Advanced Imaging Core

National Institute On Deafness And Other Communication Disorders

Investigators

Linked publications & trials

Abstract

The AIC team consists of Director Dr. Dennis C. Winkler and Dr. Julia Sokolova. This core is dedicated primarily to collaborating with and training scientists in NIDCD in studies utilizing transmission electron microscopy (TEM), soon to include focused ion-beam scanning electron microscopy (FIB-SEM) as well. The AIC also collaborates with and trains researchers from other NIH institutes. In addition, Dr. Winkler has reviewed manuscripts, grants, and various documents for NIDCD PIs, served as a reviewer for submitted journal manuscripts, written letters of recommendation, and served on several NIH committees. The electron microscopes available to the core and its users are a JEOL JEM-2100 200kV TEM and a Thermo Fisher/FEI Titan Halo 300 kV TEM with STEM and phase plate capabilities. New to the core is a TESCAN Amber FIB-SEM. With training recently completed, this scope will soon be available for use. Dr. Winkler continues to work with NIHs facility management team (ORF, DFOM, building engineers, and contractors) on lingering issues related to the Halo TEM room renovation and the NIH house chilled water systems. He works with and trains personnel in cryo-EM/ET related work as well as in the use of electron microscopes and ancillary electron microscopy equipment. He remains available for consultation regarding the former NIAMS lab space in Building 50 for others at NIH, as was the case for the recent flood in that basement area. The flood has brought about the demise of the JEOL 2200 electron microscope in the basement of Building 50, ending Dr. Winklers de facto management of the instrument for the occasional use of this scope by the AIC and NIAMS for cryo-EM work and training. Decommissioning/removal of this TEM instrument is imminent. Dr. Sokolova very recently started working with the core full time, which is an increase from the prior 75%. Much of her work is still associated with the Friedman lab but includes work with other NIDCD labs as well. Dr. Sokolovas work involves study of auditory structures using TEM (and soon FIB-SEM). Dr. Sokolova is also working on making high pressure freezing and subsequent freeze-substitution a regular sample preparation offering. This technique will be used as a substitution for chemical fixation and plunge freezing as it is highly recommended for better preservation of the native state of cellular structures and, particularly, antigenic sites for immune EM. NIDCD: Laboratory of Molecular Genetics (Dr. Thomas Friedman): Dr. Sokolova has continued the collaboration with the Friedman Lab, working closely with Dr. Inna Belyantseva on TEM (and soon FIB-SEM) projects involving the organ of Corti, focusing on the fine structure of inner and outer hair cells in wildtype and knockout (by CRISPR/CAS9 technology) mice as well as on immunolocalization of proteins of interest. She is also working with Dr. Takushi Miyoshi on a project examining methods to detect abnormal functions of deafness gene variants in hair cell stereocilia, which began as a cryoEM project with the assistance of Dr. Winkler. Additional assistance to the lab included arranging group vendor-training for the TESCAN Amber FIB-SEM, and Dragonfly Image Processing Software. Section on Sensory Cell Development & Function (Dr. Katie Kindt): Dr. Sokolova continues to work with the Kindt Lab studying zebrafish hair cells. Her contributions include specimen preparation, ultramicrotomy, staining, and TEM imaging. Auditory Development & Restoration Program (Dr. Michael Hoa): Dr. Sokolova has been working with Dr. Rafal Olszewski and Dr. Hoa on a project involving preparations for sectioning of inner ear tissue on the ultramicrotome for LM studies as well as a project on visualization of changes associated with endolymphatic hydrops in the Slc26a4-insufficiency mouse model. Section on Structural Cell Biology (Dr. Bechara Kachar): Dr. Winkler continued assisting Dr. Felipe Montecinos and Mr. Dylan Murray on TEM use until their departure from the NIDCD. Dr. Winkler worked with new lab personnel, Drs. Wonchul Shin, Shiqiong Hu, Amir Tavakoli Targhi and Mr. Tirone Derek Johnson, on cryo-EM techniques. Additional assistance to the lab included aid in using the Thermo Fisher/FEI Titan Halo TEM, as well as arranging group vendor-training for the Thermo Fisher/FEI Titan Halo TEM, the TESCAN Amber FIB-SEM, and Dragonfly Image Processing Software. Other NIH institutes: NINDS/Laboratory of Functional & Molecular Imaging: Dr. Simone Mastrogiacomos project and training with Dr. Winkler has wound down with Dr. Mastrogiacomo taking a new position outside of NIH this year. NEI/Laboratory of Retinal Cell & Molecular Biology and Molecular Structure & Functional Genomics Section: Dr. Winkler has been working with and training Drs. Vijay Camasamudram and Vatsala Sagar on structural biology cryo-EM projects looking into the structure of proteins of interest. With the retirement of Dr. Camasamudram, Dr. Sheetal Uppal has taken over his project and is revamping the protein purification. Dr. Sagars project has wound down since her promotion to Unit Chief of NEIs Structural Biology Unit. NEI continues interest in using the AIC TEMs and discussions are ongoing. As part of this, Dr. Winkler has started working with and training Dr. Noor White on the AICs JEOL 2100. NIAMS/Protein Expression Lab: Dr. Felipe Montecinos joined Drs. Elif Eren and Norman Watts in the Protein Expression Lab headed by Dr. Paul Wingfield. With the demise of the JEOL 2200 in Building 50, Dr. Montecinos has been able to start using the AICs JEOL 2100 for his cryo-EM work as he has experience using this scope as a former NIDCD lab member.