Skip to main content

Advertisement

SpringerLink
Log in

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

  • Review
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Amiry-Moghaddam M, Ottersen OP (2013) Immunogold cytochemistry in neuroscience. Nat Neurosci 16:798–804

    Article  CAS  PubMed  Google Scholar 

  • Anderson JB, Carol AA, Brown VK, Anderson LE (2003) A quantitative method for assessing co-localization in immunolabeled thin section electron micrographs. J Struct Biol 143:95–106

    Article  CAS  PubMed  Google Scholar 

  • Baddeley AJ, Gundersen HJG, Cruz-Orive LM (1986) Estimation of surface area from vertical sections. J Microsc 142:259–276

    Article  CAS  PubMed  Google Scholar 

  • Baudoin J-P, Jerome WG, Kübel C, Jonge N de (2013) Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography. PLoS One 8:e55022

  • Bendayan M (1982) Double immunocytochemical labelling applying the protein A gold technique. J Histochem Cytochem 30:81–85

    Article  CAS  PubMed  Google Scholar 

  • Bleher R, Kandela I, Meyer DA, Albrecht RM (2008) Immuno-EM using colloidal metal nanoparticles and electron spectroscopic imaging for co-localization at high spatial resolution. J Microsc 230:388–395

    Article  CAS  PubMed  Google Scholar 

  • Brandt S, Heikkonen J, Engelhardt P (2001) Multiphase method for automatic alignment of transmission electron microscope images using markers. J Struct Biol 133:10–22

    Article  CAS  PubMed  Google Scholar 

  • D’Amico F, Skarmoutsou E (2008a) Quantifying immunogold labelling in transmission immunoelectron microscopy. J Microsc 230:9–15

    Article  PubMed  Google Scholar 

  • D’Amico F, Skarmoutsou E (2008b) Clustering and colocalization in transmission immunoelectron microscopy: a brief review. Micron 39:1–6

    Article  PubMed  Google Scholar 

  • Daly LE, Bourke GJ (2000) Interpretation and uses of medical statistics. Blackwell Science, Oxford

    Book  Google Scholar 

  • Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329

    Article  PubMed Central  PubMed  Google Scholar 

  • Geiser M, Quaile O, Wenk A, Wigge C, Eigeldinger-Berthou S, Hirn S, Schäffler M, Schleh C, Mőller W, Mall MA, Kreyling WG (2013) Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease. Part Fibre Toxicol 10:19

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Geuze HJ, Slot JW, Ley P van der, Scheffer RCT (1981) Use of colloidal gold particles in double-labelling immunoelectron microscopy of ultrathin tissue sections. J Cell Biol 89:653–659

  • Griffiths G (1993) Fine structure immunocytochemistry. Springer, Heidelberg

    Book  Google Scholar 

  • Griffiths G, Lucocq JM (2014) Antibodies for immunolabeling by light and electron microscopy: not for the faint hearted. Histochem Cell Biol 142:347–360

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gundersen HJG (1977) Notes on the estimation of the numerical density of arbitrary profiles: the edge effect. J Microsc 111:219–223

    Article  Google Scholar 

  • Gundersen HJG, Jensen EB (1987) The efficiency of systematic sampling in stereology and its prediction. J Microsc 147:229–263

    Article  CAS  PubMed  Google Scholar 

  • Gundersen HJG, Østerby R (1981) Optimizing sampling efficiency of stereological studies in biology: or “do more less well!”. J Microsc 121:65–73

    Article  CAS  PubMed  Google Scholar 

  • Gundersen HJG, Jensen EBV, Kiêu K, Nielsen J (1999) The efficiency of systematic sampling in stereology: reconsidered. J Microsc 193:199–211

    Article  CAS  PubMed  Google Scholar 

  • Gupta M, Mayhew TM, Bedi KS, Sharma AK, White FH (1983) Inter-animal variation and its influence on the overall precision of morphometric estimates based on nested sampling designs. J Microsc 131:147–154

    Article  CAS  PubMed  Google Scholar 

  • Howard CV, Reed MG (2005) Unbiased stereology. Three-dimensional measurement in microscopy, 2nd edn. Garland Science/BIOS Scientific, Abingdon

    Google Scholar 

  • Kay KR, Smith C, Wright AK, Serrano-Pozo A, Pooler AM, Koffie R, Bastin ME, Bak TH, Abrahams S, Kopeikina KJ, McGuone D, Frosch MP, Gillingwater TH, Hyman BT, Spires-Jones TL (2013) Studying synapses in human brain with array tomography and electron microscopy. Nat Protoc 8:1366–1380

    Article  PubMed Central  PubMed  Google Scholar 

  • Killingsworth MC, Lai K, Wu X, Yong JLC, Lee CS (2012) Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by widefield epifluorescence, super-resolution light, and immunoelectron microscopy. J Histochem Cytochem 60:832–843

    Article  PubMed Central  PubMed  Google Scholar 

  • Klein T, Proppert S, Sauer M (2014) Eight years of single-molecule localization microscopy. Histochem Cell Biol 141:561–575

    Article  CAS  PubMed  Google Scholar 

  • Koster AJ, Klumperman J (2003) Electron microscopy in cell biology: integrating structure and function. Nat Rev Mol Cell Biol 4 (suppl):SS6–SS10

    Google Scholar 

  • Lebonvallet S, Mennesson T, Bonnet N, Girod S, Plotkowski C, Hinnrasky J, Puchelle E (1991) Semi-automatic quantitation of dense markers in cytochemistry. Histochem Cell Biol 96:245–250

    CAS  Google Scholar 

  • Loukanov A, Karnasawa N, Danev R, Shigemoto R, Nagayama K (2010) Immunolocalization of multiple membrane proteins on a carbon replica with STEM and EDX. Ultramicroscopy 110:366–374

    Article  CAS  PubMed  Google Scholar 

  • Lucocq J (1992) Quantitation of gold labelling and estimation of labelling efficiency with a stereological counting method. J Histochem Cytochem 40:1929–1936

    Article  CAS  PubMed  Google Scholar 

  • Lucocq J (1994) Quantitation of gold labelling and antigens in immunolabelled ultrathin sections. J Anat 184:1–13

    PubMed Central  CAS  PubMed  Google Scholar 

  • Lucocq J (2012) Can data provenance go the full monty? Trends Cell Biol 22:229–230

    Article  PubMed  Google Scholar 

  • Lucocq JM, Gawden-Bone C (2009) A stereological approach for estimation of cellular immunolabeling and its spatial distribution in oriented sections using the rotator. J Histochem Cytochem 57:709–719

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lucocq JM, Gawden-Bone C (2010) Quantitative assessment of specificity in immunoelectron microscopy. J Histochem Cytochem 58:917–927

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lucocq JM, Hacker C (2013) Cutting a fine figure. On the use of thin sections in electron microscopy to quantify autophagy. Autophagy 9:1–6

    Article  Google Scholar 

  • Lucocq JM, Habermann A, Watt S, Backer JM, Mayhew TM, Griffiths G (2004) A rapid method for assessing the distribution of gold labelling on thin sections. J Histochem Cytochem 52:991–1000

    Article  CAS  PubMed  Google Scholar 

  • Lucocq JM, Mayhew TM, Schwab Y, Steyer AM, Hacker C (2014) Systems biology in 3D space—enter the morphome. Trends Cell Biol. doi:10.1016/j.tcb.2014.09.008

  • Mattfeldt T, Mall G, Gharehbaghi H, Möller P (1990) Estimation of surface area and length with the orientator. J Microsc 159:301–317

    Article  CAS  PubMed  Google Scholar 

  • Mayhew TM (1991) The new stereological methods for interpreting functional morphology from slices of cells and organs. Exp Physiol 76:639–665

    Article  CAS  PubMed  Google Scholar 

  • Mayhew TM (2008) Taking tissue samples from the placenta: an illustration of principles and strategies. Placenta 29:1–14

    Article  CAS  PubMed  Google Scholar 

  • Mayhew TM (2011) Mapping the distributions and quantifying the labelling intensities of cell compartments by immunoelectron microscopy: progress towards a coherent set of methods. J Anat 219:647–660

    Article  PubMed Central  PubMed  Google Scholar 

  • Mayhew TM, Desoye G (2004) A simple method for comparing immunogold distributions in two or more experimental groups illustrated using GLUT1 labelling of isolated trophoblast cells. Placenta 25:580–584

    Article  CAS  PubMed  Google Scholar 

  • Mayhew TM, Lucocq JM (2008a) Quantifying immunogold labelling patterns of cellular compartments when they comprise mixtures of membranes (surface-occupying) and organelles (volume-occupying). Histochem Cell Biol 129:367–378

    Article  CAS  PubMed  Google Scholar 

  • Mayhew TM, Lucocq JM (2008b) Developments in cell biology for quantitative immunoelectron microscopy based on thin sections—a review. Histochem Cell Biol 130:299–313

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mayhew TM, Lucocq JM (2011) Multiple-labelling immunoEM using different sizes of colloidal gold: alternative approaches to test for differential distribution and colocalization in subcellular structures. Histochem Cell Biol 135:317–326

    Article  CAS  PubMed  Google Scholar 

  • Mayhew TM, Reith A (1988) Practical ways to correct cytomembrane surface densities for the loss of membrane images that results from oblique sectioning. In: Reith A, Mayhew TM (eds) Stereology and morphometry in electron microscopy. Problems and solutions. Hemisphere, New York

    Google Scholar 

  • Mayhew TM, Lucocq JM, Griffiths G (2002) Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections. J Microsc 205:153–164

    Article  CAS  PubMed  Google Scholar 

  • Mayhew T, Griffiths G, Habermann A, Lucocq J, Emre N, Webster P (2003) A simpler way of comparing the labelling densities of cellular compartments illustrated using data from VPARP and LAMP-1 immunogold labelling experiments. Histochem Cell Biol 119:333–341

    CAS  PubMed  Google Scholar 

  • Mayhew TM, Griffiths G, Lucocq JM (2004) Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells. Histochem Cell Biol 122:171–177

    Article  CAS  PubMed  Google Scholar 

  • Mayhew TM, Mühlfeld C, Vanhecke D, Ochs M (2009) A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs. Ann Anat 191:153–170

    Article  CAS  PubMed  Google Scholar 

  • Micheva KD, Smith SJ (2007) Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55:25–36

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Micheva KD, Busse B, Weller NC, O’Rourke N, Stephen J, Smith SJ (2010) Single synapse analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron 68:639–653

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mironov AA Jr, Mironov AA (1998) Estimation of subcellular organelle volume from ultrathin sections through centrioles with a discretized version of the vertical rotator. J Microsc 192:29–36

    Article  PubMed  Google Scholar 

  • Monteiro-Leal LH, Troster H, Campanati L, Spring H, Trendelenburg MF (2003) Gold finder: a computer method for fast automatic double gold labelling detection, counting, and color overlay in electron microscopic images. J Struct Biol 141:228–239

    Article  CAS  PubMed  Google Scholar 

  • Mühlfeld C, Mayhew TM, Gehr P, Rothen-Rutihauser B (2007) A novel quantitative method for analysing the distribution of nanoparticles between different tissue and intracellular compartments. J Aerosol Med 20:395–407

    Article  PubMed  Google Scholar 

  • Mühlfeld C, Rothen-Rutishauser B, Blank F, Vanhecke D, Ochs M, Gehr P (2008) Interactions of nanoparticles with pulmonary structures and cellular responses. Am J Physiol Lung Cell Mol Physiol 294:L817–L829

    Article  PubMed  Google Scholar 

  • Mühlfeld C, Nyengaard JR, Mayhew TM (2010) A review of state-of-the-art stereology for better quantitative 3D morphology in cardiac research. Cardiovasc Pathol 19:65–82

    Article  PubMed  Google Scholar 

  • Nickel BM, DeFranco BH, Gay VL, Murray SA (2008) Clathrin and Cx43 gap junction plaque endoexocytosis. Biochem Biophys Res Commun 374:679–682

    Article  CAS  PubMed  Google Scholar 

  • Nikonenko AG, Nikonenko IR, Skibo GG (2000) Computer simulation approach to the quantification of immunogold labelling on plasma membrane of cultured neurons. J Neurosci Meth 96:11–17

    Article  CAS  Google Scholar 

  • Nisman R, Dellaire G, Ren Y, Li R, Bazett-Jones DP (2012) Application of quantum dots as probes for correlative fluorescence, conventional, and energy-filtered transmission electron microscopy. J Histochem Cytochem 52:13–18

    Article  Google Scholar 

  • Nyengaard JR, Gundersen HJG (1992) The isector: a simple and direct method for generating isotropic, uniform random sections from small specimens. J Microsc 165:427–431

    Article  Google Scholar 

  • Nyengaard JR, Gundersen HJG (2006) Direct and efficient stereological estimation of total cell quantities using electron microscopy. J Microsc 222:182–187

    Article  CAS  PubMed  Google Scholar 

  • Oddone A, Vilanova IV, Tam J, Lakadamyali M (2014) Super-resolution imaging with stochastic single-molecule localization: concepts, technical developments, and biological applications. Microsc Res Tech 77:502–509

    Article  CAS  PubMed  Google Scholar 

  • Owen DM, Magenau A, Williamson DJ, Gaus K (2013) Super-resolution imaging by localization microscopy. Meth Mol Biol 950:81–93

    CAS  Google Scholar 

  • Petrie A, Sabin C (2000) Medical statistics at a glance. Blackwell Science, Oxford

    Google Scholar 

  • Philimonenko AA, Janáček J, Hozák P (2000) Statistical evaluation of colocalization patterns in immunogold labelling experiments. J Struct Biol 132:201–210

    Article  CAS  PubMed  Google Scholar 

  • Philimonenko VV, Philimonenko AA, Šloufová I, Hrubý M, Novotný F, Halbhuber Z, Krivjanská M, Nebesářová J, Šlouf M, Hozák P (2014) Simultaneous detection of multiple targets for ultrastructural immunocytochemistry. Histochem Cell Biol 141:229–329

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Robinson JM, Takizawa T, Vandré DD (2000) Enhanced labelling efficiency using ultrasmall immunogold probes: immunocytochemistry. J Histochem Cytochem 48:487–492

    Article  CAS  PubMed  Google Scholar 

  • Roth J (1996) The silver anniversary of gold: 25 years of the colloidal gold marker system for immunocytochemistry and histochemistry. Histochem Cell Biol 106:1–8

    Article  CAS  PubMed  Google Scholar 

  • Schmiedl A, Ochs M, Mühlfeld C, Johnen G, Brasch F (2005) Distribution of surfactant proteins in type II pneumocytes of newborn, 14-day old, and adult rats: an immunoelectron microscopic and stereological study. Histochem Cell Biol 124:465–476

    Article  CAS  PubMed  Google Scholar 

  • Sibarita J-B (2014) High-density single-particle tracking: quantifying molecule organization and dynamics at the nanoscale. Histochem Cell Biol 141:587–595

    Article  CAS  PubMed  Google Scholar 

  • Slot JW, Geuze HJ (1985) A new method for preparing gold probes for multiple-labelling cytochemistry. Eur J Cell Biol 38:87–93

    CAS  PubMed  Google Scholar 

  • Sterio DC (1984) The unbiased estimation of number and sizes of arbitrary particles using the disector. J Microsc 134:127–136

    Article  CAS  PubMed  Google Scholar 

  • Vanhecke D, Studer D, Ochs M (2007) Stereology meets electron tomography: towards quantitative 3D electron microscopy. J Struct Biol 159:443–450

    Article  PubMed  Google Scholar 

  • Vedel-Jensen EB, Gundersen HJG (1993) The rotator. J Microsc 171:35–44

    Article  Google Scholar 

  • Wacker I, Schroeder RR (2013) Array tomography. J Microsc 252:93–99

    Article  CAS  PubMed  Google Scholar 

  • Wang R, Pokhariya H, McKenna SJ, Lucocq J (2011) Recognition of immunogold markers in electron micrographs. J Struct Biol 176:151–158

    Article  PubMed  Google Scholar 

  • Webster P, Schwarz H, Griffiths G (2008) Preparation of cells and tissues for immuno EM. Meth Cell Biol 88:45–58

    Article  CAS  Google Scholar 

  • Yashchenko VV, Gavrilova OV, Rautian MS, Jakobsen KS (2012) Association of Paramecium bursaria Chlorella viruses with Paramecium bursaria cells: ultrastructural studies. Eur J Protistol 48:149–159

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

For over 40 years, I have greatly enjoyed fruitful interactions and collaborations with many colleagues, students and fellow teachers on international courses of stereology, electron microscopy and cell and molecular biology.

Author information

Authors and Affiliations

  1. School of Life Sciences, Queen’s Medical Centre, University of Nottingham, E Floor, Nottingham, NG7 2UH, UK

    Terry M. Mayhew

Authors
  1. Terry M. Mayhew
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Terry M. Mayhew.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mayhew, T.M. Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics. Cell Tissue Res 360, 43–59 (2015). https://doi.org/10.1007/s00441-014-2038-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-014-2038-y

Keywords

Search

Navigation