Advanced Imaging Core

National Institute On Deafness And Other Communication Disorders

Investigators

Linked publications, trials & patents

Abstract

Drs. Ronald S. Petralia and Ya-Xian Wang, who ran the NIDCD Advanced Imaging Core facility since its inception in October 2011, retired in December 2021. As of January 2022, Dr. Dennis C. Winkler runs the core with Dr. Julia Sokolova. This core is dedicated primarily to collaborating with and training scientists in NIDCD in studies utilizing transmission electron microscopy (TEM). The AIC also collaborates with and trains researchers from other NIH institutes. In addition, Dr. Petralia and Dr. Winkler have reviewed manuscripts, grants and various documents for NIDCD PIs, written letters of recommendation, and served on several NIH committees. With the 2019 turbo pump upgrade to the JEOL JEM-2100 TEM it is now possible to look at frozen hydrated biological specimens, and SerialEM automation scripts were installed to help facilitate the cryo-EM workflow on this microscope. Additionally, the installation of our second electron microscope, a Thermo Fisher/FEI Titan Halo 300 kV TEM, was completed this year. The scope will be dedicated largely to cryo-EM but has other features such as STEM and phase plate capabilities. Dr. Winkler oversaw the installation of the Halo and the scope is now available for use, although there are still some lingering issues with the room renovation and the NIH house chilled water systems. Dr. Winkler works with lab members within NIDCD and the NIH community, training them in EM and cryo-EM. He remains available for consultation regarding the transitioning of the former NIAMS lab space in Building 50 to others at NIH and continues to be the de facto manager for the JEOL 2200 electron microscope in Building 50 as NIDCD occasionally uses this scope for cryo-EM work and training. The best estimate for this scopes decommissioning/removal is sometime in FY 2023. Dr. Sokolova joined the core at the beginning of 2021 and is now working with the core 75% of her time; she works directly for the Friedman lab during the other 25%. Dr. Sokolova has a strong background in TEM of microorganisms. Both the work for the core and for Dr. Friedmans lab involve similar multiple methods in TEM, and Dr. Petralia and Dr. Wang actively trained Dr. Sokolova on TEM methods as used for the study of auditory structures until their retirement at the end of December 2021. Dr. Sokolova is also working on implementing high pressure freezing and subsequent freeze-substitution as a sample preparation technique with a recently acquired, previously owned Leica EM PACT 2 High Pressure Freezer. 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): Collaboration with Dr. Friedman and Dr. Inna Belyantseva is ongoing. Dr. Petralia and Dr. Wang assisted Dr. Belyantseva with tissue preparation, e.g., freeze substitution and ultramicrotomy. Dr. Sokolova now works with the Friedman lab for 25% of her time and continues to work with Dr. Belyantseva on TEM projects involving the Organ of Corti focusing on the fine structure of hair cells in wildtype and knockout (by CRISPR/CAS9 technology) mice as well as on immunolocalization of proteins of interest. The cryo-EM collaboration with Drs. Friedman and Takushi Miyoshi is ongoing. The project examines methods to detect abnormal functions of deafness gene variants in hair cell stereocilia Molecular Biology & Genetics Section (Dr. Isabelle Roux): Dr. Petralia and Dr. Wang had a major study on the structure and function of the endolymphatic sac, with preliminary work done in March 2020; this was halted abruptly due to the COVID-19 pandemic. This has continued through the first part of FY22. Section on Sensory Cell Development & Function (Dr. Katie Kindt): A new study on zebrafish hair cells involved Drs. Sokolova, Petralia, and Wang. Dr. Petralia and Dr. Wang trained Dr. Sokolova in the study of TEM on these zebrafish. Dr. Wang did part of the ultramicrotomy, and Dr. Petralia did part of the grid staining for this study and initially helped with image acquisition, while Dr. Sokolova has been doing the major part including 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): We provided training for Dr. Ranjan Sengupta, Dr. Felipe Montecinos and Mr. Dylan Murray on TEM use, traditional TEM and cryo-EM sample preparation methods, and cryo-EM techniques. Additional assistance to the lab includes aid in using the newly installed Thermo Fisher/FEI Titan Halo TEM and review of manuscripts. Other NIH institutes: NINDS/Basic Neurosciences Program and Laboratory of Functional & Molecular Imaging: Drs. Petralia and Wang had a previous study with Dr. Wei Lus lab on Neuroligin 2 that is in preparation for submission (Han et al.; delayed by the COVID-19 pandemic). For the past year Dr. Winkler has worked with Dr. Simone Mastrogiacomo, training him on negative stain and cryo-EM sample preparation, concentrating on a structural biology study of a protein of interest. NICHD/Section on Molecular Neurophysiology & Biophysics: Lab of Drs. Lin Lin and Dax Hoffman: Dr. Petralia and Dr. Wang had a study examining the association of Cav2.3 and Kv4.2 channels in the journal, Cell Reports (Murphy et al.). They have a paper in review that is a follow up study on the novel structure associated with aging dementia in DPP6-KO mice that they published in 2020. In addition, Dr. Petralia and Dr. Wang have 2 earlier studies (prior to FY2022) on zebrafish neuromast development, with the former lab of Dr. Catherine Drerup; a manuscript for one of these is now in review. NIA/Human Neuroscience Section: Dr. Petralia, Dr. Wang and Dr. Sokolova have been working with Drs. Pamela Yao and Dimitrios Kapogiannis on a small project on immunogold labeling of mitochondrial extracellular vesicles; this was taken over by Dr. Sokolova after Dr. Petralia and Dr. Wang retired in December 2021. NIMH/Section on Synapse Development Plasticity: Dr. Petralia and Dr. Wang have a total of 3 papers published in FY22 with the lab of Dr. Zheng Li based on the research that they completed before retiring in December 2021. One of these is a TEM methods paper that is based on a study on mitophagy in the amygdala and hippocampus associated with social anxiety that they recently published in the journal, Neuron (Duan et al., 2021). NHGRI/Molecular Neurogenetics Section: In the past few years, Dr. Petralia and Dr. Wang have accumulated data on studies with Drs. Nahid Tayebi and Ellen Sidransky on Parkinson and Gaucher diseases; they left these data with them, but it is not clear if any of it will be published. NEI/Molecular Mechanisms Section and Molecular Structure & Functional Genomics Section: For a little over a year, Dr. Winkler has been working with Drs. Vijay Camasamudram and Vatsala Sagar on structural biology cryo-EM projects looking into the structure of proteins of interest. Additionally, both are being trained on TEM/Cryo-EM sample prep techniques and TEM use. NEI also has expressed interest in using the AIC TEMs and discussions are ongoing. NIAMS/Protein Expression Lab: We continue discussions with Drs. Elif Eren and Norman Watts, offering them access to our electron microscopes for collaborations as their access to other electron micr