Multiplex Bioimaging of Single Cell Spatial Profiles for Precision Cancer Diagnostics and Therapeutics
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BIOE MEDIA LAB |
Digital posters for interactive cellular media and bioengineering education
Communications Biology, In Press
Mythreye Venkatesan & Ahmet F. Coskun
Posters disseminate progress reports in scientific meetings. While posters are effective in sharing results, they fail to deliver the need for visualization of dynamic biological data. Paper posters have become costly with the increasing number of conferences. Papers need to be reprinted for emerging research. Here we propose “digital posters” that repurpose digital frames from the art community. Multiple display items are designed and uploaded via the wireless network to the digital frames. Presenter waves hand over a motion sensor located at the left and the bottom of the digital frame to change the interactive display item in a predetermined order. Bioimages and multiplexed movies of cells were used as the proof-of-concept demonstration of the digital poster concept. Digital posters provide a dynamic and interactive tool for bioengineering and cellular image datasets. The fixed, low-cost set-up of digital posters makes them an ideal platform for next-generation sharing platforms.
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SPATIAL METABOLOMICS |
Ion beam subcellular tomography, BioRxiv, 2019
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.
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SPATIAL PROTEOMICS |
Subcellular localization of drug distribution by super-resolution ion beam imaging,
BioRxiv, 2019
Technologies that visualize multiple biomolecules at the nanometer scale in cells will enable deeper understanding of biological processes that proceed at the molecular scale. Current fluorescence-based methods for microscopy are constrained by a combination of spatial resolution limitations, limited parameters per experiment, and detector systems for the wide variety of biomolecules found in cells. We present here super-resolution ion beam imaging (srIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added small molecules. Uniquely, the atomic constituents of the biomolecules themselves can often be used in our system as the "tag". We visualized the subcellular localization of the chemotherapy drug cisplatin simultaneously with localization of five other nuclear structures, with further carbon elemental mapping and secondary electron visualization, down to ~30 nm lateral resolution. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. These data highlight how multiplexed super-resolution techniques, such as srIBI, will enable studies of biomolecule distributions in biologically relevant subcellular microenvironments.
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SPATIAL GENOMICS |
Dense transcript profiling in single cells by image correlation decoding,
Nature Methods 2016
Sequential barcoded fluorescent in situ hybridization (seqFISH) allows large numbers of molecular species to be accurately detected in single cells, but multiplexing is limited by the density of barcoded objects. We present correlation FISH (corrFISH), a method to resolve dense temporal barcodes in sequential hybridization experiments. Using corrFISH, we quantified highly expressed ribosomal protein genes in single cultured cells and mouse thymus sections, revealing cell-type-specific gene expression.
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SPATIAL GENOMICS |
Single-cell in situ RNA profiling by sequential hybridization,
Nature Methods 2014
In this Correspondence, we present a sequential barcoding scheme to multiplex different mRNAs.Here, the mRNAs in cells are barcoded by sequential rounds of hybridization, imaging and probe stripping. As the transcripts are fixed in cells, the corresponding fluorescent spots remain in place during multiple rounds of hybridization and can be aligned to read out a fluorophore sequence. This sequential barcode is designed to uniquely identify an mRNA
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BIOIMAGING |
Wide-field optical detection of nanoparticles using on-chip microscopy and self-assembled nanolenses,
Nature Photonics 2013
The direct observation of nanoscale objects is a challenging task for optical microscopy because the scattering from an individual nanoparticle is typically weak at optical wavelengths. Electron microscopy therefore remains one of the gold standard visualization methods for nanoparticles, despite its high cost, limited throughput and restricted field-of-view. Here, we describe a high-throughput, on-chip detection scheme that uses biocompatible wetting films to self-assemble aspheric liquid nanolenses around individual nanoparticles to enhance the contrast between the scattered and background light. We model the effect of the nanolens as a spatial phase mask centred on the particle and show that the holographic diffraction pattern of this effective phase mask allows detection of sub-100 nm particles across a large field-of-view of >20 mm2. As a proof-of-concept demonstration, we report on-chip detection of individual polystyrene nanoparticles, adenoviruses and influenza A (H1N1) viral particles.
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BIOIMAGING |
Imaging without lenses: achievements and remaining challenges of wide-field on-chip microscopy,
Nature Methods 2012
We discuss unique features of lens-free computational imaging tools and report some of their emerging results for wide-field on-chip microscopy, such as the achievement of a numerical aperture (NA) of ∼0.8–0.9 across a field of view (FOV) of more than 20 mm2 or an NA of ∼0.1 across a FOV of ∼18 cm2, which corresponds to an image with more than 1.5 gigapixels. We also discuss the current challenges that these computational on-chip microscopes face, shedding light on their future directions and applications.
