scholarly journals Raman2RNA: Live-cell label-free prediction of single-cell RNA expression profiles by Raman microscopy

2021 ◽  
Author(s):  
Koseki J. Kobayashi-Kirschvink ◽  
Shreya Gaddam ◽  
Taylor James-Sorenson ◽  
Emanuelle Grody ◽  
Johain R. Ounadjela ◽  
...  

Single cell RNA-Seq (scRNA-seq) and other profiling assays have opened new windows into understanding the properties, regulation, dynamics, and function of cells at unprecedented resolution and scale. However, these assays are inherently destructive, precluding us from tracking the temporal dynamics of live cells, in cell culture or whole organisms. Raman microscopy offers a unique opportunity to comprehensively report on the vibrational energy levels of molecules in a label-free and non-destructive manner at a subcellular spatial resolution, but it lacks in genetic and molecular interpretability. Here, we developed Raman2RNA (R2R), an experimental and computational framework to infer single-cell expression profiles in live cells through label-free hyperspectral Raman microscopy images and multi-modal data integration and domain translation. We used spatially resolved single-molecule RNA-FISH (smFISH) data as anchors to link scRNA-seq profiles to the paired spatial hyperspectral Raman images, and trained machine learning models to infer expression profiles from Raman spectra at the single-cell level. In reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs), R2R accurately (r>0.96) inferred from Raman images the expression profiles of various cell states and fates, including iPSCs, mesenchymal-epithelial transition (MET) cells, stromal cells, epithelial cells, and fibroblasts. R2R outperformed inference from brightfield images, showing the importance of spectroscopic content afforded by Raman microscopy. Raman2RNA lays a foundation for future investigations into exploring single-cell genome-wide molecular dynamics through imaging data, in vitro and in vivo.

Materials ◽  
2021 ◽  
Vol 14 (13) ◽  
pp. 3732
Author(s):  
Jeon Woong Kang ◽  
Freddy T. Nguyen ◽  
Niyom Lue

Label-free live cell imaging was performed using a custom-built high-speed confocal Raman microscopy system. For various cell types, cell-intrinsic Raman bands were monitored. The high-resolution temporal Raman images clearly delineated the intracellular distribution of biologically important molecules such as protein, lipid, and DNA. Furthermore, optical phase delay measured using quantitative phase microscopy shows similarity with the image reconstructed from the protein Raman peak. This reported work demonstrates that Raman imaging is a powerful label-free technique for studying various biomedical problems in vitro with minimal sample preparation and external perturbation to the cellular system.


2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A4-A4
Author(s):  
Anushka Dikshit ◽  
Dan Zollinger ◽  
Karen Nguyen ◽  
Jill McKay-Fleisch ◽  
Kit Fuhrman ◽  
...  

BackgroundThe canonical WNT-β-catenin signaling pathway is vital for development and tissue homeostasis but becomes strongly tumorigenic when dysregulated. and alter the transcriptional signature of a cell to promote malignant transformation. However, thorough characterization of these transcriptomic signatures has been challenging because traditional methods lack either spatial information, multiplexing, or sensitivity/specificity. To overcome these challenges, we developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler (DSP) RNA assays. Using these methods, we sought to spatially profile and compare gene expression signatures of tumor niches with high and low CTNNB1 expression.MethodsAfter screening 120 tumor cores from multiple tumors for CTNNB1 expression by the RNAscope assay, we identified melanoma as the tumor type with the highest CTNNB1 expression while prostate tumors had the lowest expression. Using the RNAscope Multiplex Fluorescence assay we selected regions of high CTNNB1 expression within 3 melanoma tumors as well as regions with low CTNNB1 expression within 3 prostate tumors. These selected regions of interest (ROIs) were then transcriptionally profiled using the GeoMx DSP RNA assay for a set of 78 genes relevant in immuno-oncology. Target genes that were differentially expressed were further visualized and spatially assessed using the RNAscope Multiplex Fluorescence assay to confirm GeoMx DSP data with single cell resolution.ResultsThe GeoMx DSP analysis comparing the melanoma and prostate tumors revealed that they had significantly different gene expression profiles and many of these genes showed concordance with CTNNB1 expression. Furthermore, immunoregulatory targets such as ICOSLG, CTLA4, PDCD1 and ARG1, also demonstrated significant correlation with CTNNB1 expression. On validating selected targets using the RNAscope assay, we could distinctly visualize that they were not only highly expressed in melanoma compared to the prostate tumor, but their expression levels changed proportionally to that of CTNNB1 within the same tumors suggesting that these differentially expressed genes may be regulated by the WNT-β-catenin pathway.ConclusionsIn summary, by combining the RNAscope ISH assay and the GeoMx DSP RNA assay into one joint workflow we transcriptionally profiled regions of high and low CTNNB1 expression within melanoma and prostate tumors and identified genes potentially regulated by the WNT- β-catenin pathway. This novel workflow can be fully automated and is well suited for interrogating the tumor and stroma and their interactions.GeoMx Assays are for RESEARCH ONLY, not for diagnostics.


2018 ◽  
Vol 115 (52) ◽  
pp. 13204-13209 ◽  
Author(s):  
José Juan-Colás ◽  
Ian S. Hitchcock ◽  
Mark Coles ◽  
Steven Johnson ◽  
Thomas F. Krauss

Cell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein-secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro, and real-time analysis of specific single-cell signaling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet desialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.


2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.


Author(s):  
Kenneth H. Hu ◽  
John P. Eichorst ◽  
Chris S. McGinnis ◽  
David M. Patterson ◽  
Eric D. Chow ◽  
...  

ABSTRACTSpatial transcriptomics seeks to integrate single-cell transcriptomic data within the 3-dimensional space of multicellular biology. Current methods use glass substrates pre-seeded with matrices of barcodes or fluorescence hybridization of a limited number of probes. We developed an alternative approach, called ‘ZipSeq’, that uses patterned illumination and photocaged oligonucleotides to serially print barcodes (Zipcodes) onto live cells within intact tissues, in real-time and with on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in-vitro wound healing, live lymph node sections and in a live tumor microenvironment (TME). In all cases, we discovered new gene expression patterns associated with histological structures. In the TME, this demonstrated a trajectory of myeloid and T cell differentiation, from periphery inward. A variation of ZipSeq efficiently scales to the level of single cells, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.


2016 ◽  
Vol 113 (42) ◽  
pp. E6352-E6361 ◽  
Author(s):  
Shalin B. Mehta ◽  
Molly McQuilken ◽  
Patrick J. La Riviere ◽  
Patricia Occhipinti ◽  
Amitabh Verma ◽  
...  

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.


2021 ◽  
Author(s):  
Sanshiro Kanazawa ◽  
Hironori Hojo ◽  
Shinsuke Ohba ◽  
Junichi Iwata ◽  
Makoto Komura ◽  
...  

Abstract Although multiple studies have investigated the mesenchymal stem and progenitor cells (MSCs) that give rise to mature bone marrow, high heterogeneity in their morphologies and properties causes difficulties in molecular separation of their distinct populations. In this study, by taking advantage of the resolution of the single cell transcriptome, we analyzed Sca-1 and PDGFR-α fraction in the mouse bone marrow tissue. The single cell transcriptome enabled us to further classify the population into seven populations according to their gene expression profiles. We then separately obtained the seven populations based on candidate marker genes, and specified their gene expression properties and epigenetic landscape by ATAC-seq. Our findings will enable to elucidate the stem cell niche signal in the bone marrow microenvironment, reconstitute bone marrow in vitro, and shed light on the potentially important role of identified subpopulation in various clinical applications to the treatment of bone- and bone marrow-related diseases.


2018 ◽  
Author(s):  
Efthymios Motakis ◽  
Diana H.P. Low

AbstractModern high-throughput single-cell technologies facilitate the efficient processing of hundreds of individual cells to comprehensively study their morphological and genomic heterogeneity. Fluidigm’s C1 Auto Prep system isolates fluorescence-stained cells into specially designed capture sites, generates high-resolution image data and prepares the associated cDNA libraries for mRNA sequencing. Current statistical methods focus on the analysis of the gene expression profiles and ignore the important information carried by the images. Here we propose a new direction for single-cell data analysis and develop CONFESS, a customized cell detection and fluorescence signal estimation model for images coming from the Fluidigm C1 system. Applied to a set of HeLa cells expressing fluorescence cell cycle reporters, the method predicted the progression state of hundreds of samples and enabled us to study the spatio-temporal dynamics of the HeLa cell cycle. The output can be easily integrated with the associated single-cell RNA-seq expression profiles for deeper understanding of a given biological system. CONFESS R package is available at Bioconductor (http://bioconductor.org/packages/release/bioc/html/CONFESS.html).


Author(s):  
Ália dos Santos ◽  
Natalia Fili ◽  
David S. Pearson ◽  
Yukti Hari-Gupta ◽  
Christopher P. Toseland

ABSTRACTMechanobiology is focused on how the physical forces and the mechanical properties of proteins, cells and tissues contribute to physiology and disease. While the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. The broad applicability and versatility of our approach has been demonstrated through in vitro force-induced enzymatic activity and conformation changes, along with force-induced receptor activation and their downstream signalling pathways in live cells. Overall, our methodology allows for the first-time ensemble biochemical and cell-based assays to be performed under force, in high throughput format. This novel approach would substantially add to the mechano-biological toolbox and increase the availability of mechanobiology measurements.


Sign in / Sign up

Export Citation Format

Share Document