Sample Preparation

Miller, James M.

doi:10.1002/9780471980582.ch14

Cited by 1 publication

(1 citation statement)

References 62 publications

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“…The most widely used heavy metals are based on osmium, which has a high affinity to lipids; enabling the staining of the outer membrane and the intracellular organelles. Other metals are uranyl acetate, which binds to and stabilizes various cellular structures (DNA, RNA, and proteins) (Hirsch and Fedorko, 1968;Williams et al, 2008;Tapia et al, 2012;Ruwin, 2013), or combining the heavy metal Osmium with various salts (for more details, see the Methods section) (Culling et al, 1985). Finally, staining can be achieved by the osmium tetroxide-thiocarbohydrazide (OTOTO) combination (Tapia et al, 2012), which uses thiocarbohydrazide as a mediator to create binding between two osmium molecules to create longer osmium molecular chains (Friedman and Ellisman, 1981;Lešer et al, 2009;Rolls, 2012;Atkins et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

SEM/FIB Imaging for Studying Neural Interfaces

Henn

Atkins

Markus

et al. 2019

Developmental Neurobiology

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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.

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