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Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

ABSTRACT: Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA's multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA's analysis is strongly correlated with sequencing data (Pearson's r = 0.96) and was further benchmarked using RNAscope^TM and LGC Stellaris^TM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.

SUBMITTER: Vu T

PROVIDER: S-EPMC8748653 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

Vu Tam T Vallmitjana Alexander A Gu Joshua J La Kieu K Xu Qi Q Flores Jesus J Zimak Jan J Shiu Jessica J Hosohama Linzi L Wu Jie J Douglas Christopher C Waterman Marian L ML Ganesan Anand A Hedde Per Niklas PN Gratton Enrico E Zhao Weian W

Nature communications 20220110 1

Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and li ...[more]

PMID: 35013281

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Project description:PurposePrevious efforts at increasing spatial resolution have relied on decreasing focal spot and or detector element size. Many "super resolution" methods require physical movement of a component of the imaging system. This work describes a method for achieving spatial resolution on a scale smaller than the detector pixel without motion of the object or detector.MethodsWe introduce a weighting of the photon energy spectrum on a length scale smaller than a single pixel using a physical filter that can be placed between the focal spot and the object, between the object and the detector, or integrated into the x-ray source or detector. We refer to the method as sub pixel encoding (SPE). We show that if one acquires multiple measurements (i.e. x-ray projections), information can be synthesized at a spatial scale defined by the spectrum modulation, not the detector element size. Specifically, if one divides a detector pixel into n sub regions, and m photon-matter interactions are present, the number of x-ray measurements needed to solve for the detector response of each sub region is mxn. We discuss realizations of SPE using multiple x-ray spectra with an energy integrating detector, a single spectra with a photon counting detector, and the single photon-matter interaction case. We demonstrate the feasibility of the approach using a simulated energy integrating detector with a detector pitch of 2 mm for 80-140 kV medical and 200-600 kV industrial applications. Phantoms used for both example SPE realization had some features only a 1 mm detector could resolve. We calculate the covariance matrix of SPE output to characterize the and noise propagation and correlation of our test examples.ResultsThe mathematical foundation of SPE is provided, with details worked out for several detector types and energy ranges. Two numerical simulations were provided to demonstrate feasibility. In both the medical and industrial simulations, some phantom features were only observable with the 1 mm and SPE synthesized 2 mm detector, while the 2 mm detector was not able to visualize them. Covariance matrix analysis demonstrated negative diagonal terms for both example cases.ConclusionsThe concept of encoding object information at a length scale smaller than a single pixel element, and then retrieving that information was introduced. SPE simultaneously allows for an increase in spatial resolution and provides "dual energy" like information about the underlying photon-matter interactions.

Dataset Information

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

Publications

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

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