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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

Electron tomography of a 250 nm thick section. A tilt series (three stills are shown in A, B and C) between +/- 60° with an increment of 1° provides the information for a volume reconstruction by weighted backprojection (D) of an in vitro grown alveolar epithelial cell (cell line A549), exposed to polystyrene NP prior to chemical fixation. The 2 nm thin slice (E, at a depth of 58 nm in the section) reveals a crisper and clearer depiction of the polystyrene NP than the 250 nm thick Epon section (F). Arbitrary digital slices can be made (G, position shown by the two arrowheads in D) in order to provide unquestionable recognition of the NP. Bars = 250 nm in A-D, 50 nm in E-G. Software based 3D rendering offers a way for segmentation according to the interpretation of the user and allows full perspective freedom (H and I). Moreover, clipping planes can partially dissect the scene (J), segmented objects can be omitted (the membrane surrounding the NP in K) and specific quantitative information on rendered objects obtained. Blue: nanoparticle, shades of green: rough endoplasmic reticulum (RER), orange: plasma membrane, transparent white: membrane surrounding the NP, red: ribosomes (Ri) on the RER. Me = membrane, NP = nanoparticle, PM = plasma membrane.
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Figure 6: Electron tomography of a 250 nm thick section. A tilt series (three stills are shown in A, B and C) between +/- 60° with an increment of 1° provides the information for a volume reconstruction by weighted backprojection (D) of an in vitro grown alveolar epithelial cell (cell line A549), exposed to polystyrene NP prior to chemical fixation. The 2 nm thin slice (E, at a depth of 58 nm in the section) reveals a crisper and clearer depiction of the polystyrene NP than the 250 nm thick Epon section (F). Arbitrary digital slices can be made (G, position shown by the two arrowheads in D) in order to provide unquestionable recognition of the NP. Bars = 250 nm in A-D, 50 nm in E-G. Software based 3D rendering offers a way for segmentation according to the interpretation of the user and allows full perspective freedom (H and I). Moreover, clipping planes can partially dissect the scene (J), segmented objects can be omitted (the membrane surrounding the NP in K) and specific quantitative information on rendered objects obtained. Blue: nanoparticle, shades of green: rough endoplasmic reticulum (RER), orange: plasma membrane, transparent white: membrane surrounding the NP, red: ribosomes (Ri) on the RER. Me = membrane, NP = nanoparticle, PM = plasma membrane.

Mentions: Visualization by ET allows the analysis of NP shape, volume and surface in 3D. This greatly helps with the characterization of NP and the discrimination between genuine particles and agglomerated NP. Furthermore, ET is projected to become a useful tool to study contact sites between NP and macromolecules in detail. Such insights will potentially improve our understanding of NP entry mechanisms into cells, processing by the cellular machinery and where NP related toxicity originates. One exciting topic also relates to the fact that ET extends the application of a number of stereological tools (e.g. the optical disector, see below) to the electron microscopic level [127]. Particularly, for stereological purposes using ET no segmentation process is needed making the quantification independent of the observer preference. Finally, it needs to be emphasized that ET is independent of sample processing: it can be carried out on materials embedded in epoxy or methacrylate resins as well as cryosections and fully hydrated cryosections and allows to be combined with immuno TEM. Figure 6 provides an example of a 3D reconstruction of a NP in its cellular environment.


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)

Electron tomography of a 250 nm thick section. A tilt series (three stills are shown in A, B and C) between +/- 60° with an increment of 1° provides the information for a volume reconstruction by weighted backprojection (D) of an in vitro grown alveolar epithelial cell (cell line A549), exposed to polystyrene NP prior to chemical fixation. The 2 nm thin slice (E, at a depth of 58 nm in the section) reveals a crisper and clearer depiction of the polystyrene NP than the 250 nm thick Epon section (F). Arbitrary digital slices can be made (G, position shown by the two arrowheads in D) in order to provide unquestionable recognition of the NP. Bars = 250 nm in A-D, 50 nm in E-G. Software based 3D rendering offers a way for segmentation according to the interpretation of the user and allows full perspective freedom (H and I). Moreover, clipping planes can partially dissect the scene (J), segmented objects can be omitted (the membrane surrounding the NP in K) and specific quantitative information on rendered objects obtained. Blue: nanoparticle, shades of green: rough endoplasmic reticulum (RER), orange: plasma membrane, transparent white: membrane surrounding the NP, red: ribosomes (Ri) on the RER. Me = membrane, NP = nanoparticle, PM = plasma membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Electron tomography of a 250 nm thick section. A tilt series (three stills are shown in A, B and C) between +/- 60° with an increment of 1° provides the information for a volume reconstruction by weighted backprojection (D) of an in vitro grown alveolar epithelial cell (cell line A549), exposed to polystyrene NP prior to chemical fixation. The 2 nm thin slice (E, at a depth of 58 nm in the section) reveals a crisper and clearer depiction of the polystyrene NP than the 250 nm thick Epon section (F). Arbitrary digital slices can be made (G, position shown by the two arrowheads in D) in order to provide unquestionable recognition of the NP. Bars = 250 nm in A-D, 50 nm in E-G. Software based 3D rendering offers a way for segmentation according to the interpretation of the user and allows full perspective freedom (H and I). Moreover, clipping planes can partially dissect the scene (J), segmented objects can be omitted (the membrane surrounding the NP in K) and specific quantitative information on rendered objects obtained. Blue: nanoparticle, shades of green: rough endoplasmic reticulum (RER), orange: plasma membrane, transparent white: membrane surrounding the NP, red: ribosomes (Ri) on the RER. Me = membrane, NP = nanoparticle, PM = plasma membrane.
Mentions: Visualization by ET allows the analysis of NP shape, volume and surface in 3D. This greatly helps with the characterization of NP and the discrimination between genuine particles and agglomerated NP. Furthermore, ET is projected to become a useful tool to study contact sites between NP and macromolecules in detail. Such insights will potentially improve our understanding of NP entry mechanisms into cells, processing by the cellular machinery and where NP related toxicity originates. One exciting topic also relates to the fact that ET extends the application of a number of stereological tools (e.g. the optical disector, see below) to the electron microscopic level [127]. Particularly, for stereological purposes using ET no segmentation process is needed making the quantification independent of the observer preference. Finally, it needs to be emphasized that ET is independent of sample processing: it can be carried out on materials embedded in epoxy or methacrylate resins as well as cryosections and fully hydrated cryosections and allows to be combined with immuno TEM. Figure 6 provides an example of a 3D reconstruction of a NP in its cellular environment.

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