title: Optimizing imaging of three-dimensional multicellular tumor spheroids with fluorescent reporter proteins using confocal microscopy.
abstract: Tumor spheroids more faithfully mimic tumor biology than monolayer cultures and require three-dimensional microscopy. Our goal in this study was to overcome the limitations of signal to noise ratio that have traditionally limited three-dimensional imaging to depths of 100 microm or less. We studied the expression of hypoxia-inducible factor 1alpha (HIF-1alpha), the main regulator of cellular hypoxic response in C6 glioma spheroids. In our spheroids, red fluorescent protein is expressed constitutively and green fluorescent protein is expressed conditionally under control of a HIF-1alpha promoter. In this article, we show a series of optimizations that allowed us to obtain excellent quality confocal microscopy images at imaging depths of up to 320 microm. The combined use of special objectives, glass-bottomed culture dishes, and depth-dependent laser output modulation extended our depth range beyond previously accepted limits. This allowed us to image up to the equator of spheroids of 650 microm diameter, allowing interrogation of HIF-1alpha expression from the spheroid periphery to its hypoxic center.
authors.affiliation: Department of Experimental Diagnostic Imaging, The University of Texas, M. D. Anderson Cancer Center, Houston, 77030, USA.
authors.forename: Lucia
authors.initials: L
authors.lastname: le Roux
authors.forename: Andrei
authors.initials: A
authors.lastname: Volgin
authors.forename: David
authors.initials: D
authors.lastname: Maxwell
authors.forename: Katashi
authors.initials: K
authors.lastname: Ishihara
authors.forename: Juri
authors.initials: J
authors.lastname: Gelovani
authors.forename: Dawid
authors.initials: D
authors.lastname: Schellingerhout
mesh_terms: D001932:Brain Neoplasms / Q000235:genetics / Q000473:pathology; D003952:Diagnostic Imaging; D017930:Genes, Reporter; D005910:Glioma / Q000235:genetics / Q000473:pathology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D049408:Luminescent Agents / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D049408:Luminescent Agents; D008164:Luminescent Proteins; D011506:Proteins; D000097573:Red Fluorescent Protein