Invited review
A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs

https://doi.org/10.1016/j.aanat.2008.11.001Get rights and content

Summary

Detecting, localising and counting ultrasmall particles and nanoparticles in sub- and supra-cellular compartments are of considerable current interest in basic and applied research in biomedicine, bioscience and environmental science. For particles with sufficient contrast (e.g. colloidal gold, ferritin, heavy metal-based nanoparticles), visualization requires the high resolutions achievable by transmission electron microscopy (TEM). Moreover, if particles can be counted, their spatial distributions can be subjected to statistical evaluation. Whatever the level of structural organisation, particle distributions can be compared between different compartments within a given structure (cell, tissue and organ) or between different sets of structures (in, say, control and experimental groups). Here, a portfolio of stereology-based methods for drawing such comparisons is presented. We recognise two main scenarios: (1) section surface localisation, in which particles, exemplified by antibody-conjugated colloidal gold particles or quantum dots, are distributed at the section surface during post-embedding immunolabelling, and (2) section volume localisation (or full section penetration), in which particles are contained within the cell or tissue prior to TEM fixation and embedding procedures. Whatever the study aim or hypothesis, the methods for quantifying particles rely on the same basic principles: (i) unbiased selection of specimens by multistage random sampling, (ii) unbiased estimation of particle number and compartment size using stereological test probes (points, lines, areas and volumes), and (iii) statistical testing of an appropriate null hypothesis. To compare different groups of cells or organs, a simple and efficient approach is to compare the observed distributions of raw particle counts by a combined contingency table and chi-squared analysis. Compartmental chi-squared values making substantial contributions to total chi-squared values help identify where the main differences between distributions reside. Distributions between compartments in, say, a given cell type, can be compared using a relative labelling index (RLI) or relative deposition index (RDI) combined with a chi-squared analysis to test whether or not particles preferentially locate in certain compartments. This approach is ideally suited to analysing particles located in volume-occupying compartments (organelles or tissue spaces) or surface-occupying compartments (membranes) and expected distributions can be generated by the stereological devices of point, intersection and particle counting. Labelling efficiencies (number of gold particles per antigen molecule) in immunocytochemical studies can be determined if suitable calibration methods (e.g. biochemical assays of golds per membrane surface or per cell) are available. In addition to relative quantification for between-group and between-compartment comparisons, stereological methods also permit absolute quantification, e.g. total volumes, surfaces and numbers of structures per cell. Here, the utility, limitations and recent applications of these methods are reviewed.

Introduction

Currently, a great deal of pure and applied research in science and medicine involves using high-resolution bio-imaging tools to examine the spatiotemporal distributions of nano-sized particles (otherwise known as nanoparticles) within cells, tissues and organs. Nanoparticles have at least one dimension less than 100 nm long and may be environmental or ambient (e.g. ultrafine particles, UFPs), biological (e.g. ferritin), manufactured (e.g. titanium dioxide particles, colloidal gold particles and block co-polymer micelles) or engineered (e.g. quantum dots QDs). In general, these particles serve a variety of purposes including vehicles for drug delivery, tumour targeting, toxicological testing, health-hazard screening, diagnosis and immunocytochemistry. Moreover, their properties influence their utility: for instance, QDs are crystalline probes which fluoresce when hit by laser or ultraviolet light and, by conjugation with antibodies or receptor ligands, can be used to target cells and tissues. Other particles, e.g. colloidal gold, ferritin and titanium dioxide particles, are heavy-metal based and provide good contrast for visualizing by TEM.

Nanoparticles seem to differ from larger particles (more than 200 nm in size) in the mechanisms by which they enter cells and translocate within them (Geiser et al., 2005). For example, both ambient UFPs and manufactured nanoparticles can enter different types of cell by non-endocytic and actin-independent mechanisms (Kreyling et al., 2002; Oberdörster et al., 2002, Oberdörster et al., 2005; Kapp et al., 2004; Geiser et al., 2005; Rothen-Rutishauser et al., 2006, Rothen-Rutishauser et al., 2007; Peters et al., 2006; Porter et al., 2006). Once internalised, they may elicit a number of biological responses that include generation of reactive oxygen species, enhanced expression of inflammatory cytokines and DNA strand breaks (Gonzalez-Flecha, 2004; Donaldson et al., 2005; Muller et al., 2005; Vinzents et al., 2005; Schins and Knaapen, 2007; Mühlfeld et al., 2008). The spatial and temporal distributions of such particles are of considerable interest in environmental and occupational medicine and pharmaceutical research. An important issue is whether their translocation within cells and tissues is restrained or, alternatively, essentially random (e.g. as the result of diffusion).

Often, the spatial distribution of particles in thin sections of cells and tissues is based on the qualitative inspection of TEM images or counts of numbers of particles observed in different compartments. More rigorous investigation requires better sampling and quantitative approaches in order to answer fundamental questions: for example, (1) Is the distribution of particles within a cell or tissue random or is there evidence of non-random (i.e. preferential) localization?, (2) Are there changes in observed distribution patterns between cell types or, over time, in the same cell type?, and (3) What is the number of particles per compartment or per cell?

Recently, efficient stereological methods for estimating the ultrastructural composition of cells and their subcellular compartments have been reviewed (Nyengaard and Gundersen, 2006; Ochs, 2006; Weibel et al., 2007). In addition, a resurgence of interest in immunoelectron microscopy, which is used to localize interesting molecules (often, but not exclusively, protein antigens) with suitable particulate markers (often colloidal gold conjugates), has seen the development of more robust sampling, stereological estimation and statistical procedures for evaluating the intracellular distributions of antigens labelled using colloidal gold particles (Lucocq, 1994; Griffiths et al., 2001; Mayhew et al., 2002, Mayhew et al., 2003, Mayhew et al., 2004; Lucocq et al., 2004; Mayhew and Desoye, 2004; Mayhew, 2007; Mayhew and Lucocq, 2008a, Mayhew and Lucocq, 2008b). The use of stereology in combination with molecular methods has also made it possible to determine immunolabelling efficiency for organelle or membrane compartments (Griffiths and Hoppeler, 1986; Lucocq, 1992, Lucocq, 1994). These new developments may also be applicable to quantifying nanoparticles in general. Though virus particles are usually larger than 100 nm in size, the methods might also be used to study the distributions of viruses across different parts of cell membrane surfaces.

Recent applications of stereology-based methods have expressed the packing density of particles within each cell compartment as a relative labelling index (RLI, applicable to immunogold particles) or relative deposition index (RDI, suitable for other nanoparticles). These indices express the degree to which a compartment exhibits preferential localisation in comparison to the theoretical situation of random localisation (Mayhew et al., 2002, Mayhew et al., 2003, Mayhew et al., 2004; Mayhew, 2007; Mühlfeld et al., 2007b, Mühlfeld et al., 2007a; Mayhew and Lucocq, 2008b). For drawing comparisons between particle distributions in different groups of cells, raw particle counts can be analysed and distributional shifts can be detected. A practical benefit of this approach is that its utility is not restricted by the nature of compartments (organelles or membranes or cytoskeletal filaments or tubules).

The aim of this article is to review the availability, utility, limitations and applications of stereological methods for quantifying particles in TEM thin sections. We begin by recognising two main scenarios, distinguished by the degree to which particles are dispersed throughout the section thickness:

  • 1.

    Section surface localization: In this scenario, particles (exemplified by colloidal gold particles or QDs conjugated to protein A or secondary antibodies), are distributed at the section surface during post-embedding immunolabelling (see Figure 1 and Griffiths, 1993; Lucocq, 1994).

  • 2.

    Section volume localization or full penetration: In contrast, this scenario (see Figure 2) is one in which particles are contained throughout the cell or tissue prior to TEM fixation, embedding and thin-sectioning procedures. This occurs when nanoparticles are internalised by intact cells in vitro or by the intact organism in vivo via alternative routes including mouth, skin, gills or upper respiratory tract (e.g. Geiser et al., 2005).

There is an intermediate scenario in which there is partial penetration of the section. Again, this may be encountered in certain immunogold-labelling studies (Griffiths and Hoppeler, 1986; Lucocq, 1994).

Section snippets

TEM thin sectioning

The aim of cutting semithin and ultrathin sections as a prelude to TEM is to reveal the internal structure of a specimen (cell, tissue and organ) at a sufficiently high level of resolution. In practice, optimal lateral resolution in TEM images is attained by cutting ultrathin (50–100 nm thick) sections. One consequence of this is that there is loss of dimensional information in the ultrastructural image. This occurs because the real 3-dimensional (3D) structures do not, in general, reveal their

Multistage random sampling

Whatever quantitation is to be pursued, the first requirement is that the specimens must be selected in a sufficiently rigorous fashion by some form of random sampling (Mayhew, 2008). Random sampling affords every part of the specimen the same chance of being selected and this is important regardless of the nature of the compartments (volume- or surface-occupying) being investigated (Mayhew, 2008). It can also allow every orientation of the specimen the same chance of being selected. Indeed,

Defining compartments

In most instances, the compartments of interests will be volumes or surfaces. For instance, in immunogold-labelling studies, intracellular compartments might comprise organelles (volume-occupying) or membranes (surface-occupying). The same categorisation of compartments may also obtain when studying the distribution of nanoparticles between tissue compartments within an organ.

In immunoEM studies, antigens of interest may pass between different types of compartment and, when the aim is to test

Relative quantification

In immunoEM, molecules of interest can be localised on sections with antibodies whose distributions are visualised by using gold particles (Figure 1). Post-embedding immunolabelling combined with resin sections seems to restrict particles to the cut surface of the section, but use of frozen sections (cryosections) may lead to deeper penetration (Bendayan, 1984; Griffiths and Hoppeler, 1986; Stierhof and Schwarz, 1989). Whilst most applications of QDs involve fluorescence immunolabelling (Arya

Concluding remarks

Compared to even the most advanced LM-based techniques, TEM stands out because it provides an “open view” at high resolution on cells and tissues by visualising the complete structural context in which nanoparticles are embedded. In short, TEM permits localization and not merely visualization. In addition, electron-dense nanoparticles are ideally suited for quantification at the EM level. The value of quantitative microscopy based on sound stereological principles has been recognized in many

Acknowledgements

TMM is grateful to the Medical Research Council and Anatomical Society of Great Britain & Ireland for continued research funding. The work of MO has been funded by the Deutsche Forschungsgemeinschaft (DFG 23/7-3 and 23/8-1) and the Swiss National Science Foundation (SNF 326000-113159, 3100A0-116417 and 316000-121390). The work of CM has been funded by the research program of the Faculty of Medicine, Georg–August–University Göttingen and the Swiss National Science Foundation (SNF 3100A0-118420).

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