Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography.
Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2013-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3561407?pdf=render |
| _version_ | 1819092177619255296 |
|---|---|
| author | Jean-Pierre Baudoin W Gray Jerome Christian Kübel Niels de Jonge |
| author_facet | Jean-Pierre Baudoin W Gray Jerome Christian Kübel Niels de Jonge |
| author_sort | Jean-Pierre Baudoin |
| collection | DOAJ |
| description | Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters. |
| first_indexed | 2024-12-21T22:51:28Z |
| format | Article |
| id | doaj.art-435501567da84c4dac0463c91fa81f51 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T22:51:28Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-435501567da84c4dac0463c91fa81f512022-12-21T18:47:34ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0181e5502210.1371/journal.pone.0055022Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography.Jean-Pierre BaudoinW Gray JeromeChristian KübelNiels de JongeNanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.http://europepmc.org/articles/PMC3561407?pdf=render |
| spellingShingle | Jean-Pierre Baudoin W Gray Jerome Christian Kübel Niels de Jonge Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography. PLoS ONE |
| title | Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography. |
| title_full | Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography. |
| title_fullStr | Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography. |
| title_full_unstemmed | Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography. |
| title_short | Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography. |
| title_sort | whole cell analysis of low density lipoprotein uptake by macrophages using stem tomography |
| url | http://europepmc.org/articles/PMC3561407?pdf=render |
| work_keys_str_mv | AT jeanpierrebaudoin wholecellanalysisoflowdensitylipoproteinuptakebymacrophagesusingstemtomography AT wgrayjerome wholecellanalysisoflowdensitylipoproteinuptakebymacrophagesusingstemtomography AT christiankubel wholecellanalysisoflowdensitylipoproteinuptakebymacrophagesusingstemtomography AT nielsdejonge wholecellanalysisoflowdensitylipoproteinuptakebymacrophagesusingstemtomography |