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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Smith EA,McDermott G,Do M,Leung K,Panning B,Le Gros MA,Larabell CA

doi

10.1016/j.bpj.2014.09.011

subject

Has Abstract

pub_date

2014-10-21 00:00:00

pages

1988-1996

issue

8

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(14)00949-7

journal_volume

107

pub_type

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