Tags

Type your tag names separated by a space and hit enter

SEM/FIB Imaging for Studying Neural Interfaces.

Abstract

Tissue and neural engineering for various regenerative therapies are rapidly growing fields. Of major interest is studying the complex interface between cells and various 3D structures by scanning electron microscopy with focused ion beam. Notwithstanding its unrivaled resolution, the optimal fixation, dehydration, and staining protocols of the samples while preserving the complex cell interface in its natural form, are highly challenging. The aim of this work was to compare and optimize staining and sample drying procedures in order to preserve the cells in their "life-like state" for studying the cell interface with either 3D well-like structures or gold-coated mushroom-shaped electrodes. The process involved chemical fixation using a combination of glutaraldehyde and formaldehyde, followed by gentle drying techniques in which we compared four methods: (critical point drying, hexamethyldisiloxane, repeats of osmium tetroxide-thiocarbohydrazide [OTOTO], and resin) in order to determine the method that best preserves the cell and cell interface morphology. Finally, to visualize the intracellular organelles and membrane, we compared the efficacy of four staining techniques: osmium tetroxide, osmium tetroxide and salts, osmium and uranyl acetate, and OTOTO. Experiments were performed on embryonic stem cell-derived photoreceptor precursors, neural cells, and a human retinal pigment epithelial cell line, which revealed that the optimal processing combination was resin drying and OTOTO staining, as manifested by preservation of cell morphology, the lowest percentage of cellular protrusion breakage as well as a high-quality image. The obtained results pave the way for better understanding the cell interface with various structures for enhancing various biomedical applications.

Authors+Show Affiliations

Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel. Bar-Ilan Institute for Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, Israel.Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel. Bar-Ilan Institute for Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, Israel.Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel. Bar-Ilan Institute for Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, Israel.Bar-Ilan Institute for Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, Israel. Department of Chemistry, Bar-Ilan Institute for Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, Israel.Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel. Faculty of Life Science, School of Optometry and Vision Science, Bar-Ilan University, Ramat Gan, Israel.Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel. Bar-Ilan Institute for Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, Israel. Faculty of Life Science, School of Optometry and Vision Science, Bar-Ilan University, Ramat Gan, Israel.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31228876

Citation

Henn, Itai, et al. "SEM/FIB Imaging for Studying Neural Interfaces." Developmental Neurobiology, 2019.
Henn I, Atkins A, Markus A, et al. SEM/FIB Imaging for Studying Neural Interfaces. Dev Neurobiol. 2019.
Henn, I., Atkins, A., Markus, A., Shpun, G., Barad, H. N., Farah, N., & Mandel, Y. (2019). SEM/FIB Imaging for Studying Neural Interfaces. Developmental Neurobiology, doi:10.1002/dneu.22707.
Henn I, et al. SEM/FIB Imaging for Studying Neural Interfaces. Dev Neurobiol. 2019 Jun 22; PubMed PMID: 31228876.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - SEM/FIB Imaging for Studying Neural Interfaces. AU - Henn,Itai, AU - Atkins,Ayelet, AU - Markus,Amos, AU - Shpun,Gal, AU - Barad,Hannah-Noa, AU - Farah,Nairouz, AU - Mandel,Yossi, Y1 - 2019/06/22/ PY - 2019/01/30/received PY - 2019/05/11/revised PY - 2019/06/15/accepted PY - 2019/6/23/pubmed PY - 2019/6/23/medline PY - 2019/6/23/entrez KW - 3D scaffolds KW - SEM/FIB KW - cellular membrane KW - electron microscope KW - fixation KW - interface KW - staining JF - Developmental neurobiology JO - Dev Neurobiol N2 - Tissue and neural engineering for various regenerative therapies are rapidly growing fields. Of major interest is studying the complex interface between cells and various 3D structures by scanning electron microscopy with focused ion beam. Notwithstanding its unrivaled resolution, the optimal fixation, dehydration, and staining protocols of the samples while preserving the complex cell interface in its natural form, are highly challenging. The aim of this work was to compare and optimize staining and sample drying procedures in order to preserve the cells in their "life-like state" for studying the cell interface with either 3D well-like structures or gold-coated mushroom-shaped electrodes. The process involved chemical fixation using a combination of glutaraldehyde and formaldehyde, followed by gentle drying techniques in which we compared four methods: (critical point drying, hexamethyldisiloxane, repeats of osmium tetroxide-thiocarbohydrazide [OTOTO], and resin) in order to determine the method that best preserves the cell and cell interface morphology. Finally, to visualize the intracellular organelles and membrane, we compared the efficacy of four staining techniques: osmium tetroxide, osmium tetroxide and salts, osmium and uranyl acetate, and OTOTO. Experiments were performed on embryonic stem cell-derived photoreceptor precursors, neural cells, and a human retinal pigment epithelial cell line, which revealed that the optimal processing combination was resin drying and OTOTO staining, as manifested by preservation of cell morphology, the lowest percentage of cellular protrusion breakage as well as a high-quality image. The obtained results pave the way for better understanding the cell interface with various structures for enhancing various biomedical applications. SN - 1932-846X UR - https://www.unboundmedicine.com/medline/citation/31228876/SEM/FIB_Imaging_for_Studying_Neural_Interfaces L2 - https://doi.org/10.1002/dneu.22707 DB - PRIME DP - Unbound Medicine ER -