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:

Image	David Z. He, MD, PhD Professor of Neuroscience
Image	Michael Nichols, PhD Professor of Physics
Image	D. David. Smith, PhD Professor of Biochemistry

The AVT Core offers a broad range of services:

Electrophysiology (directed by David He)
Molecular Biology (directed by David He)
Imaging (directed by Michael Nichols)
Mass Spectrometry (directed by David Smith)

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.

Electrophysiology Services

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:

two complete sets of TDT RZ6 for measuring auditory and vestibular function (evoked potentials and otoacoustic emissions) from rodents.
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.

Fig. 1: Setup for auditory function examination

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

Molecular Biology Services

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.

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

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

Advanced Imaging Services

Instrumentation is currently located in room 326 of the Criss II building and room 407 of the Criss I building at Creighton University.

The current instrumentation includes:

the Leica TCS SP8 multiphoton laser scanning confocal microscope with 5 visible laser lines and a Spectra-Physics Mai Tai DeepSee tunable ultrafast laser for multiphoton excitation (Fig. 3A).
Nikon TI-E Inverted Yokogawa spinning disk confocal microscope capable of operating at very low light levels and 100 frames/s full field of view (Fig. 3B). The high sensitivity of this microscope is due to Andor iXon EMCCD camera. This microscope also has a temperature-controlled hydrated stage incubator for long-term observations of living cells over multiple days.
One LSM 700 and one LSM 710 upright Laser scanning confocal microscopes in a dedicated imaging lab on the 4th floor of Criss I (Fig. 3C,D).

*Directed by Mike Nichols

Mass Spectrometry Services

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.