title: Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography.
abstract: 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.
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors: Jean-Pierre
authors: JP
authors: Baudoin
authors: W Gray
authors: WG
authors: Jerome
authors: Christian
authors: C
authors: Kübel
authors: Niels
authors: N
authors: de Jonge
mesh_terms: D002460:Cell Line; D006046:Gold / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D053768:Metal Nanoparticles; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D021381:Protein Transport
chemical_list: D008077:Lipoproteins, LDL; D006046:Gold
references: Ann Anat. 2009 Apr;191(2):153-70
references: 19135344
references: Nat Protoc. 2011 Jun;6(6):845-58
references: 21637203
references: J Histochem Cytochem. 2000 Apr;48(4):471-80
references: 10727288
references: J Microsc. 1977 May;110(1):51-7
references: 409847
references: Clin Lipidol. 2010 Dec 1;5(6):853-865
references: 21643524
references: Curr Opin Cell Biol. 2009 Feb;21(1):89-96
references: 19185480
references: Chem Commun (Camb). 2009 Sep 7;(33):5009-11
references: 19668832
references: Cell. 2011 Apr 29;145(3):341-55
references: 21529710
references: Trends Biochem Sci. 2009 Feb;34(2):60-70
references: 19101147
references: J Struct Biol. 1996 Jan-Feb;116(1):71-6
references: 8742726
references: Histochem Cell Biol. 2009 Sep;132(3):253-62
references: 19649648
references: J Struct Biol. 2006 Jul;155(1):63-73
references: 16713294
references: J Struct Biol. 2011 Jun;174(3):552-62
references: 21440635
references: J Histochem Cytochem. 1995 Oct;43(10):1071-8
references: 7560885
references: Nat Methods. 2009 Jan;6(1):21-3
references: 19116610
references: Histochem Cell Biol. 2011 Jan;135(1):1-9
references: 21113611
references: Microsc Microanal. 2003 Feb;9(1):54-67
references: 12597787
references: Biotechnology. 1992;24:384-6
references: 1422046
references: Ultramicroscopy. 2008 Dec;109(1):70-80
references: 18977089
references: Nat Biotechnol. 2004 Feb;22(2):198-203
references: 14704683
references: PLoS Biol. 2011 Apr;9(4):e1001041
references: 21483721
references: J Cell Sci. 1995 Nov;108 ( Pt 11):3443-9
references: 8586656
references: J Struct Biol. 2007 Sep;159(3):507-22
references: 17689263
references: Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2159-64
references: 19164524
references: J Struct Biol. 2008 Mar;161(3):298-313
references: 18069000
references: Microsc Microanal. 2002 Apr;8(2):81-93
references: 12533238
references: Nanotoxicology. 2011 Sep;5(3):381-92
references: 20846020
references: Nature. 2011 Feb 17;470(7334):374-7
references: 21289625
references: Bioconjug Chem. 2002 Jan-Feb;13(1):3-6
references: 11792172
references: Nanomedicine. 2010 Feb;6(1):161-9
references: 19447206
references: Structure. 2012 May 9;20(5):759-66
references: 22579245
references: Nature. 2003 Mar 13;422(6928):216-25
references: 12634795
references: J Cell Sci. 1997 Sep;110 ( Pt 17):2027-40
references: 9378754
references: Science. 2007 May 25;316(5828):1153-8
references: 17525330
references: J Struct Biol. 2007 Sep;159(3):381-91
references: 17600727
references: J Microsc. 2011 Sep;243(3):273-83
references: 21770941
references: Nano Lett. 2011 Apr 13;11(4):1733-8
references: 21410218
references: Science. 2002 Nov 8;298(5596):1209-13
references: 12424373
references: Nanomedicine (Lond). 2010 Nov;5(9):1447-57
references: 21128725
references: Int J Mol Sci. 2009 Feb;10(2):441-91
references: 19333416
references: J Struct Biol. 2009 Mar;165(3):157-68
references: 19114107
references: Microsc Microanal. 2008 Apr;14(2):138-49
references: 18312718
references: Nature. 2003 Mar 6;422(6927):37-44
references: 12621426
references: Nat Rev Mol Cell Biol. 2007 Aug;8(8):622-32
references: 17637737
references: Bioessays. 2008 Jun;30(6):606-10
references: 18478541
references: Nanotechnology. 2008 May 7;19(18):
references: 21494419
references: Nat Methods. 2009 Oct;6(10):729-31
references: 19718033
references: Nano Rev. 2010;1:
references: 22110850
references: Br J Pharmacol. 2009 May;157(2):195-206
references: 19309362
references: Ultramicroscopy. 2010 Aug;110(9):1114-9
references: 20542380
references: J Cell Sci. 2000 May;113 ( Pt 9):1515-24
references: 10751143