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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

Conventional TEM of polystyrene nanoparticles. This figure demonstrates the impossibility to distinguish between NP and cellular structures by conventional TEM unambiguously. In A, five polystyrene NP (NP!) with a mean diameter of 78 nm are observed next to an A549 cell. Once taken up by the cells, they may have an appearance as shown in B. It is very likely that the spherical structures in B (NP?) are not NP but vesicular structures like caveolae. CC = Clathrin coated vesicle; PM = Plasma membrane; AJ = Adherens junction. Chemical fixation, Epon embedding, 40–70 nm sections. Bar = 1 μm (A and B are at identical magnification).
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Figure 3: Conventional TEM of polystyrene nanoparticles. This figure demonstrates the impossibility to distinguish between NP and cellular structures by conventional TEM unambiguously. In A, five polystyrene NP (NP!) with a mean diameter of 78 nm are observed next to an A549 cell. Once taken up by the cells, they may have an appearance as shown in B. It is very likely that the spherical structures in B (NP?) are not NP but vesicular structures like caveolae. CC = Clathrin coated vesicle; PM = Plasma membrane; AJ = Adherens junction. Chemical fixation, Epon embedding, 40–70 nm sections. Bar = 1 μm (A and B are at identical magnification).

Mentions: Conventional TEM in NP research is frequently used to characterize particle structure [18,91-93], to demonstrate the intracellular localization of NP [28,94-96] and less frequently to assess the morphology of tissue or cell samples [28,96]. Its popularity is partly explained by the high resolution and because it is established in many laboratories. However, particles that are shown in TEM figures are often agglomerated structures with diameters of far more than 100 nm. For this reason, too low magnifications are used, which do not allow the identification of particles in the range of 10–20 nm or even less. Furthermore, it is often ignored that NP can sometimes be indistinguishable from cellular structures in the same size range. For example, electron dense particles (e.g. titanium dioxide) may have similarity with glycogen granules, mitochondrial matrix granules or ribosomes whereas electron lucent particles (e.g. polystyrene) may be confused with spherical vesicular structures [97]. Therefore, there is a potential risk that a technical bias is present either due to the fact that cellular structures are mistaken for NP or vice versa. Using only conventional TEM this bias cannot be overcome (Figure 3).


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)

Conventional TEM of polystyrene nanoparticles. This figure demonstrates the impossibility to distinguish between NP and cellular structures by conventional TEM unambiguously. In A, five polystyrene NP (NP!) with a mean diameter of 78 nm are observed next to an A549 cell. Once taken up by the cells, they may have an appearance as shown in B. It is very likely that the spherical structures in B (NP?) are not NP but vesicular structures like caveolae. CC = Clathrin coated vesicle; PM = Plasma membrane; AJ = Adherens junction. Chemical fixation, Epon embedding, 40–70 nm sections. Bar = 1 μm (A and B are at identical magnification).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211502&req=5

Figure 3: Conventional TEM of polystyrene nanoparticles. This figure demonstrates the impossibility to distinguish between NP and cellular structures by conventional TEM unambiguously. In A, five polystyrene NP (NP!) with a mean diameter of 78 nm are observed next to an A549 cell. Once taken up by the cells, they may have an appearance as shown in B. It is very likely that the spherical structures in B (NP?) are not NP but vesicular structures like caveolae. CC = Clathrin coated vesicle; PM = Plasma membrane; AJ = Adherens junction. Chemical fixation, Epon embedding, 40–70 nm sections. Bar = 1 μm (A and B are at identical magnification).
Mentions: Conventional TEM in NP research is frequently used to characterize particle structure [18,91-93], to demonstrate the intracellular localization of NP [28,94-96] and less frequently to assess the morphology of tissue or cell samples [28,96]. Its popularity is partly explained by the high resolution and because it is established in many laboratories. However, particles that are shown in TEM figures are often agglomerated structures with diameters of far more than 100 nm. For this reason, too low magnifications are used, which do not allow the identification of particles in the range of 10–20 nm or even less. Furthermore, it is often ignored that NP can sometimes be indistinguishable from cellular structures in the same size range. For example, electron dense particles (e.g. titanium dioxide) may have similarity with glycogen granules, mitochondrial matrix granules or ribosomes whereas electron lucent particles (e.g. polystyrene) may be confused with spherical vesicular structures [97]. Therefore, there is a potential risk that a technical bias is present either due to the fact that cellular structures are mistaken for NP or vice versa. Using only conventional TEM this bias cannot be overcome (Figure 3).

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