title: Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.
abstract: A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.
authors: South Western Sydney Clinical School, Faculty of Medicine, University of New South Wales Australia; School of Medicine, Western Sydney University; Correlative Microscopy Group, Ingham Institute for Applied Medical Research; Electron Microscopy Laboratory, Department of Anatomical Pathology, Sydney South West Pathology Service, New South Wales Health Pathology; murray.killingsworth@sswahs.nsw.gov.au.
authors: Murray C
authors: MC
authors: Killingsworth
authors: Correlative Microscopy Group, Ingham Institute for Applied Medical Research; Electron Microscopy Laboratory, Department of Anatomical Pathology, Sydney South West Pathology Service, New South Wales Health Pathology; School of Medical Sciences, Faculty of Medicine, University of New South Wales Australia.
authors: Yuri V
authors: YV
authors: Bobryshev
mesh_terms: D005453:Fluorescence; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D053758:Nanoparticles / Q000648:ultrastructure*; D045663:Quantum Dots / Q000648:ultrastructure*; D018036:Selenium Compounds / Q000737:chemistry*
chemical_list: D018036:Selenium Compounds