ion irradiation and is based on in situ experiments, performed at the HVEM-Tandem User Facility at Argonne National Laboratory. This Facility interfaces a 2 MV Tandem ion accelerator and a 0.6 MV ion implanter to a 1.2 MeV AEI high voltage electron microscope, which allows a wide variety of in situ ion beam experiments to be performed with simultaneous irradiation and electron microscopy or diffraction.At elevated temperatures, U3Si exhibits the ordered AuCu3 structure. On cooling below 1058 K, the intermetallic transforms, evidently martensitically, to a body-centered tetragonal structure (alternatively, the structure may be described as face-centered tetragonal, which would be fcc except for a 1 pet tetragonal distortion). Mechanical twinning accompanies the transformation; however, diferences between electron diffraction patterns from twinned and non-twinned martensite plates could not be distinguished.

To Eliminate Curtain Effect of FIB TEM Samples by a Combination of Sample Dicing and Backside Milling

2014 ◽  
Author(s):  
Jian-Shing Luo ◽  
Hsiu Ting Lee
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Low Cost ◽  
Ion Beam ◽  
Sample Preparation Method ◽  
Site Specific ◽  
Dual Beam ◽  
Transmission Electron

Abstract Several methods are used to invert samples 180 deg in a dual beam focused ion beam (FIB) system for backside milling by a specific in-situ lift out system or stages. However, most of those methods occupied too much time on FIB systems or requires a specific in-situ lift out system. This paper provides a novel transmission electron microscopy (TEM) sample preparation method to eliminate the curtain effect completely by a combination of backside milling and sample dicing with low cost and less FIB time. The procedures of the TEM pre-thinned sample preparation method using a combination of sample dicing and backside milling are described step by step. From the analysis results, the method has applied successfully to eliminate the curtain effect of dual beam FIB TEM samples for both random and site specific addresses.

Investigations of Leakage Paths in Sub-0.35μm DRAM Products Using Advanced Focused Ion Beam Techniques

1998 ◽  
Author(s):  
H. Lorenz ◽  
C. Engel
Keyword(s):  
Focused Ion Beam ◽  
Cell Function ◽  
Dielectric Breakdown ◽  
Ion Beam ◽  
Electrical Characterization ◽  
Floating Gate ◽  
Leakage Path ◽  
Contrast Technique ◽  
Voltage Contrast

Abstract Due to the continuously decreasing cell size of DRAMs and concomitantly diminishing thickness of some insulating layers new failure mechanisms appear which until now had no significance for the cell function. For example high resistance leakage paths between closely spaced conductors can lead to retention problems. These are hard to detect by electrical characterization in a memory tester because the involved currents are in the range of pA. To analyze these failures we exploit the very sensitive passive voltage contrast of the Focused Ion Beam Microscope (FIB). The voltage contrast can further be enhanced by in-situ FIB preparations to obtain detailed information about the failure mechanism. The first part of this paper describes a method to detect a leakage path between a borderless contact on n-diffusion and an adjacent floating gate by passive voltage contrast achieved after FIB circuit modification. In the second part we will demonstrate the localization of a DRAM trench dielectric breakdown. In this case the FIB passive voltage contrast technique is not limited to the localization of the failing trench. We can also obtain the depth of the leakage path by selective insitu etching with XeF2 stopped immediately after a voltage contrast change.

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