"Rinse and trickle": a protocol for TEM preparation and investigation of inorganic fibers from biological material

Our plain-English summary of "Rinse and trickle": a protocol for TEM preparation and investigation of inorganic fibers from biological material, by R. Vigliaturo, S. Capella, C. Rinaudo, et al (Inhalation Toxicology, 2016).

The study

• Authors: R. Vigliaturo, S. Capella, C. Rinaudo, E. Belluso
• Journal: Inhalation Toxicology
• Published: 2016;28(8):357-63
• Cited by: 4 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

The purpose of this work is to define a sample preparation protocol that allows inorganic fibers and particulate matter extracted from different biological samples to be characterized morphologically, crystallographically and chemically by transmission electron microscopy-energy dispersive spectroscopy (TEM-EDS). The method does not damage or create artifacts through chemical attacks of the target material. A fairly rapid specimen preparation is applied with the aim of performing as few steps as possible to transfer the withdrawn inorganic matter onto the TEM grid. The biological sample is previously digested chemically by NaClO. The salt is then removed through a series of centrifugation and rinse cycles in deionized water, thus drastically reducing the digestive power of the NaClO and concentrating the fibers for TEM analysis. The concept of equivalent hydrodynamic diameter is introduced to calculate the settling velocity during the centrifugation cycles. This technique is applicable to lung tissues and can be extended to a wide range of organic materials. The procedure does not appear to cause morphological damage to the fibers or modify their chemistry or degree of crystallinity. The extrapolated data can be used in interdisciplinary studies to understand the pathological effects caused by inorganic materials.

In plain English

This laboratory methods study set out to define a way of preparing biological tissue so that inorganic fibers in it could be examined by transmission electron microscopy (TEM) without the preparation damaging them. The authors used autopsy lung tissue of two kinds, one with a low fiber load and one with a high fiber load, with four samples of each. The tissue was dissolved in sodium hypochlorite (bleach), and the fibers were then concentrated and rinsed by repeated centrifugation. Three versions of the method were compared. With the basic "Rinse and Trickle" version, no fiber or bundle seen was totally free of organic matter, and chrysotile bundles were found trapped in organic material. Adding two sonic baths ("Sparkle Rinse and Trickle") left hardly any organic residue on the fibers. Adding hydrogen peroxide caused almost total loss of material and damaged the microscope grid. The authors concluded that the sonic-bath version is the better approach. The study did not aim to count fibers and gives no information about the people the tissue came from.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory methods-development study comparing three versions of a sample preparation protocol for transmission electron microscopy with energy dispersive spectroscopy (TEM-EDS) and electron diffraction (SAED)
• Who was studied: Lung tissue obtained from autopsies, of two kinds: tissue with a low fiber load (M_I) and tissue with a high fiber load (C_I, used as a control). Four samples of each type were used, each 0.1 g, and procedures were done in duplicate. The article does not say where or when the tissue was collected or how many people it came from.
• Exposure: Not an exposure study. The article does not describe the asbestos exposure of the people the tissue came from. What was tested was laboratory treatment of the tissue. The tissue was digested in 13% sodium hypochlorite (NaClO) at 60 degrees, then centrifuged and rinsed. Three versions were compared: plain Rinse and Trickle (R&T), the same with two sonication steps (SR&T), and the same with hydrogen peroxide added (HR&T). Fibers observed included chrysotile, tremolite and other amphibole fibers.
• Main results:
  • Digesting tissue in 20 mL of NaClO was more efficient than digesting it in 10 mL.
  • With the basic R&T approach, no fiber or bundle seen in the C_Id and M_Id samples was totally free of organic matter.
  • With the R&T approach, the low-load sample M_Id contained long chrysotile bundles trapped in organic material.
  • With the SR&T approach (two sonic baths added), hardly any organic residue was found on the fibers, and no fibers or bundles were incorporated into organic clusters.
  • The HR&T approach (hydrogen peroxide added) could not be checked by TEM for any sample. Almost all of the material was lost, and the peroxide attacked the carbon layer of the TEM grid.
  • In both the C_Id and M_Id samples, magnesium and silicon were regularly detected by EDS in the area surrounding the fibers.
  • Chemical analyses of clean areas of chrysotile fibers showed no chlorine or sodium. The authors used this as a check that the fibers had been well rinsed.
• The authors' conclusion: The authors concluded that the SR&T approach clearly increases the quality of the specimen for TEM study and is the better approach for investigating inorganic fibers in biological tissue. They also stated that the procedure does not appear to damage the fibers' shape or change their chemistry or crystallinity.
• Limitations the authors state:
  • The authors state that the study's aim was not to measure the amounts of fiber for statistical purposes, so it gives no fiber counts or concentrations.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Chrysotile.

Medical terms in this study, explained in our Lexicon: Transmission Electron Microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), TEM/EDS.

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