title: Quantitatively imaging chromosomes by correlated cryo-fluorescence and soft x-ray tomographies.
abstract: Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.
authors.affiliation: Department of Anatomy, University of California at San Francisco, San Francisco, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; National Center for X-ray Tomography, Advanced Light Source, Berkeley, California.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Smith
authors.affiliation: Department of Anatomy, University of California at San Francisco, San Francisco, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; National Center for X-ray Tomography, Advanced Light Source, Berkeley, California.
authors.forename: Gerry
authors.initials: G
authors.lastname: McDermott
authors.affiliation: Department of Anatomy, University of California at San Francisco, San Francisco, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; National Center for X-ray Tomography, Advanced Light Source, Berkeley, California.
authors.forename: Myan
authors.initials: M
authors.lastname: Do
authors.affiliation: Department of Anatomy, University of California at San Francisco, San Francisco, California.
authors.forename: Karen
authors.initials: K
authors.lastname: Leung
authors.affiliation: Biochemistry and Biophysics Department, University of California at San Francisco, San Francisco, California.
authors.forename: Barbara
authors.initials: B
authors.lastname: Panning
authors.affiliation: Department of Anatomy, University of California at San Francisco, San Francisco, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; National Center for X-ray Tomography, Advanced Light Source, Berkeley, California. Electronic address: mark.legros@ucsf.edu.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Le Gros
authors.affiliation: Department of Anatomy, University of California at San Francisco, San Francisco, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; National Center for X-ray Tomography, Advanced Light Source, Berkeley, California. Electronic address: carolyn.larabell@ucsf.edu.
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Larabell
mesh_terms: D045744:Cell Line, Tumor; D041321:Chromosomes, Human, X / Q000648:ultrastructure*; D015925:Cryopreservation / Q000379:methods; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D041622:Tomography, Optical / Q000379:methods*; D014056:Tomography, X-Ray / Q000379:methods*