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2020 Notre Dame Integrated Imaging Annual Report Distributed July of 2021

The Integrated Imaging Facility

• Bradley D. Smith, Director of the Notre Dame Integrated Imaging Facility

Sarah Chapman, Senior Assistant Director of the Notre Integrated Imaging Facility

Alexander Mukasyan, Director of Advanced Electron Microscopy

Maksym Zhukovskyi, Program Director of Advanced Electron Microscopy

Tatyana Orlova, Senior Research Technician

Sara Cole, Director of Optical Microscopy

Theresa Bollinger, Administrator for the entire NDIIF facility
The mission of the Notre Dame Integrated Imaging Faciltiy (NDIIF) is to provide an integrated suite of sophisticated microscopes and imaging stations that enable expert users to attack the most complex modern research problems and, equally important, resident professional staff, including technicians and research specialists, to guide the non-expert users.

Sarah Chapman is the point person for InVivo imaging, histology, sample preparation and image analysis and is working in close collaboration with her NDIIF counterparts within the Optical Microscopy Facility (Galvin building) and the Advanced Electron Microscopy Facility, in the Stinson-Remick building. This mode of operation is beneficial to the community as it provides extended support hours, wider expertise, and access to more equipment than each individual investigator can afford on their own. The EM branch of the facility is staffed by Alex Mukasyan (director and SEM expert) and Maksym Zhukovskyi (program director specializing in TEM). Tatyana Orlova is responsible for instrumentation training and scheduling. The OM branch of the facility is staffed by Sara Cole (director) and Sarah Chapman (serving as a technician). All faculty and staff report directly to Bradley Smith, who serves as an executive director under the management of Bob Bernhard. For administrative questions, contact Theresa Bollinger.

Mode of operation

Located within the basement floor of Galvin building, the InVivo Imaging and Optical Microscopy facility is accessible 24/7, and users can reserve time on each instrument by using an online calendar. The histology core is located on the ground floor of Galvin and is available to users throughout the working hours of M-F, 9-5 and by request on weekends. The Advanced Electron Microscopy facility located in the basement of Stinson-Remick is accessible 24/7 and is also available to users via an online calendar.

For light microscopy, sample preparation and image acquisition are typically handled by the end-users, with support and training provided by Sara Cole (imaging acquisition, data analysis, and specimen preparation). For electron microscopy (EM), most of the sample processing and imaging is done in-house by Tatyana Orlova to ensure consistent and reproducible conditions. Maksym Zhukovskyi is fully trained for sample processing and imaging on our TEM instruments. Sarah Chapman is fully trained for sample preparation of soft and biological materials for both SEM and TEM imaging.

After an initial orientation during which the project is discussed and the best approach is decided upon, users receive hands-on training on the equipment and/or software best suited to their goals, followed by continuous support when required. Additional training and support is offered to the community in different ways: (1) on-site assistance and training on equipment owned by individual investigators; (2) a yearly workshop on microscopy covering light and electron microscopy, image analysis, and sample processing, organized by the NDIIF.

Optical microscopy

The Optical Microscopy Core (OMC) provides researchers with the opportunity to acquire informative single images and three-dimensional reconstructions of fluorescently labeled cells and tissues, either fixed or live. Our devices can accommodate samples mounted on either slides or in Petri dishes. Depending on the device used, images can include up to four separate channels with emission spectra ranging from those of DAPI to far-red fluorophores. The OMC operates seven modern microscopes, each optimized for specific applications: (1) The Nikon AZ 100 is a multi-purpose zoom macroscope that has multiple methods for one to observe samples (fixed slides, to small organisms) either in brightfield (top lit or backlit), epi-fluorescence, or Nomarski DIC. (2) The Nikon 90i is ideal for fluorescent and/or bright-field images of whole tissues and small organisms. Three single filters allow for detection of fluorescence in the widely used ranges of DAPI, FITC, and TRITC. (3) The DeltaVision’s greatest strength is its ability to obtain long-term time-lapse three-dimensional images with a minimum of photodamage on live cells. Then with its deconvolution algorithm, redirects all out of focus light to become in focus for sharp images. (4) The Nikon CSU-X1 spinning disk system is a field scanning confocal microscope that is faster, brighter and more efficient allowing for multimode imaging of both live and fixed samples using lower power lasers and shorter exposure times. (5) The Nikon C2+ Confocal is on an upright microscope base and operates with the same software that the A1R confocal uses. This allows researchers to image their samples from above, when an inverted scope is not practical for their samples.(6) With both fixed and live samples, the Nikon A1R is capable of generating high-resolution confocal images in up to four separate channels. Z-series of images can be used to generate three-dimensional renderings of samples. It is also capable of preforming FRET (fluorescence resonant energy transfer) analysis. Finally, the Bruker Luxendo MuVi SPIM lightsheet system is ideal for long-term, three-dimensional fluorescence imaging of organisms, tissue explants and 3D cell culture systems. This microscope can be used for live, fixed and optically cleared samples. High-end computer workstations with imaging software (Nikon Element, Bitplane Imaris, Metamorph, and ImageJ) are also available.

The OMC has a total of 71 registered users in 29 laboratories. At 2,761 instrument hours, overall usage has increased significantly since last fiscal year. In part this can be contributed to an increase of grant funding. Nearly 90% of the total usage is contributed by The Center for Zebrafish Research and Biological Sciences. Additional contributors include Chemistry and Biochemistry, Electrical Engineering, Aerospace and Mechanical Engineering, Harper Cancer Research Institute, Civil Engineering and Geological Sciences, Center for Rare and Neglected Disease, Chemical and Biomolecular Engineering, and Physics. The A1R confocal microscope was used primarily at an average of 30 hours per week. The Nikon C2 confocal has helped to alleviate this high usage and added additional availability. In addition, the facility has acquired a light sheet microscope and exploring other advanced imaging capabilities. Featuring a horizontal setup, the MuVi SPIM LS is designed to image large volumes of living objects very fast. The unique 4-axis concept with its two-sided illumination facilitates four orthogonal views of the sample without the need for rotation. Updates on the addition of these instruments as well as additional analysis tools will be forthcoming soon!!

Advanced Electron microscopy

The Advanced Electron Microscopy Core (AEM) integrates a unique bundle of state-of-the-art FEI tools that enable expert users to attack the most complex modern research problems from micron to Angstrom spatial resolution. The facility houses a (1) Magellan 400 allowing for digital field emission scanning electron microscopy at record high spatial resolution of 0.6nm, (2) The Helios G4 UX is a fully digital, Extreme High Resolution (XHR) Field Emission Scanning Electron Microscope (FE SEM) equipped with Focused Ion Beam (FIB) technology., (3) Titan 80-300 which enables sub-angstrom, atomic-scale discovery and exploration in both EM and STEM modes over a wide range of materials and operating conditions is now retired and is being replaced with a Spectra 300 TEM microscope with capabilities that are unique to the field of Electron Microscopy revolutionizing the imaging capabilities at the University of Notre Dame and beyond, and (4) a JEOL 2011, a TEM 80-200kV, 0.14nm resolution, a workhorse for materials and biological samples.

The advanced electron microscopy branch of the facility processes specimens from start to finish: fixation, embedding, cutting, ultra-fine sectioning, staining, and imaging. The AEM has a total of 111 registered users in 53 laboratories. At 1,839 instrument hours, overall usage has decreased since the last fiscal year. In part, this is due to the shut down of the Titan for replacement and room renovation as well as the pandemic shutdown of the facility for nearly three months. Over 60% of usage is contributed by Electrical Engineering and Chemical and Biomolecular Engineering. Other contributing resources include Aerospace and Mechanical Engineering, Chemistry and Biochemistry, Physics, Radiation Laboratory, ND Nano, Harper Cancer Research Institute, Civil Engineering and Geological Sciences, Biological Sciences, and an additional 1% of resources contributed by external customers. The Helios FIB was used the most heavily averaging approximately 64 hours per month. The Magellan and Titan TEM were used almost equally averaging 35 hours per month throughout 2020. The facility purchased the Helios G4 UX SEM/FIB instrument as an upgrade for the Helios FIB to offer users further advanced imaging capabilities. This instrument is now fully installed and operational in the facility. The new Spectra 300 TEM instrument will be installed and in operation in the late fall of 2021. The University anticipates this instrument to provide a unique competitive advantage that is exclusive to the University of Notre Dame as well as the greater region. More information regarding this instrument will be forthcoming!

InVivo Imaging Facility

The InVivo Imaging facility provides a non-invasive approach to observe various disease and biological conditions in living systems. This facility currently has seven instruments under management spanning eight modalities available for direct user access. The facility is located with the basement of Galvin and provides (1) an Albira, a small animal PET/SPECT/CT imaging station, (2) a Bruker Xtreme instrument, optical and X-ray small animal imaging system, (3) an IVIS Lumina, a benchtop system for 2D bioluminescence, standard fluorescence and Cerenkov imaging, (4) EchoMRI, a body composition analysis device for live animals, measuring fat, lean, water, and bone mineral content, (5) ICON MRI, an easy-to-use 1 Tesla desktop MRI scanner for small rodents like mice and rats, (6) MARS Medipix Spectral X-ray CT, which promises to revolutionize diagnostic imaging by quantifying the elements and compounds of a sample in a single scan. It is the first commercially available 3D spectral (multi-energy) scanner to produce in vivo images with anatomic and molecular quantification at a fraction of the cost, time, and radiation dose of traditional molecular imaging, such as PET or SPECT (7) a ScanCo VivaCT 80 Micro X-ray CT, an InVivo microCT scanner that offers the largest field-of-view (80mm) and the best resolution (5um) in its category and is hence the most versatile scanner for the in-vivo X-ray micro computed-tomography imaging of mice, rats, and extremities of larger animals up to small rabbits. The strength of the InVivo facility is optical and CT imaging.

The InVivo facility serves 31 users in 11 different laboratories. In 2020 the facility was used for 578 hours averaging just over 48 hours a month which is significantly lower than the usage anticipated for a facility of this capacity. The facility has taken this decrease of usage seriously and is working towards improvements in the facility and have already begun to see an increase in usage during the first half of 2021. The pandemic of 2020 significantly hurt Animal Research labs and thus the usage in the facility. 50% of the total usage of resources is contributed by Biological Sciences and Chemistry and Biochemistry. Other contributing resources include Chemical and Biomolecular Engineering, Harper Cancer Research Institute, Indiana University School of Med- SB, Center for Rare and Neglected Disease, and Aerospace and Mechanical Engineering. The ScanCo VivaCT80 remains the most used instrument in the facility and to expand imaging modality, the facility acquired an additional optical imaging instrument with advanced imaging capabilities. The SPECTRAL AMI HT acquires quantitative images from a diverse set of applications including well plates, plants and small animals. The imaging system contains a high performance cooled CCD camera to record the image collected by a large aperture lens with automation for filter and field of view selection. Luminescence and fluorescence modalities are combined into the system utilizing the latest technologies. This instrument is quickly becoming a workhorse instrument within the core.

Histology and Tissue preparation

Sarah Chapman continues to provide sample processing, training and services to the facility’s users, both for light and electron microscopy applications. She spends a significant amount of time training users in various techniques such as rodent perfusion, cryopreservation, cryosectioning, immunofluorescence, and tissue clearing. During the past year, she trained staff, processed approximately 190 samples per month and provided services for 29 users in 18 different laboratories.

The NDIIF is working towards educational seminars and webinars in partnership with leading industry experts! More to come regarding opportunity to attend in the early spring of 2022!!

Goals for 2021

-continue improving communications and making sure that faculty needs are being addressed and valued

-ensure that we are committed to better science and a competitive source for facility and instrumentation grants

-work towards providing better means of education to our existing users and recruiting new users (helping the University as a whole understand the tools that are available to them and helping to apply it to their research to expand capabilities for visibility and funding efforts)

Created By
Sarah Chapman
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