RT Journal Article
SR Electronic
T1 Ion beam subcellular tomography
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 557728
DO 10.1101/557728
A1 Ahmet F. Coskun
A1 Guojun Han
A1 Shih-Yu Chen
A1 Xavier Rovira Clavé
A1 Sizun Jiang
A1 Christian M. Schürch
A1 Yunhao Bai
A1 Chuck Hitzman
A1 Garry P. Nolan
YR 2019
UL http://biorxiv.org/content/early/2019/02/22/557728.abstract
AB Multiplexed ion beam imaging (MIBI) has been previously used to profile multiple parameters in two dimensions in single cells within tissue slices. Here, a mathematical and technical framework for three-dimensional subcellular MIBI is presented. We term the approach ion beam tomography (IBT) wherein ion beam images are acquired iteratively across successive, multiple scans and later compiled into a 3D format. For IBT, cells were imaged at 0.2-4 pA ion current across 1,000 axial scans. Consecutive subsets of ion beam images were binned over 3 to 20 slices (above and below) to create a resolved image, wherein binning was incremented one slice at a time to yield an enhanced multi-depth data without loss of depth resolution. Algorithmic deconvolution, tailored for ion beams, was then applied to the transformed ion image series using a hybrid deblurring algorithm and an ion beam current-dependent point-spread function. Three-dimensional processing was implemented by segmentation, mesh, molecular neighborhoods, and association maps. In cultured cancer cells and tissues, IBT enabled accessible visualization of three-dimensional volumetric distributions of genomic regions, RNA transcripts, and protein factors with 65-nm lateral and 5-nm axial resolution. IBT also enabled label-free elemental mapping of cells, allowing “point of source” cellular component measurements not possible for most optical microscopy targets. Detailed multiparameter imaging of subcellular features at near macromolecular resolution should now be made possible by the IBT tools and reagents provided here to open novel venues for interrogating subcellular biology.