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Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy.

Mühlfeld C, Rothen-Rutishauser B, Vanhecke D, Blank F, Gehr P, Ochs M - Part Fibre Toxicol (2007)

Bottom Line: While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale.Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach.Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Anatomy, University of Bern, Baltzerstrasse 2, CH-3000 Bern 9, Switzerland. muehlfeld@ana.unibe.ch.

ABSTRACT
Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions.This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

No MeSH data available.


Related in: MedlinePlus

Immuno TEM of rat lung labeled for caveolin-1. Caveolae are cholesterol-rich regions of the plasma membrane involved in endocytosis. One of the constituting proteins of caveolae is caveolin-1 which was labeled here using newborn rat lung tissue fixed by instillation of 4% PFA, 0.1% GA in 0.2 M Hepes buffer. After freeze-substitution and embedding in acrylic resin (Table 1), ultrathin sections (40–70 nm) were cut and mounted on formvar-coated Ni mesh grids. Immunogold labeling was performed according to standard protocols [99]. The primary antibody was a rabbit anti-caveolin-1 antibody (BD Biosciences, Pharmingen, Germany) diluted 1:50. The secondary antibody was a goat-anti-rabbit antibody coupled to 10 nm gold particles (British Biocell, Cardiff, United Kingdom). A strong signal is found for caveolae in capillary endothelium and alveolar epithelium. Unspecific background labeling was weak (note the gold particle in the interstitium) but not completely absent. Immunogold labeling requires good knowledge about the biology of the target antigen and the specificity of the antibody. Before going to the TEM level, one is well advised to perform pilot light microscopic experiments. CL = Capillary lumen; EC = Endothelial cell; IC = Interstitial cell; AEI = Alveolar epithelial type I cell; AL = Alveolar lumen. Bar = 1 μm.
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Figure 4: Immuno TEM of rat lung labeled for caveolin-1. Caveolae are cholesterol-rich regions of the plasma membrane involved in endocytosis. One of the constituting proteins of caveolae is caveolin-1 which was labeled here using newborn rat lung tissue fixed by instillation of 4% PFA, 0.1% GA in 0.2 M Hepes buffer. After freeze-substitution and embedding in acrylic resin (Table 1), ultrathin sections (40–70 nm) were cut and mounted on formvar-coated Ni mesh grids. Immunogold labeling was performed according to standard protocols [99]. The primary antibody was a rabbit anti-caveolin-1 antibody (BD Biosciences, Pharmingen, Germany) diluted 1:50. The secondary antibody was a goat-anti-rabbit antibody coupled to 10 nm gold particles (British Biocell, Cardiff, United Kingdom). A strong signal is found for caveolae in capillary endothelium and alveolar epithelium. Unspecific background labeling was weak (note the gold particle in the interstitium) but not completely absent. Immunogold labeling requires good knowledge about the biology of the target antigen and the specificity of the antibody. Before going to the TEM level, one is well advised to perform pilot light microscopic experiments. CL = Capillary lumen; EC = Endothelial cell; IC = Interstitial cell; AEI = Alveolar epithelial type I cell; AL = Alveolar lumen. Bar = 1 μm.

Mentions: If coated NP are labeled by immunogold particles it may be interesting to determine their exact localization. For example, early and late endosomes as well as lysosomes cannot be distinguished from each other by their morphology alone. The double-labeling technique (e.g. using 5 nm golds for the NP and 15 nm golds for the organelle) may represent an interesting approach to perform co-localization studies. In those cases where NP can be identified by EFTEM (see below) immunogold labeling of organelles or cytoskeletal proteins offers a detailed analysis of how NP are associated with cellular proteins. Finally, exposure of cells to NP may result in alterations of the cellular distribution of a particular antigen [102] which can be studied by quantitative immuno TEM [103]. An example of sufficient immunogold labeling for an organelle suspicious of being involved in active cellular NP uptake is shown in Figure 4.


Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy.

Mühlfeld C, Rothen-Rutishauser B, Vanhecke D, Blank F, Gehr P, Ochs M - Part Fibre Toxicol (2007)

Immuno TEM of rat lung labeled for caveolin-1. Caveolae are cholesterol-rich regions of the plasma membrane involved in endocytosis. One of the constituting proteins of caveolae is caveolin-1 which was labeled here using newborn rat lung tissue fixed by instillation of 4% PFA, 0.1% GA in 0.2 M Hepes buffer. After freeze-substitution and embedding in acrylic resin (Table 1), ultrathin sections (40–70 nm) were cut and mounted on formvar-coated Ni mesh grids. Immunogold labeling was performed according to standard protocols [99]. The primary antibody was a rabbit anti-caveolin-1 antibody (BD Biosciences, Pharmingen, Germany) diluted 1:50. The secondary antibody was a goat-anti-rabbit antibody coupled to 10 nm gold particles (British Biocell, Cardiff, United Kingdom). A strong signal is found for caveolae in capillary endothelium and alveolar epithelium. Unspecific background labeling was weak (note the gold particle in the interstitium) but not completely absent. Immunogold labeling requires good knowledge about the biology of the target antigen and the specificity of the antibody. Before going to the TEM level, one is well advised to perform pilot light microscopic experiments. CL = Capillary lumen; EC = Endothelial cell; IC = Interstitial cell; AEI = Alveolar epithelial type I cell; AL = Alveolar lumen. Bar = 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Immuno TEM of rat lung labeled for caveolin-1. Caveolae are cholesterol-rich regions of the plasma membrane involved in endocytosis. One of the constituting proteins of caveolae is caveolin-1 which was labeled here using newborn rat lung tissue fixed by instillation of 4% PFA, 0.1% GA in 0.2 M Hepes buffer. After freeze-substitution and embedding in acrylic resin (Table 1), ultrathin sections (40–70 nm) were cut and mounted on formvar-coated Ni mesh grids. Immunogold labeling was performed according to standard protocols [99]. The primary antibody was a rabbit anti-caveolin-1 antibody (BD Biosciences, Pharmingen, Germany) diluted 1:50. The secondary antibody was a goat-anti-rabbit antibody coupled to 10 nm gold particles (British Biocell, Cardiff, United Kingdom). A strong signal is found for caveolae in capillary endothelium and alveolar epithelium. Unspecific background labeling was weak (note the gold particle in the interstitium) but not completely absent. Immunogold labeling requires good knowledge about the biology of the target antigen and the specificity of the antibody. Before going to the TEM level, one is well advised to perform pilot light microscopic experiments. CL = Capillary lumen; EC = Endothelial cell; IC = Interstitial cell; AEI = Alveolar epithelial type I cell; AL = Alveolar lumen. Bar = 1 μm.
Mentions: If coated NP are labeled by immunogold particles it may be interesting to determine their exact localization. For example, early and late endosomes as well as lysosomes cannot be distinguished from each other by their morphology alone. The double-labeling technique (e.g. using 5 nm golds for the NP and 15 nm golds for the organelle) may represent an interesting approach to perform co-localization studies. In those cases where NP can be identified by EFTEM (see below) immunogold labeling of organelles or cytoskeletal proteins offers a detailed analysis of how NP are associated with cellular proteins. Finally, exposure of cells to NP may result in alterations of the cellular distribution of a particular antigen [102] which can be studied by quantitative immuno TEM [103]. An example of sufficient immunogold labeling for an organelle suspicious of being involved in active cellular NP uptake is shown in Figure 4.

Bottom Line: While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale.Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach.Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Anatomy, University of Bern, Baltzerstrasse 2, CH-3000 Bern 9, Switzerland. muehlfeld@ana.unibe.ch.

ABSTRACT
Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions.This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

No MeSH data available.


Related in: MedlinePlus