Annual Newsletter

 Ndiif Newsletter

This newsletter summarizes current capabilities and highlights usage trends to date for 2018. The NDIIF operates as a recharge facility and revises user fees every July 1.

Questions about NDIIF policies should be directed to the Director or any member of the steering committee. For technical and scheduling questions, see the relevant NDIIF staff members listed throughout the website. 

Sincerely

Professor Bradley Smith, Director of NDIIF

Winter 2018

NDIIF News Brief
  1. Midwest Imaging and Microanalysis Workshop at Notre Dame: The fifth annual workshop, held May 2018, was very successful with a focus on electron beam technologies. The event will be repeated mid May 2019 and will include a focus on Biological Imaging.
  2. NDIIF Awards for Best Imaging Publications in 2017: 
    • The Best Electron Microscopy Imaging Publication Award for 2017 was awarded to Yuliya Klymenko, a Post-Doctoral Fellow with Dr. Sharon Stack in the Department of Chemistry and Biochemisty.
    • The Best Biological Imaging Publication Award for 2017 was awarded to Jeremiah Zartman, an Assistant Professor in the Department of Chemical and Biomolecular Engineering.
    • Image submission for Best Imaging Publication Award in 2018 is open now and will end mid-April, 2019. 
  3. CTSI Core Facility: Affiliation with CTSI as a core research facility was renewed. For more information please visit:  http://www.indianactsi.org

Funding opportunity and grants are continually available through CTSI.  Please inquire for further information.  NDIIF staff is available to assist in budget preparation and to develop scope of work.

  1. Advanced Electron Microscopy Core: a new Helios G4 PFIB UXe FIB was purchased and is scheduled to arrive in late March, 2019.
  2. Optical Microscopy Core: a Nikon CSU Spinning Disk System was purchased and will be housed at Harper Cancer Research Institute under the management of the NDIIF.  This instrument is scheduled to arrive in late January.

Established in 2008, the NDIIF provides an integrated suite of sophisticated microscopes and imaging stations that enable the expert users to attack the most complex modern research problems and, equally important, the resident professional staff (technicians and research specialists) to guide the non-expert users

 

Advanced Microscopy                  Optical Microscopy                         Histology                             InVivo Imaging

Feature Stories

Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis.

Hook, P., Brito-Robinson, T., Narcisco, C., Kim, O., Goodson, R.I.,Weisel, J.W., Alber, M.S., and Zartman. J.J. Biomedical Optics Express, 2017, 8 (8), 3671-3686.

Zartman Highres

Our understanding of three-dimensional high-resolution clot structure remains incomplete since most knowledge has come from studies of relatively small clots or thrombi, due to the optical impenetrability of clots beyond a few cell layers in depth.  In this publication, we report an optimized optical clearing method termed cCLOT that renders large whole blood clots transparent and allows confocal imaging close to one millimeter inside the clot.  We have tested this method by investigating the 3D structure of clots made from reconstituted pre-labeled blood components yielding new information about the effects of clot contraction on erythrocytes.  Although it has been shown recently that erythrocytes are compressed to form polyhedrocytes during clot contraction, observations of this phenomenon have been impeded by the inability to easily image inside clots.  We validated the utility of cCLOT by demonstrating a quantitative structural difference in the fibrin network appearance when clot contraction is impaired pharmacologically with blebbistatin, which targets platelets’ non-muscle myosin.  This treatment is consistent with lower fibrin density and connectivity across clots when contraction is compromised.  We also measured erythrocyte volumes at different depths inside clots and showed the volume remains the same during contraction, suggesting that contraction depends solely on reducing extracellular space.  This finding indicates that clot contraction is not due to osmotic changes in erythrocytes but rather due to a higher compaction of the cells.  As an efficient and non-destructive method, cCLOT represents a powerful research tool in studying blood clot structure and mechanisms controlling clot morphology. 

Kim Oncogeneslide

 

 Cadherdrin composition and multicellular aggregate invasion in organotypic models of epithelial ovarian cancer intraperitoneal metastasis.

Klymenko Y, Kim O, Loughran E, Yang J, Lombard R, Alber M, Stack MS. Oncogene. 2017 Oct; 36(42):5840.doi:10.1038/onc.2017.171.

Nikon A1R­MP multiphoton confocal microscope (Notre Dame Integrated Imaging Facility) was employed to monitor in real time and at high resolution the dynamics of N­cadherin­ expressing (Ncad+) and E­cadherin ­expressing (Ecad+) ovarian cancer cells (green/red) within a three-­dimensional extracellular collagen matrix (blue).  Continuous live cell z-stack imaging using fluorescence and reflectance confocal modes enabled comprehensive characterization of the metastasis ­associated behavior of single cells and multicellular aggregates as well as remodeling of the surrounding collagen.  To maintain physiologically relevant conditions during imaging, the environmental chamber of A1R­MP was applied.  Three-­dimensional image-­based quantitative analysis using Nikon software revealed that Ncad+ cells were much more efficient invaders than Ecad+ cells by forming an extensive, spatial cellular network via tip-­like, cell-­cell junctions within the collagen matrix.  In co-­cultures of Ncad+ and Ecad+ cells, cadherin­dependent sorting did not promote collective cell migration.  After 7 days of incubation, Ecad+ cells remained superficially localized, while Ncad+ cells invaded deep inside the matrix.  Furthermore, cancer cell invasion was successfully abrogated by blocking the Ncad extracellular domain, thus delineating the importance of Ncad expression for peritoneal seeding by metastasizing cells and suggesting intraperitoneal delivery of Ncad ­blocking molecules as a possible therapeutic approach to suppress epithelial ovarian cancer metastasis.  

2017 August 11 Jbc 32 Cropped

 

Post-translational modification of the membrane type 1 matrix metalloproteinase (MT1-MMP) cytoplasmic tail impacts ovarian cancer multicellular aggregate dynamics

Jing Yang, William C. Kasberg, Angelo Celo, Zhong Liang, Kristal Quispe and M. Sharon Stack. The Journal of Biological Chemistry. 2017 Aug 11:292(32):13111-13121. Doi: 10.1074/jbc.M117.800904. Epub 2017 Jun 27.

Metastatic ovarian cancer (Ovca) cells are shed as multicellular aggregates (MCAs) into ascites fluid, which anchors peritoneally to send metastases.  Yang at al. show that post-translational phosphorylation of matrix metalloproteinase-14, expresses on Ovca cells, and promotes the adhesion to and dispersal of MCAs on peritoneal mesothelial monolayers and peritoneal explants.  These data identify a mechanism for regulation of Ovca metastatic success.  This work is published in The Journal of Biological Chemistry (Cover image) in August 11, 2017.

Klymenko Neoplasiaslide

 

 Heterogeneous cadherin expression and multicellular aggregate dynamics in ovarian cancer dissemination

Klymenko Y, Johnson J, Bos B, Lombard R, Campbell L, Loughran E, Stack MS. Neoplasia. 2017 Jul 1;19(7):549-63. DOI: 10.1016/j.neo.2017.04.002.

Epithelial ovarian cancer (EOC) spreads via shedding of malignant cells and multicellular aggregates (MCAs) from the primary tumor into peritoneal cavity, with subsequent peritoneal adhesion as a key early event in metastatic seeding. Field emission scanning electron microscopy (FEI‐Magellan 400, Notre Dame Integrated Imaging Facility) allowed us to evaluate the contribution of cellular cadherin composition toMCA phenotype by visualizing surface topography of 3‐dimensional MCAs grown in vitro using the panel of EOC cell lines, with high resolution and wide magnification range. We were able to identify striking cadherin‐dependent differences in aggregate surface ultrastructure. In particular, cells expressing mesenchymal‐type N‐cadherin (Ncad+) formed solid, smooth, cohesive spheroids, while cells expressing epithelial‐type E‐cadherin (Ecad+) formed loosely clustered aggregates covered by uniform microvilli. An intermediate surface phenotype was displayed by the hybrid cadherin (Ecad+/Ncad+) cell lines. Subsequent genetic manipulation of cadherin expression in EOC cells altered MCA surface morphology (assessed bySEM) and resulted in changes in cell adhesion, migration, invasion and proliferation (assessed by functional assays later in the study). Collectively, these findings support the hypothesis that MCA cadherin composition impacts intraperitoneal cell and MCA dynamics and thereby affects ultimate metastatic success. 


 Jhu 2017awardnominationslides Analyticalchem

 

Whole-cell Pseudomonas aeruginosa localized surface plasmon resonance aptasensor

Hu, J., Fu, K., Bohn, P.W. Anal. Chem. 2018, 90,2326-2332. Doi: 10.1021/acs.analchem.7b04800.

A whole-­cell biosensor was developed capable of direct and specific detection of intact bacteria in a healthcare setting. A schematic illustration of our sensor chip with legend is shown in Figure 1a. Using a field­ emission scanning electron microscope (FEI Magellan 400), we characterized sensor chips containingAu nano-triangle arrays (Figure 1b) and verified the capture of bacterial cells onto the modified sensor surface(Figure 1c). These SEM images provided key information of the sensor chip fabrication and bacterial binding. Specifically, Au nano-triangles were measured to have in-­plane width of 210.5 ± 9.1 nm, out-­of-­plane height of 47.6 ± 0.4 nm, and tip­-to-­tip distance of 121.3 ± 7.7 nm. Overall, our bacterial sensing platform is label-­free, rapid (~3 h for detection), sensitive (down to a single bacterium level), selective (Pseudomonas strain PAO1), semi­-quantitative with a clinically relevant dynamic range, as well as robust exhibiting over 2 months shelf life at ambient conditions. This versatile sensing platform should be extendable to a wide range of supermolecular analytes, such as bacteria and viruses, and it has potential to be used as an advanced pathogen diagnostic platform to combat infectious diseases.

Gpszakmany Nomination2017 Bestimage Slide

 

THz Wave detection by antenna-coupled nanoscale thermoelectric converters

G.P. Szakmany, A.O. Orlov, G.H. Bernstein and W. Porod. IEEE Transactions on Terahertz Science and Technology, vol. 7, no. 5, pp. 582-585, Sept. 2017. Doi: 10.1109/TTHZ.2017.2715420.

Using the high­ resolution imaging capability of the Magellan SEM allows us to inspect various nanowires used for antenna­ coupled nanoscale thermoelectric converters. Using these images, we can study the integrity of the center section (10 um long, 100 nm wide) of a dipole antenna and its contact to the hot junction of our single ­metal nanothermocouples that are constructed from 50­ and 250­nm wide Ni wire segments. We were able to image the junctions and the nanowires and identify any fabrication errors that influenced our electrical and optical measurement results.


 

Yizhou Zhang Image Wap

 Block polymer membranes functionalized with nanoconfrimed polyelectrolyte brushes achieve sub-nanometer selectivity

Zhang, Y., Mulvenna, R. A., Qu, S., Boudouris, B.W., Philllip, W.A. ACS Macro Lett.,2017, 6(7), pp 726-732. Doi: 10.1021/acsmacrolett,7b00278.

The images discussed below were obtained using the Magellan 400 Scanning Electron Microscope and the associated Bruker Energy Dispersive X-Ray Spectrometer feature.  We reported the fabrication of a nanofiltration membrane from self-assembled polystyrene-b-polyisoprene-b-poly(N,N-dimethylacrylamide) block  polymer precursors.  After  covalently  bonding  polyelectrolytes brushes along the pore wall of the membrane, an exceptionally size-selective separation device capable of gating solutes with only an 8 Å difference in size was generated.  Since both the nanostructure and pore wall chemistry of  the  membrane were  critical to its performance, these  features  were characterized using scanning electron microscopy(SEM) and energy dispersive spectroscopy(EDS).  SEM micrographs of the surface demonstrated a  high  density of well-defined, nanoscale  pores while SEM micrographs of  the membrane  cross-section highlighted a 500-nm  thick,  asymmetric architecture that reduced  the hydraulic resistance  at  the  interface  between the block  polymer  and  its  support.  In  combination, these  structural features produced a membrane with high throughput. Furthermore, because the chemical modification with polyelectrolyte  brushes  was  crucial  to  realizing  sub-nm  selectivity, EDS was  used  to  demonstrate the localized enrichment of oxygen in the blockpolymer—an observation that was consistent with the covalent attachment  of  brushes  occurring exclusively  in  the block  polymer  layer.  These  high-quality  images provided  critical support to  our  claims that the  combination  of  well-defined  nanostructure  and tailored chemical features resulted in a highly selective nanofiltration device. 


 

Nomination For Best Imaging Publication Kaiyu

 Ion selective redox cycling in zero-dimensional nanopore electrode arrays at low ionic strength

Kaiyu Fu, Donghoon Han, Chaoxiong Ma, an Paul W. Bohn. Nanoscale, 2017, 9, 5164-5171. Doi: 10.1039/C7NR00206H.

In this research project, the nominee developed a robust approach to fabricate high density nanopore­electrode arrays at large scale through the state-­of-­art equipment at the NDNF, as shown in Figure 1(A) and (B). These miniscule cavities are incredibly powerful and have the capacity to detect single­ molecules, study single ­reaction events, and be used for applications ranging from DNA sequencing to measuring the transfer of individual electrons. By using Helios FIB at NDIIF, we successfully imaged those tiny nanopores in great details (Figure 1(C) and (D)). The nominee’s group are among a few groups around the world whom are interested in, as well as have the ability to make the most exacting nanoscale measurements possible, i.e. manipulating a single molecule or a single reaction to define the chemical principles in nature. More importantly, these nanoscale electrodes can also be used in the fabrication of lab-­on-­a-­chip devices for advanced diagnostics and therapeutics. Beside the image from this paper, the nominee published around ten papers, which at least four of them are related to this research topic, over the last two years (Small, 2018, DOI:10.1002/smll.201703248.; ACS Central Science, 2017, 4, 20.; ACS Applied Materials & Interfaces, 2017, 9, 24908.;Faraday discussions, 2016, 193, 51.). Overall, this multidisciplinary research will definitely attract wide publicity across the Notre Dame Research Community in the near future (see the News reported by ND Research, NDIIF, ND Nano, NDAD&T, ND Science, and ND Chem.).

Bailey Scirepslide

 

Chemical analysis of morphological changes in lysophosphatidic acid-treated ovarian cancer cells

Bailey, K.A., Klymenko, Y., Feist, P.E., Hummon, A.B., Stack, M.S., and Schultz, Z.D. (2017). Scientific reports, 7(1), 15295. Doi: 10.1038/s41598-017-15547-7.

Ovarian cancer metastasis occurs as tumor cells and multicellular aggregates (MCA) exfoliate from the tumor origin directly into the peritoneal cavity. This process is accompanied by elevated levels of the bioactive lipid lysophosphatidic acid (LPA) in the intraperitoneal ascetic fluid. Scanning electron microscopy (SEM) enables visualization of surface detail and complexity inaccessible by light microscopy due to high resolution at high magnification and depth of field, enabling us to investigate morphological changes occurring on the surface of ovarian cancer cells/MCAs in response to LPA exposure. SEM micrographs acquired with a field emission SEM FEI‐Magellan 400 (Notre Dame Integrated Imaging Facility) showed distinct shedding of microvilli‐like features upon treatment with LPA, which would not be visible with any other microscopic technique. Further analysis of MCAs and cellular surface “sheddings” using multiplex coherent an‐Stokes Raman scattering imaging confirmed the loss of the surface protein signal and increase in lipid composition on the surface of LPA‐treated cells, whereas, subsequent mass spectrometry on the cellular “sheddings” identified a large list of membrane and intracellular proteins in the shed samples.

Dhu Slide For Ndiif

 

 Preparation of hyperstar polymers with encapsulate Au25(SR)18 clusters as recyclable catalysts for nitrophenol reduction

D. Hu; S. Jin; Y Shi; X. Wang; R.W. Graff; W. Liu; M. Zhu; H. Gao. Nanoscale 2017, 9, 3629-3636.

This paper developed a robust approach to prepare a hybrid nanomaterials, i.e., hyperstar polymer-Au25(SR)18 nanocomposite,for catalysis.  The core–shell structured hyperstar polymers were synthesized using atom transfer radical polymerization of three functional monomers in sequence before loading hundreds of 1 nm Au25(SR)18 nanoclusters into the core domain through ligand exchange. The obtained hyperstar-Au25(SR)18 nanocomposites showed great stability with no size change after a three-month shelf storage. They were used as efficient catalysts for the catalytic reduction of 4-nitrophenol by NaBH4, showing convenient recovery and reuse without losing catalytic efficiency.The transmission electron microscopy (TEM) image clearly demonstrates the encapsulated Au25(SR)18 nanoclusters in the core domain of hyperstar polymers, which was the key features to exhibit the materials stability and catalytic efficiency.

 

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