Auditory & Vestibular Technology (AVT) Core

The Auditory & Vestibular Technology (AVT) Core is established to provide infrastructure to support Research Project Leaders and principle investigators associated with the Dr. Richard J. Bellucci Translational Hearing Center to conduct auditory and vestibular research across the full range of experimental model systems, from single molecule analysis to whole organism models.

The AVT Core is located within the Criss Building complex of Creighton University School of Medicine and is under the direction of the following co-Core Directors:

Image	Molly McDevitt, PhD Mass Spectrometry
Image	Anthony Stender, PhD Imaging Specialist and Imaging Core Manager
Image	Michael Nichols, PhD AVT Core Director - Professor of Physics
Image	D. David. Smith, PhD Professor of Biochemistry
Image	Sarath Vijayakumar, PhD Electrophysiology, Molecular Biology

The AVT Core offers a broad range of services:

Electrophysiology (Sarath Vijayakumar)
Molecular Biology (Sarath Vijayakumar)
Imaging (Anthony Stender)
Mass Spectrometry (Molly McDevitt)

The AVT Core facilities and its personnel can also enhance the scope of technical options, foster collaboration for multidisciplinary research, play an important role to prepare talented new investigators submitting new research proposals, and in the technical training of graduate students and post-doctoral fellows. The AVT Core will continue to incorporate new methodologies as Core options to provide leading-edge technologies to center investigators.

Sub Cores

Electrophysiology (Sarath Vijayakumar)
- Contact Sarath Vijayakumar (SarathVijayakumar@creighton.edu)

Molecular Biology (Sarath Vijayakumar)
- Contact Sarath Vijayakumar (SarathVijayakumar@creighton.edu)

Imaging (Anthony Stender)
- Contact Anthony Stender (AnthonyStender@creighton.edu)

Mass Spectrometry (Molly McDevitt)
- Contact Molly McDevitt (MollyMcDevitt@creighton.edu)

Electrophysiology Services

The electrophysiology facilities are composed of the main facility on the Creighton campus (Rooms 304 and 334 in the Criss I Building) and two satellite facilities at BTNRH and UNMC.
- The electrophysiology facility offers these services:
Auditory Brainstem Response (ABR)
Distortion Product Otoacoustic Emission (DPOAE)
Wideband Acoustic Immitance (WAI). Coming soon...
Endocochlear Potential (EP)
Cochlear Microphonics (CM)
Vestibular sensory Evoked Potential (VsEP)
Video oculography for VOR/OKR
Current Instruments include:
TDT RZ6 system for measuring auditory evoked potentials and otoacoustic emissions.
Axopatch 200B intergrating patch clamp amplifiers and 1440A Digidata boards for recording cochlear potentials (CM, CAP, EP) from small mammals and microphonic response from zebrafish.
National Instruments (NI) based system for vestibular evoked potentials.
ISCAN 200 based system for VOR/OKR

*Directed by Sarath Vijayakumar

Molecular Biology Services

Located in Room 209 of Criss I building, the facility is equipped with instruments commonly used for molecular biology and genomic experiments.

Milliplex Analyzer developed by Luminex uses multiplex xMAP bead technology that allows for teh simultaneous analysis of a broad selection of analytes (e.g. cytokines, hormones and adipokines) across a wide range of disease states, including metabolic disease, inflammation, neurodegenerative disease, toxicity, cancer and more. With the Milliplex system, you can:

Quantitate up to 100 protein targets from culture media, sera, and other matrices simultaneously.
Automatically analyze up to 96 samples in 60 minutes (general format: 88 sample wells + 8 standard wells)
Dramatically increase the amount of useful data

The QuantStudio 3 Real-Time PCR System in designed for users who need an affordable, easy-to-use real-time PCR system that doesn't compromise performance and quality. The simplified Design and Analysis software is ideal for both first-time and experienced users. Using Proven OptiFlex technology (featuring 4 coupled channels and white LED) and featuring three independent Veriflex temperature zones, the QuantStudio 3 system enables improved data accuracy and sensitivity for a broad range of genomic applications. Applied Biosystems ProFlex thermocycler is also available for routine PCR applications.

The ChemiDoc MP Imaging System is a full-feature instrument for imaging and analyzing gels and western blots. It is designed to address multiplex fluorescent western blotting, chemiluminescence detection, general gel documentation applications, and stain-free technology imaging needs. Features include:

All-in-one flexible imaging - get precise, reproducible fluorescent, chemiluminescence, and colorimetric gel and blot detection, analysis and documentation in a single system
Multiplex fluorescent western blotting - detect up to three proteins simultaneously and eliminate the need for stripping and reprobing
Ease of use - features include automatic selection of optimal light source by application, auto focus, auto exposure, and preview features ensure optimal images. Learn in minutes with on screen help.
Stain-free protein normalization - stain-free imaging permits the normalization of bands to total protein in both in gels and blots; eliminates the need for housekeeping proteins
Sample Trays and Smart Tray Technology - three sample trays available to cover diverse imaging applications. Smart Tray Technology automatically recognizes the application-specific trays and adjusts imaging parameters and software options accordingly

The 10X Chromium Controller uses advanced microfluidics to perform single cel activity in multiple dimensions, including gene expression, cell surface proteins, immune clonotype, antigen specificity, and chromatin accessibility. It is compatible with Chromium Singe Cell products, including Single Cell Gene Expression, Single Cell Immune Profiling, and Single Cell ATAC.

Process 1-8 samples per run
Collect 500-10,000 cells per sample
Libraries produced are compatible with Illumina NextSeq 2000 in the innovative Genomics and Bioinformatics core

*Directed by Sarath Vijayakumar

Advanced Imaging Services

We offer multiple advanced research microscopes that are available for imaging-based experiments. We also provide a selection of sophisticated software packages that can be utilized for image analysis. Please inquire if you have specific questions about any of these instruments or about gaining access to them. We are happy to provide training as well as to consult with users about their experimental and analysis needs. New users must receive official training and sign a user agreement prior to gaining access to the facility's tools or its reservation systems.

Instrumentation:

The Advanced Imaging Core hosts three confocal laser scanning microscopes from Zeiss. All three microscopes offer DIC imaging capabilities and popular objective magnifications (10X, 20X, 40X, 63X). The inverted LSM 700 offers four excitation wavelengths (405, 488, 555, 639 nm), while our upright LSM 710 has 7 laser lines (405, 458, 488, 514, 561, 594, 633), 2 PMT’s, and a 32-channel detector. The facility also has a Zeiss LSM 980 with Airyscan, which offers high resolution, improved sensitivity, and capabilities for live cell imaging. Finally, the AVT- Advanced Imaging Core manages a Zeiss PALM CombiSystem, which enables users to perform microdissection and micromanipulation of samples on standard glass slides within a single microscope. These microscopes can be found in rooms 407, 332, and 312 of the Criss Complex.

The Advanced Imaging team also manages several specialized microscopes associated with CU-IBIF, Creighton University’s Integrated Biomedical Imaging Facility, located in rooms 324, 325, 326C, 376, and 382A of the Criss Complex.

First is an inverted Nikon Ti-2 confocal microscope with a Yokogawa spinning disk, a Hamamatsu Orca Flash camera, and an incubated stage. This microscope is capable of fast full-frame imaging, and it can perform Z-stacks at rates 10 times faster than a typical point scanning confocal. This microscope is equipped with Live Super-Resolution, which enables the high-speed imaging that is required for live cell experiments. It is also an ideal choice for extended time-lapse imaging experiments.

Next is an upright Leica SP8 confocal microscope that is suitable for live animal imaging and can support accessories for electrophysiology measurements. In addition to visible-range lasers, it has a tunable, pulsed, near-infrared Ti:S laser for multiphoton excitation of UV/Visible fluorophores and second harmonic imaging. Non-descanned Super HyD detectors and a Becker and Hickl SPC 830 Time Correlated single photon counting system enable fluorescence lifetime imaging, fluorescence correlations spectroscopy, and lifetime-based FRET techniques. The Leica has spectral detectors that enable spectral imaging.

Third is a home-built Total-Internal Reflection Fluorescence Microscope. This instrument offers a through-the-prism beam path to reduce noise from scattered laser light, and it incorporates three objective options (10X, 20X, 40X) and four laser lines (405, 488, 552, and 647 nm). Images are captured with an Andor iXon EMCCD scientific camera.

The team also manages an inverted ImageXpress Micro 4 widefield microscope from Molecular Devices, equipped with an incubation chamber. The ImageXpress enables users to conduct preprogrammed experiments over extended time periods at high-throughput. In addition, CU-IBIF is also the home to two other widefield microscopes for basic imaging needs.

Software Packages Available:

The Advanced Imaging Core provides a selection of analysis tools for the THC and users of our facility. We have two workstations that run the Imaris Microscopy Image Analysis software, which is helpful in processing 3D and 4D images. A standalone workstation is available for utilizing Nikon’s NIS Elements and Leica’s LAS X software. Users interested in knowing more about these software packages and accessing them are asked to reach out to the team. We are happy to discuss your image processing needs with you, and we have experience in using a variety of other programs as well for analysis and processing, including ImageJ, MATLAB, Python, and R.

Mass Spectrometry Services

The Mass Spectrometry Core offers two mass spectrometers and provides a selection of software to be utilized for the analysis of mass spectrometry-based experiments. Current capabilities include mass determination, proteomics, and absolute quantitation of small molecules. Please inquire with Molly McDevitt (mollymcdevitt@creighton.edu) if you have specific questions regarding the capabilities of the Mass Spectrometry Core. New users must sign a user agreement prior to running samples. At this time, no training for the individual use of instruments will be provided. However, we will happily provide software training so users can complete their own analysis of any mass spectrometry data, if desired.

Instrumentation

The Mass Spectrometry Core houses two mass spectrometers. Both can be found in Criss II Room 303.

The Bruker ultrafleXtreme is a MALDI-TOF/TOF mass spectrometer with resolving power up to 40,000 over a wide range of masses and up to 1 ppm mass accuracy. It is equipped with both MS and MS/MS capabilities, exhibiting high data acquisition speeds for both. It is ideal for mass determination and structure identification for a variety of biomolecules, including proteins, peptides, lipids, and glycans.

The Thermo Q Exactive is a hybrid quadrupole Orbitrap mass spectrometer which combines high-performance quadrupole precursor selection with high-resolution, accurate mass (HR/AM) Orbitrap detection. It boasts a resolving power of up to 140,000 FWHM and mass accuracy below 1 ppm. Fast scan speeds, the HR/AM capabilities, the capacity for MS/MS, and versatility of the instrument make it ideal for the identification and quantitation of small molecules, peptides, and proteins.

In addition to the two mass spectrometers, Criss II Room 303 is also home to two different liquid chromatography (LC) systems. Used in conjunction with the Thermo Q Exactive, both LC systems enable separation of analytes prior to analysis, reducing sample complexity and resulting in lower detection limits.

The Thermo Vanquish Flex Binary UHPLC system is comprised of a biocompatible, binary, high-pressure gradient mixing pump, an autosampler, and a heated column compartment. With a pressure limit of 1000 bar and flow rate range of 50 µL – 8 mL/min, it is suited for a variety of analyses, including absolute quantitation of small molecules, lipidomics, and metabolomics.

The Waters ACQUITY M-Class Chromatography System provides nanoflow technology with the straightforward setup found in an analytical-scale UPLC system. Nanoflow technology, adds increased sensitivity compared to analytical-scale UPLC systems and is ideal for analyses in which sample is limited. With a pressure limit of 15,000 psi and capable of flow rates as low as 200 nL/min, it is best suited for bottom-up proteomics, including data dependent acquisition, targeted analysis, and affinity-purification mass spectrometry.

Software Packages:

The Mass Spectrometry Core provides a selection of analysis tools for users of our facility:

· fleXAnalysis

· Xcalibur Software Package

· TraceFinder 4.1

· Proteome Discoverer 3.1

For users that are interested in doing their own data analysis, please reach out to the team and we will be happy to provide training.

*Directed by David Smith and assisted by Molly McDevitt

Acknowledgement Statement for AVT Core Papers

When publishing a research paper that relied on equipment used within the AVT Core Facility, please remember to acknowledge the core. Proper acknowledgment is critical for tracking research papers and meets the requirements of our funding agencies. We recommend that you use the statement below that best fits your paper (you may simply copy and paste this content into the Acknowledgments section of your paper):

If Imaging Core was used:

This research was partially conducted at the Auditory and Vestibular Technology Core (AVT) at Creighton University, Omaha, NE (RRID:SCR_023866). This facility is supported by the Creighton University School of Medicine and grants GM103427 and GM139762 from the National Institute of General Medical Science (NIGMS), a component of the National Institutes of Health (NIH). This investigation is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or NIH.

If Imaging Core was not used:

This research was partially conducted at the Auditory and Vestibular Technology Core (AVT) at Creighton University, Omaha, NE. This facility is supported by the Creighton University School of Medicine and grants GM103427 and GM139762 from the National Institute of General Medical Science (NIGMS), a component of the National Institutes of Health (NIH). This investigation is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or NIH.