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While there have been numerous efforts to minimize these factors during EM observations [6], [9] [13], none of the studies have fully resolved the lack of specific labeling methods or concerns of perturbations induced during the harsh sample preparation procedures required for electron microscopy.
However, interpretation of the results from these methods is limited, as the harsh sample preparation frequently leads to structural impairment of the biological specimens [ 2,5].
In addition, EM imaging can only be performed on fixed samples and often requires harsh sample preparation techniques that can disrupt native protein structures.
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Application of a gas chromatography/mass spectrometry (GC/MS) assay for MDA has indicated that the commonly used TBARS assay overestimates the actual MDA levels by more than 10-fold, possibly resulting from cross reactivity with other aldehydes and the harsh conditions used in sample preparation [ 26].
While improving the operational simplicity of sample preparation, the SLIDE also eliminates the need for dilutive and/or harsh washing steps during which analyte can be lost, the need for immiscible fluids, or complicated manifolds for collecting and purifying analyte from a complex sample.
Samples were prepared for digestion using the filter-assisted sample preparation method (https://doi.org/10.1038/nmeth.1322).1322
However, the conventional mild sample preparation is not necessarily preferable because the results in Figure 2 show that longer treatments and harsher conditions are beneficial to determine inner-matrix contamination and do not affect the bacteria.
Y.C. assisted in sample preparation.
L.S. participated in sample preparation.
J.M.K. developed sample preparation protocols.
Staff of the CARF assisted sample preparation.
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