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Auditory & Vestibular Technology (AVT) Core

The Auditory & Vestibular Technology (AVT) Core is established to provide infrastructure to support Research Project Leaders and principal investigators associated with the 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:

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Dr. Anthony S. Stender

Anthony Stender, PhD
Imaging Specialist and CU IBIF Core Manager

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Michael Nichols, PhD
Michael Nichols, PhD
AVT Core Director - Professor of Physics
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​​​​​​​D. David. Smith, PhD
D. David. Smith, PhD
Professor of Biochemistry

 

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.

The electrophysiology facilities are composed of the main facility (Fig. 1) on the Creighton campus (Rooms 304 and 334 in the Criss I Building) and two satellite facilities at BTNRH and UNMC. The electrophysiology facilities offer both non-invasive (ABR, VEP, CAEP and OAE recording) and invasive electrophysiological procedures (cochlear potentials).

Current instruments include:

  1. two complete sets of TDT RZ6 for measuring auditory and vestibular function (evoked potentials and otoacoustic emissions) from rodents.
  2. two Axopatch 200B integrating patch clamp amplifiers and 1440A Digidata boards for recording cochlear potentials (CM, CAP, EP) from small mammals and microphonic response from zebrafish.
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Auditory function examination equipment

 

Fig. 1: Setup for auditory function examination

*Directed by David He and assisted by Mr. Huizhan Liu.

Located in Room 211 of Criss I building, the facility is equipped for techniques commonly used for molecular biological investigations. In addition to commonly used equipment for molecular biology such genotyping, qPCR and in situ hybridization, the facility also has a 10x Genomics for single-cell RNA-sequencing (Fig. 2).

The services offered by this core include PCR, q-PCR, in situ hybridization, RNAScope in situ hybridization, RNA extraction, quality examination, and preparation for RNA-seq and DNA-sequencing.

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10x Genomics

Fig. 2: 10x Genomics for single-cell RNA-seq.​

*Directed by David He and assisted by Mr. Huizhan Liu.

AVT Advanced Imaging Services section

We offer multiple advanced research microscopes that are available for confocal, high-resolution, or widefield imaging.  We also provide a selection of sophisticated software packages that can be utilized for image analysis.  The facilities that house the resources for the Advanced Imaging Core are located in Criss I, II, and III.   Please inquire if you have specific questions about these instruments or about gaining access to them.  We are happy to provide training to users as well as to consult with users about their experimental and analysis needs involving microscopy and imaging.

Instrumentation:

Our primary imaging labs for the THC are in Criss I.  We manage three confocal laser scanning microscopes (one upright LSM 700, one inverted LSM 700, and one upright LSM 710) for users who wish to perform fixed cell or live cell imaging.  The inverted LSM 700 has a stage incubator and water immersion objectives, which are advantageous for live cell imaging.  The LSM 710 has a 34-channel detector and 7 laser lines (405, 458, 488, 514, 561, 594, 633).  All three of these microscopes offer DIC imaging capabilities and valuable options for objectives (10X, 20X, 40X, 63X, 100X).   In addition, the imaging core also manages a Zeiss PALM CombiSystem, which enables users to perform microdissection and micromanipulation of samples on standard glass slides within a single microscope.  The THC is also awaiting the arrival of an additional advanced microscope, a Zeiss LSM 980, at the end of 2022.

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Zeiss

*Directed by Mike Nichols and assisted by Anthony Stender

The Advanced Imaging team also manages several other instruments that are available to the THC through CU-IBIF, Creighton’s Integrated Biomedical Imaging Facility. The first is a Nikon epifluorescence microscope with a Hamamatsu Orca Flash camera that is designed for widefield imaging in either fluorescence or brightfield mode.  This microscope has a Sutter lamp which provides UV excitation and can rapidly switch between two excitation wavelengths, thereby enabling ratiometric fluorescence imaging of Calcium probes (for example, FURA).  The next is a Nikon confocal microscope with a Yokogawa spinning disk, a Hamamatsu Orca Flash camera, and a heated, incubated stage.  This confocal microscope is capable of fast full-frame imaging at speeds up to 1000 frames/second, and it can perform Z-stacks at rates 10 times faster than a point scanning confocal.  This microscope is equipped with Live Super-Resolution, thus enabling the high-speed imaging which is ideal for live cell experiments.  It is also an ideal choice for extended time-lapse imaging experiments.

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Nikon

Also available is a Leica SP8 microscope.  This instrument is an upright microscope with a large, heavy-duty stage that is suitable for live animal imaging and can support accessories for simultaneous electrophysiology measurements.  In addition to visible wavelength laser lines for confocal microscopy, it also 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 on this microscope. All of the confocal detectors are spectral detectors, enabling spectral imaging that can be used in combination with time-series and z-stacks.

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LEICA

A Total-Internal Reflection Fluorescence Microscope is also available through the Advanced Imaging facility.  This instrument was home-built using an Olympus microscope body and offers a through-the-prism beam path to reduce noise from scattered laser light.  This microscope currently incorporates three objective options (10X, 20X, 40X) and four laser lines (405, 488, 552, and 647 nm).  Images are captured on the TIRF with an Andor iXon EMCCD scientific camera.

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TIRF

Finally, the team also manages a recently acquired ImageXpress Micro 4 from Molecular Devices, which is equipped with an incubation chamber that allows for imaging of live cells on either microscope slides or well plates.  The ImageXpress enables users to conduct preprogrammed experiments over extended time periods at high-throughput while utilizing multi-color widefield imaging.

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ImageXpress

Software Packages Available:

We also provide a selection of analysis resources 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 in order to post-process microscope images, whether the data were saved as native Nikon or Leica files or in one of the more common file types such as TIF.  In addition, users can gain access to our Volocity 3D Image Analysis workstation, either in person or remotely for image analysis.  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.

This room is also the location of two Imaris workstations for analyzing microscope images, and a Zeiss Axioskop microscope for basic widefield imaging.

The Mass Spectrometry facility is located in room 303 of the Criss II building.

Current instrument includes a Q Exactive™ hybrid Quadrupole-Orbitrap™ mass spectrometer from Thermo Scientific™ (Fig. 4). This is a remarkable instrument with a resolving power of up to 140,000 FWHM and a mass accuracy of less than 1 ppm. For LC-MS experiments, the Q Exactive™ is coupled to a Vanquish™ Flex Binary UHPLC System comprised of a biocompatible, binary, high pressure gradient mixing pump, autosampler and heated column compartment. The Q Exactive™ is capable of LC-MS-MS experiments employing collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) of mass ions to obtain amino acid sequences and quantitative data from isobaric tagged proteolytic fragments.

Alternatively, an EASY-nLC™ 1200 instrument, capable of flow rates as low as 100 nL/min, is available for nLC-MS experiments.

Data analysis will be aided by the Proteome Discoverer 2.3, Tracefinder 4.1 and Compound Discoverer 3.0 software packages.

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Mass spectrometer
 
Fig. 4: Mass spectrometer

 

*Directed by David Smith and assisted by Molly McDevitt