The invention of a scanning electron microscopy (SEM) pushed the imaging methods and allowed for the observation of cell details with a high resolution. Currently, SEM appears as an extremely useful tool to analyse the morphology of biological samples. The aim of this paper is to provide a set of guidelines for using SEM to analyse morphology of prokaryotic and eukaryotic cells, taking as model cases Escherichia coli bacteria and B-35 rat neuroblastoma cells. Herein, we discuss the necessity of a careful sample preparation and provide an optimised protocol that allows to observe the details of cell ultrastructure (≥ 50 nm) with a minimum processing effort. Highlighting the versatility of morphometric descriptors, we present the most informative parameters and couple them with molecular processes. In this way, we indicate the wide range of information that can be collected through SEM imaging of biological materials that makes SEM a convenient screening method to detect cell pathology.

Assessing fish liver status is common in aquaculture nutrition assays. This often implies determining hepatocytes profile areas in routine thin (5-7 μm) histological sections. However, there are theoretical problems using planar morphometry in thin sections: inherent sampling cells biases, too small numbers of sampled cells, under/overestimation of size, measuring size as areas when cells are three-dimensional (3D) entities. The gold standard for assessing/validate cell size is stereology using thick sections (20-40 μm). Here, we estimated the volume of hepatocytes and their nuclei by the nucleator and optical disector stereological probes (in thick sections), and, innovatively, in thin sections too (using single-section disectors). The liver of common carp eating feed containing either low or high level of lipids was targeted. Results were compared with prior profile areas from planar morphometry using thin sections, and with profile areas estimated here with the two-dimensional (2D) nucleator. Ratios between nucleus and cell/cytoplasm (N/C) areas and volumes were calculated and compared. There was high positive correlation between volumes in thin and thick sections (r = .85 to .89; p < .001), empirically validating the single-section disector. Strong correlations existed between profile-derived versus 2D-nucleator areas (r = .74 to .83; p < .001). There was systematic underestimation of cells and nucleus size using planar morphometry. The N/C ratios derived from the 2D-nucleator data were higher than those from planar morphometry. Despite theoretical premises for using simple planar morphometry in thin sections are flawed, our results support that such morphometry on carp/fish hepatocytes may offer some valid biological conclusions. Anyway, we advanced guidelines for implementing proper methods.

The potential for developmental neurotoxicity (DNT) of environmental chemicals may be evaluated using specific test guidelines from the US Environmental Protection Agency or the Organisation for Economic Cooperation and Development (OECD). These guidelines generate neurobehavioral, neuropathological, and morphometric data that are evaluated by regulatory agencies globally. Data from these DNT guideline studies, or the more recent OECD extended one-generation reproductive toxicity guideline, play a pivotal role in children\'s health risk assessment in different world areas. Data from the same study may be interpreted differently by regulatory authorities in different countries resulting in inconsistent evaluations that may lead to inconsistencies in risk assessment decisions internationally, resulting in regional differences in public health protection or in commercial trade barriers. These issues of data interpretation and reporting are also relevant to juvenile and pre-postnatal studies conducted more routinely for pharmaceuticals and veterinary medicines. There is a need for development of recommendations geared toward the operational needs of the regulatory scientific reviewers who apply these studies in risk assessments, as well as the scientists who generate DNT data sets. The workshops summarized here draw upon the experience of the authors representing government, industry, contract research organizations, and academia to discuss the scientific issues that have emerged from diverse regulatory evaluations. Although various regulatory bodies have different risk management decisions and labeling requirements that are difficult to harmonize, the workshops provided an opportunity to work toward more harmonized scientific approaches for evaluating DNT data within the context of different regulatory frameworks. Five speakers and their coauthors with neurotoxicology, neuropathology, and regulatory toxicology expertise discussed issues of variability, data reporting and analysis, and expectations in DNT data that are encountered by regulatory authorities. In addition, principles for harmonized evaluation of data were suggested using guideline DNT data as case studies.