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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

Chemical and physical fixation of the lung. Alveolar epithelial type II cells were studied by cTEM either after chemical (A and B) or after physical (C and D) fixation. The overview in A shows a well-preserved type II cell from a newborn rat lung fixed by instillation of 1.5% GA, 1.5% PFA in Hepes buffer and processed according to Table 1. Lamellar bodies (LB), nucleus (Nu) and mitochondria (Mt) are well preserved. At a higher magnification, details of the endoplasmic reticulum (ER) as well as an ER related multivesicular transport vesicle (MvTV) can be visualized. The overview in C shows a well-preserved type II cell from an adult rat lung. A small piece of tissue was cut from the whole lung, put in a syringe with 1-hexadecene and air was extracted from the tissue block by negative pressure. Afterwards, the specimen was high-pressure frozen (Leica EMPact 2.0, Leica, Vienna, Austria), freeze-substituted with acetone containing 1% osmium tetroxide (AFS 2.0, Leica, Vienna, Austria) and embedded in epoxy resin. Most likely due to the lack of uranyl acetate during freeze-substitution the lamellar bodies are not well preserved, with almost complete loss of the surfactant material, only the limiting membrane can be seen. However, the ultrastructure of other organelles like multivesicular bodies (MvB) is highly increased (D) due to the excellent preservation of the membrane structures (Me). Since this is the first description of high-pressure frozen lung tissue, systematic studies are needed to determine the ideal processing both for conventional and immuno TEM. Bars = 1 μm (A, C), 250 nm (B, D).
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Figure 2: Chemical and physical fixation of the lung. Alveolar epithelial type II cells were studied by cTEM either after chemical (A and B) or after physical (C and D) fixation. The overview in A shows a well-preserved type II cell from a newborn rat lung fixed by instillation of 1.5% GA, 1.5% PFA in Hepes buffer and processed according to Table 1. Lamellar bodies (LB), nucleus (Nu) and mitochondria (Mt) are well preserved. At a higher magnification, details of the endoplasmic reticulum (ER) as well as an ER related multivesicular transport vesicle (MvTV) can be visualized. The overview in C shows a well-preserved type II cell from an adult rat lung. A small piece of tissue was cut from the whole lung, put in a syringe with 1-hexadecene and air was extracted from the tissue block by negative pressure. Afterwards, the specimen was high-pressure frozen (Leica EMPact 2.0, Leica, Vienna, Austria), freeze-substituted with acetone containing 1% osmium tetroxide (AFS 2.0, Leica, Vienna, Austria) and embedded in epoxy resin. Most likely due to the lack of uranyl acetate during freeze-substitution the lamellar bodies are not well preserved, with almost complete loss of the surfactant material, only the limiting membrane can be seen. However, the ultrastructure of other organelles like multivesicular bodies (MvB) is highly increased (D) due to the excellent preservation of the membrane structures (Me). Since this is the first description of high-pressure frozen lung tissue, systematic studies are needed to determine the ideal processing both for conventional and immuno TEM. Bars = 1 μm (A, C), 250 nm (B, D).

Mentions: A prerequisite for transmission electron microscopy is that all material entering the microscope has to be fixed in one way or another. Fixation of cells and tissues aims to preserve the specimens as close to the living state as possible. As further outlined, different electron microscopic techniques as well as specific questions of a particular study significantly influence the choice of the fixation and embedding method. Currently, there are two major approaches to fix biological samples, viz. chemical or physical fixation. For the lung as an entire organ, there is no routine alternative approach to chemical fixation to date but for restricted tissue samples, such as larger airways or cell cultures, physical fixation offers an excellent alternative. The following paragraphs as well as Figure 1 provide an overview on different possible methods, their impact for the different TEM techniques and relevant references. Nevertheless, one will have to evaluate the usefulness of a specific protocol for each particular study. For this reason, Weibel et al. [36] have introduced a number of very instructive external and internal standards. In Figure 2, we provide a chemically and a physically fixed specimen for comparison between both methods.


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)

Chemical and physical fixation of the lung. Alveolar epithelial type II cells were studied by cTEM either after chemical (A and B) or after physical (C and D) fixation. The overview in A shows a well-preserved type II cell from a newborn rat lung fixed by instillation of 1.5% GA, 1.5% PFA in Hepes buffer and processed according to Table 1. Lamellar bodies (LB), nucleus (Nu) and mitochondria (Mt) are well preserved. At a higher magnification, details of the endoplasmic reticulum (ER) as well as an ER related multivesicular transport vesicle (MvTV) can be visualized. The overview in C shows a well-preserved type II cell from an adult rat lung. A small piece of tissue was cut from the whole lung, put in a syringe with 1-hexadecene and air was extracted from the tissue block by negative pressure. Afterwards, the specimen was high-pressure frozen (Leica EMPact 2.0, Leica, Vienna, Austria), freeze-substituted with acetone containing 1% osmium tetroxide (AFS 2.0, Leica, Vienna, Austria) and embedded in epoxy resin. Most likely due to the lack of uranyl acetate during freeze-substitution the lamellar bodies are not well preserved, with almost complete loss of the surfactant material, only the limiting membrane can be seen. However, the ultrastructure of other organelles like multivesicular bodies (MvB) is highly increased (D) due to the excellent preservation of the membrane structures (Me). Since this is the first description of high-pressure frozen lung tissue, systematic studies are needed to determine the ideal processing both for conventional and immuno TEM. Bars = 1 μm (A, C), 250 nm (B, D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Chemical and physical fixation of the lung. Alveolar epithelial type II cells were studied by cTEM either after chemical (A and B) or after physical (C and D) fixation. The overview in A shows a well-preserved type II cell from a newborn rat lung fixed by instillation of 1.5% GA, 1.5% PFA in Hepes buffer and processed according to Table 1. Lamellar bodies (LB), nucleus (Nu) and mitochondria (Mt) are well preserved. At a higher magnification, details of the endoplasmic reticulum (ER) as well as an ER related multivesicular transport vesicle (MvTV) can be visualized. The overview in C shows a well-preserved type II cell from an adult rat lung. A small piece of tissue was cut from the whole lung, put in a syringe with 1-hexadecene and air was extracted from the tissue block by negative pressure. Afterwards, the specimen was high-pressure frozen (Leica EMPact 2.0, Leica, Vienna, Austria), freeze-substituted with acetone containing 1% osmium tetroxide (AFS 2.0, Leica, Vienna, Austria) and embedded in epoxy resin. Most likely due to the lack of uranyl acetate during freeze-substitution the lamellar bodies are not well preserved, with almost complete loss of the surfactant material, only the limiting membrane can be seen. However, the ultrastructure of other organelles like multivesicular bodies (MvB) is highly increased (D) due to the excellent preservation of the membrane structures (Me). Since this is the first description of high-pressure frozen lung tissue, systematic studies are needed to determine the ideal processing both for conventional and immuno TEM. Bars = 1 μm (A, C), 250 nm (B, D).
Mentions: A prerequisite for transmission electron microscopy is that all material entering the microscope has to be fixed in one way or another. Fixation of cells and tissues aims to preserve the specimens as close to the living state as possible. As further outlined, different electron microscopic techniques as well as specific questions of a particular study significantly influence the choice of the fixation and embedding method. Currently, there are two major approaches to fix biological samples, viz. chemical or physical fixation. For the lung as an entire organ, there is no routine alternative approach to chemical fixation to date but for restricted tissue samples, such as larger airways or cell cultures, physical fixation offers an excellent alternative. The following paragraphs as well as Figure 1 provide an overview on different possible methods, their impact for the different TEM techniques and relevant references. Nevertheless, one will have to evaluate the usefulness of a specific protocol for each particular study. For this reason, Weibel et al. [36] have introduced a number of very instructive external and internal standards. In Figure 2, we provide a chemically and a physically fixed specimen for comparison between both methods.

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