Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics

Abstract

Biological systems span multiple levels of structural organisation from the macroscopic, via the microscopic, to the nanoscale. Therefore, comprehensive investigation of systems biology requires application of imaging modalities that reveal structure at multiple resolution scales. Nanomorphomics is the part of morphomics devoted to the systematic study of functional morphology at the nanoscale and an important element of its achievement is the combination of immunolabelling and transmission electron microscopy (TEM). The ultimate goal of quantitative immunocytochemistry is to estimate numbers of target molecules (usually peptides, proteins or protein complexes) in biological systems and to map their spatial distributions within them. Immunogold cytochemistry utilises target-specific affinity markers (primary antibodies) and visualisation aids (e.g., colloidal gold particles or silver-enhanced nanogold particles) to detect and localise target molecules at high resolution in intact cells and tissues. In the case of post-embedding labelling of ultrathin sections for TEM, targets are localised as a countable digital readout by using colloidal gold particles. The readout comprises a spatial distribution of gold particles across the section and within the context of biological ultrastructure. The observed distribution across structural compartments (whether volume- or surface-occupying) represents both specific and non-specific labelling; an assessment by eye alone as to whether the distribution is random or non-random is not always possible. This review presents a coherent set of quantitative methods for testing whether target molecules exhibit preferential and specific labelling of compartments and for mapping the same targets in two or more groups of cells as their TEM immunogold-labelling patterns alter after experimental manipulation. The set also includes methods for quantifying colocalisation in multiple-labelling experiments and mapping absolute numbers of colloidal gold particles across compartments at specific positions within cells having a point-like inclusion (e.g., centrosome, nucleolus) and a definable vertical axis. Although developed for quantifying colloidal gold particles, the same methods can in principle be used to quantify other electron-dense punctate nanoparticles, including quantum dots.

Morphomics: An integral part of systems biology of the human placenta

“…Examples are provided by fluorophores, ferritin, lectins and colloidal gold (in immunoelectron microscopy), silver grains (in autoradiography) and electron-dense reaction products (in enzyme histochemistry). In these situations, counting affords a valuable way of quantifying specific labelling intensities providing there is a countable digital readout [116].…”

“…numbers of colloidal gold particles, other metal nanoparticles or quantum dots. With a coherent set of principles for quantifying immunolabelling within cells, stereological sampling and estimation procedures can be utilized to estimate local intensities of labelling/co-labelling and labelling distributions across different compartments [116].…”

Quantitative techniques for imaging cells and tissues

“…Heterogeneity in refinement of the quantitative methods is reflected in the various contributions of this issue of Cell & Tissue Research . For example, as elucidated in the contributions of Geuna and Herrera-Rincon (2015), Kubinova and Janáček (2015), and Mayhew (2015), proper counting of particles in sectioned tissues has been worked out over several decades, culminating with the design of the tools of stereology (Haug, 1986; Coggeshall and Lekan, 1996; Evans et al, 2004), but it took nearly a century and considerable effort by a large number of investigators to perfect the design-based approaches after the first cells were counted under the light microscope in the late 1800s (Blinkov and Glezer, 1968; Haug, 1986). Currently, the key stereological method papers enjoy exceptionally high citation rates, although the actual usage still lags behind, as revealed in the contribution by Geuna and Herrera-Rincon (2015).…”

“…The advantages and disadvantages of this simple, yet elegant approach are compared with stereology, including practical hands-on issues. We complete the “particle counting tool kit” with the contribution by Mayhew (2015). He explains how stereological methods are used for tissues at the ultrastructural level, again including several hands-on, practical examples that guide the reader through each of the steps.…”

Neurostereology protocol for unbiased quantification of neuronal injury and neurodegeneration

“…Modern design-based stereology has been an important and efficient tool in many applications of optical and confocal microscopy. There is a multitude of uses for stereology in many biomedical fields including histology, bone and neuroanatomy to accurately quantify the number of cells, the length of fibers, and the area and volume of biological structures or regions (Mayhew, 2014 ; Bronoosh et al, 2015 ).…”