Quantifying Golgi structure using EM: combining volume-SEM and stereology for higher throughput.

Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5-10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell-cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.

Nanoparticle suspensions enclosed in methylcellulose: a new approach for quantifying nanoparticles in transmission electron microscopy.

Nanoparticles are of increasing importance in biomedicine but quantification is problematic because current methods depend on indirect measurements at low resolution. Here we describe a new high-resolution method for measuring and quantifying nanoparticles in suspension. It involves premixing nanoparticles in a hydrophilic support medium (methylcellulose) before introducing heavy metal stains for visualization in small air-dried droplets by transmission electron microscopy (TEM). The use of methylcellulose avoids artifacts of conventional negative stain-TEM by (1) restricting interactions between the nanoparticles, (2) inhibiting binding to the specimen support films and (3) reducing compression after drying. Methylcellulose embedment provides effective electron imaging of liposomes, nanodiscs and viruses as well as comprehensive visualization of nanoparticle populations in droplets of known size. These qualities facilitate unbiased sampling, rapid size measurement and estimation of nanoparticle numbers by means of ratio counting using a colloidal gold calibrant. Specimen preparation and quantification take minutes and require a few microliters of sample using only basic laboratory equipment and a standard TEM.

Antibodies for immunolabeling by light and electron microscopy: not for the faint hearted.

Reliable antibodies represent crucial tools in the arsenal of the cell biologist and using them to localize antigens for immunocytochemistry is one of their most important applications. However, antibody-antigen interactions are much more complex and unpredictable than suggested by the old 'lock and key' analogy, and the goal of trying to prove that an antibody is specific is far more difficult than is generally appreciated. Here, we discuss the problems associated with the very complicated issue of trying to establish that an antibody (and the results obtained with it) is specific for the immunolabeling approaches used in light or electron microscopy. We discuss the increasing awareness that significant numbers of commercial antibodies are often not up to the quality required. We provide guidelines for choosing and testing antibodies in immuno-EM. Finally, we describe how quantitative EM methods can be used to identify reproducible patterns of antibody labeling and also extract specific labeling distributions.

Cutting a fine figure: On the use of thin sections in electron microscopy to quantify autophagy.

Over the last few years, two guideline articles have been published with recommendations for assessing autophagy. These articles contained advice on quantification of autophagy by electron microscopy and proposed using thin slices for quantifying autophagic structures. Here, we expand on what can and cannot be quantified using single 2D slices and give some suggestions for efficient and minimally biased approaches for quantifying this fascinating and important process. We recommend that the journal Autophagy follow other journals in demanding stringent random sampling design and application of unbiased design-based quantification when reviewing submitted manuscripts.

Quantitative assessment of specificity in immunoelectron microscopy.

In immunoelectron microscopy (immuno-EM) on ultrathin sections, gold particles are used for localization of molecular components of cells. These particles are countable, and quantitative methods have been established to estimate and evaluate the density and distribution of "raw" gold particle counts from a single uncontrolled labeling experiment. However, these raw counts are composed of two distinct elements: particles that are specific (specific labeling) and particles that are not (nonspecific labeling) for the target component. So far, approaches for assessment of specific labeling and for correction of raw gold particle counts to reveal specific labeling densities and distributions have not attracted much attention. Here, we discuss experimental strategies for determining specificity in immuno-EM, and we present methods for quantitative assessment of (1) the probability that an observed gold particle is specific for the target, (2) the density of specific labeling, and (3) the distribution of specific labeling over a series of compartments. These methods should be of general utility for researchers investigating the distribution of cellular components using on-section immunogold labeling.

A stereological approach for estimation of cellular immunogold labeling and its spatial distribution in oriented sections using the rotator.

Particulate gold labeling applied to ultrathin sections is a powerful approach for locating cellular proteins and lipids on thin sections of cellular structures and compartments. Effective quantitative methods now allow estimation of both density and distribution of gold labeling across aggregate organelles or compartment profiles. However, current methods generally use random sections of cells and tissues, and these do not readily present the information needed for spatial mapping of cellular quantities of gold label. Yet spatial mapping of gold particle labeling becomes important when cells are polarized or show internal organization or spatial shifts in protein/lipid localization. Here we have applied a stereological approach called the rotator to estimate cellular gold label and proportions of labeling over cellular compartments at specific locations related to a chosen cell axis or chosen cellular structures. This method could be used in cell biology for mapping cell components in studies of protein translocation, cell polarity, cell cycle stages, or component cell types in tissues.

Efficient quantitative morphological phenotyping of genetically altered organisms using stereology.

Genetically modified organisms present the challenge of quantifying structures and functions in organs, tissues and cells. Morphological investigation is greatly facilitated by taking sections in MRI, CAT scanning, histological preparations or EM, and powerful unbiased quantitative tools called stereology can use these sections in a sampling based approach to measure volume, number surface and length. Stereological tools have become methods of choice in the fields of neurobiology, nephrology and cell biology and allow accurate unbiased description of intact organs, tissues, cells and organelles. Stereology has yet to be applied widely in the field of transgenics. Here I provide an overview of stereological methods and explain how they represent a powerful addition to the transgenic biologists armoury of techniques.

A rapid method for assessing the distribution of gold labeling on thin sections.

Particulate gold labeling on ultrathin sections is in widespread use for antigen localization at the EM level. To extend the usefulness of gold labeling technology, we are evaluating different methods for sampling and estimating quantities of gold labeling. Here we present a simple, rapid, and unbiased method for assessing the relative pool sizes of immunogold labeling distributed over different cell compartments. The method uses a sampling approach developed for stereology in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. From these readouts, gold particles are counted and assigned to identifiable cell structures to construct a gold labeling frequency distribution of those labeled compartments. Here we use ultrathin cryosections labeled for a range of different proteins and for a signaling lipid. We show by scanning labeled sections at the electron microscope that counting 100-200 particles on each of two grids is sufficient to obtain a reproducible and rapid assessment of the pattern of labeling proportions over 10-16 compartments. If more precise estimates of labeling proportions over individual compartments are required (e.g., to achieve coefficients of error of 10-20%), then 100-200 particles need to be counted over each compartment of interest.