title: An efficient and reproducible process for transmission electron microscopy (TEM) of rare cell populations.
abstract: Transmission electron microscopy (TEM) provides ultra-structural details of cells at the sub-organelle level. However, details of the cellular ultrastructure, and the cellular organization and content of various organelles in rare populations, particularly in the suspension, like hematopoietic stem cells (HSCs) remained elusive. This is mainly due to the requirement of millions of cells for TEM studies. Thus, there is a vital requirement of a method that will allow TEM studies with low cell numbers of such rare populations. We describe an alternative and novel approach for TEM studies for rare cell populations. Here we performed a TEM study from 10,000 HSC cells with relative ease. In particular, tiny cell pellets were identified by Evans blue staining after PFA-GA fixation. The cell pellet was pre-embedded in agarose in a small microcentrifuge tube and processed for dehydration, infiltration and embedding. Semi-thin and ultra-thin sections identified clusters of numerous cells per sections with well preserved morphology and ultrastructural details of golgi complex and mitochondria. Together, this method provides an efficient, easy and reproducible process to perform qualitative and quantitative TEM analysis from limited biological samples including cells in suspension.
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Pathology Department, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA. Electronic address: Sachin.kumar@cchmc.org.
authors.forename: Sachin
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Electron microscopy Unit, Pathology Department, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA.
authors.forename: Georgianne
authors.initials: G
authors.lastname: Ciraolo
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Pathology Department, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA.
authors.forename: Ashwini
authors.initials: A
authors.lastname: Hinge
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Pathology Department, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA. Electronic address: Marie-Dominique.Filippi@cchmc.org.
authors.forename: Marie-Dominique
authors.initials: MD
authors.lastname: Filippi
mesh_terms: D000818:Animals; D006056:Golgi Apparatus / Q000648:ultrastructure; D006412:Hematopoietic Stem Cells / Q000648:ultrastructure*; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008867:Microtomy; D008928:Mitochondria / Q000648:ultrastructure; D015203:Reproducibility of Results; D012685:Sepharose; D016610:Tissue Embedding; D016707:Tissue Fixation
chemical_list: D012685:Sepharose
keywords: Rare population; Stem cell; Transmission electron microscopy; Ultra structure