A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs
- PMID: 19135344
- DOI: 10.1016/j.aanat.2008.11.001
A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs
Abstract
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.
Similar articles
-
Quantifying immunogold localization patterns on electron microscopic thin sections of placenta: recent developments.Placenta. 2009 Jul;30(7):565-70. doi: 10.1016/j.placenta.2009.04.013. Epub 2009 May 28. Placenta. 2009. PMID: 19481258 Review.
-
A simple method for comparing immunogold distributions in two or more experimental groups illustrated using GLUT1 labelling of isolated trophoblast cells.Placenta. 2004 Jul;25(6):580-4. doi: 10.1016/j.placenta.2003.12.002. Placenta. 2004. PMID: 15135242
-
Quantifying immunogold localization on electron microscopic thin sections: a compendium of new approaches for plant cell biologists.J Exp Bot. 2011 Aug;62(12):4101-13. doi: 10.1093/jxb/err176. Epub 2011 Jun 1. J Exp Bot. 2011. PMID: 21633081
-
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. 2002 Feb;205(Pt 2):153-64. doi: 10.1046/j.0022-2720.2001.00977.x. J Microsc. 2002. PMID: 11879430
-
Mapping the distributions and quantifying the labelling intensities of cell compartments by immunoelectron microscopy: progress towards a coherent set of methods.J Anat. 2011 Dec;219(6):647-60. doi: 10.1111/j.1469-7580.2011.01438.x. Epub 2011 Oct 17. J Anat. 2011. PMID: 21999926 Free PMC article. Review.
Cited by
-
In vivo Biodistribution and Clearance of Magnetic Iron Oxide Nanoparticles for Medical Applications.Int J Nanomedicine. 2023 Jul 26;18:4067-4100. doi: 10.2147/IJN.S415063. eCollection 2023. Int J Nanomedicine. 2023. PMID: 37525695 Free PMC article. Review.
-
Method for nanoparticles uptake evaluation based on double labeled fluorescent cells scanned in enhanced darkfield microscopy.Biomed Opt Express. 2023 May 18;14(6):2796-2810. doi: 10.1364/BOE.490136. eCollection 2023 Jun 1. Biomed Opt Express. 2023. PMID: 37342715 Free PMC article.
-
Sleeping Beauty transposon system for GDNF overexpression of entrapped stem cells in fibrin hydrogel in a rat model of Parkinson's disease.Drug Deliv Transl Res. 2023 Jun;13(6):1745-1765. doi: 10.1007/s13346-023-01289-9. Epub 2023 Feb 28. Drug Deliv Transl Res. 2023. PMID: 36853436 Free PMC article.
-
Quantitative Imaging Metrics for the Assessment of Pulmonary Pathophysiology: An Official American Thoracic Society and Fleischner Society Joint Workshop Report.Ann Am Thorac Soc. 2023 Feb;20(2):161-195. doi: 10.1513/AnnalsATS.202211-915ST. Ann Am Thorac Soc. 2023. PMID: 36723475 Free PMC article.
-
Optical Microscopy Systems for the Detection of Unlabeled Nanoparticles.Int J Nanomedicine. 2022 May 13;17:2139-2163. doi: 10.2147/IJN.S355007. eCollection 2022. Int J Nanomedicine. 2022. PMID: 35599750 Free PMC article. Review.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources