scholarly journals Multiplexed imaging of high density libraries of RNAs with MERFISH and expansion microscopy

2017 ◽  
Author(s):  
Guiping Wang ◽  
Jeffrey R. Moffitt ◽  
Xiaowei Zhuang
Keyword(s):  
Single Molecule ◽  
Detection Efficiency ◽  
High Abundance ◽  
Total Density ◽  
Accurate Identification ◽  
Rna Molecules ◽  
Wide Range ◽  
Multiplexed Imaging ◽  
Labeling Approach

AbstractAs an image-based single-cell transcriptomics approach, multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows hundreds to thousands of RNA species to be identified, counted and localized in individual cells while preserving the native spatial context of RNAs. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule FISH (smFISH) to read out these barcodes. The accuracy of RNA identification relies on spatially separated signals from individual RNA molecules, which limits the density of RNAs that can be measured and makes the multiplexed imaging of a large number of high-abundance RNAs challenging. Here we report an approach that combines MERFISH and expansion microscopy to substantially increase the total density of RNAs that can be measured. Using this approach, we demonstrate accurate identification and counting of RNAs, with a near 100% detection efficiency, in a ~130-RNA library composed of many high-abundance RNAs, the total density of which is more than 10 fold higher than previously reported. In parallel, we demonstrate the combination of MERFISH with immunofluorescence. These advances increase the versatility of MERFISH and will facilitate its application of a wide range of biological problems.

scholarly journals High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

2016 ◽  
Vol 113 (50) ◽  
pp. 14456-14461 ◽  
Author(s):  
Jeffrey R. Moffitt ◽  
Junjie Hao ◽  
Dhananjay Bambah-Mukku ◽  
Tian Lu ◽  
Catherine Dulac ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Single Molecule ◽  
High Performance ◽  
Detection Efficiency ◽  
Tissue Samples ◽  
Spatially Resolved ◽  
Wide Range ◽  
Cellular Context ◽  
Target Binding

Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background—from off-target binding of FISH probes and cellular autofluorescence—can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.

scholarly journals Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4–P6 RNA by smFRET

2015 ◽  
Vol 43 (2) ◽  
pp. 172-178 ◽  
Author(s):  
Namita Bisaria ◽  
Daniel Herschlag
Keyword(s):  
Single Molecule ◽  
Specific Binding ◽  
Specific Interaction ◽  
Monovalent Cations ◽  
Folding Kinetics ◽  
Rna Molecules ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Monovalent Ion

Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4–P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na+ and K+, to the RNA tetraloop–tetraloop receptor (TL–TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state.

scholarly journals ProbeDealer is a convenient tool for designing probes for highly multiplexed fluorescence in situ hybridization

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mengwei Hu ◽  
Bing Yang ◽  
Yubao Cheng ◽  
Jonathan S. D. Radda ◽  
Yanbo Chen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Molecule ◽  
Probe Design ◽  
Powerful Method ◽  
Rna Fish ◽  
Technological Advances ◽  
Multiplexed Imaging ◽  
One Stop

AbstractFluorescence in situ hybridization (FISH) is a powerful method to visualize the spatial positions of specific genomic loci and RNA species. Recent technological advances have leveraged FISH to visualize these features in a highly multiplexed manner. Notable examples include chromatin tracing, RNA multiplexed error-robust FISH (MERFISH), multiplexed imaging of nucleome architectures (MINA), and sequential single-molecule RNA FISH. However, one obstacle to the broad adoption of these methods is the complexity of the multiplexed FISH probe design. In this paper, we introduce an easy-to-use, versatile, and all-in-one application called ProbeDealer to design probes for a variety of multiplexed FISH techniques and their combinations. ProbeDealer offers a one-stop shop for multiplexed FISH design needs of the research community.

scholarly journals NAD tagSeq reveals that NAD+-capped RNAs are mostly produced from a large number of protein-coding genes in Arabidopsis

2019 ◽  
pp. 201903683 ◽  
Author(s):  
Hailei Zhang ◽  
Huan Zhong ◽  
Shoudong Zhang ◽  
Xiaojian Shao ◽  
Min Ni ◽  
...  
Keyword(s):  
Single Molecule ◽  
Enzymatic Reaction ◽  
Accurate Identification ◽  
Protein Coding ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Protein Coding Genes ◽  
Oxford Nanopore ◽  
Almost All ◽  
Identification And Quantification

The 5′ end of a eukaryotic mRNA transcript generally has a 7-methylguanosine (m7G) cap that protects mRNA from degradation and mediates almost all other aspects of gene expression. Some RNAs in Escherichia coli, yeast, and mammals were recently found to contain an NAD+ cap. Here, we report the development of the method NAD tagSeq for transcriptome-wide identification and quantification of NAD+-capped RNAs (NAD-RNAs). The method uses an enzymatic reaction and then a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq can allow more accurate identification and quantification of NAD-RNAs, as well as reveal the sequences of whole NAD-RNA transcripts using single-molecule RNA sequencing. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis were produced by at least several thousand genes, most of which are protein-coding genes, with the majority of these transcripts coming from <200 genes. For some Arabidopsis genes, over 5% of their transcripts were NAD capped. Gene ontology terms overrepresented in the 2,000 genes that produced the highest numbers of NAD-RNAs are related to photosynthesis, protein synthesis, and responses to cytokinin and stresses. The NAD-RNAs in Arabidopsis generally have the same overall sequence structures as the canonical m7G-capped mRNAs, although most of them appear to have a shorter 5′ untranslated region (5′ UTR). The identification and quantification of NAD-RNAs and revelation of their sequence features can provide essential steps toward understanding the functions of NAD-RNAs.

scholarly journals FISIK: Framework for the Inference of in Situ Interaction Kinetics from single-molecule imaging data

2019 ◽  
Author(s):  
Luciana R. de Oliveira ◽  
Khuloud Jaqaman
Keyword(s):  
Single Molecule ◽  
Molecular Interaction ◽  
Model Parameters ◽  
Single Molecule Imaging ◽  
Imaging Data ◽  
Tracking Errors ◽  
Spatiotemporal Resolution ◽  
Wide Range ◽  
Interaction Kinetics

AbstractRecent experimental and computational developments have been pushing the limits of live-cell single-molecule imaging, enabling the monitoring of inter-molecular interactions in their native environment with high spatiotemporal resolution. However, interactions are captured only for the labeled subset of molecules, which tends to be a small fraction. As a result, it has remained a challenge to calculate molecular interaction kinetics, in particular association rates, from single-molecule tracking data. To overcome this challenge, we developed a mathematical modeling-based Framework for the Inference of in Situ Interaction Kinetics from single-molecule imaging data (termed “FISIK”). FISIK consists of (1) devising a mathematical model of molecular movement and interactions, mimicking the biological system and data-acquisition setup, and (2) estimating the unknown model parameters, including molecular association and dissociation rates, by fitting the model to experimental single-molecule data. Due to the stochastic nature of the model and data, we adapted the method of indirect inference for model calibration. We validated FISIK using a series of tests, where we simulated trajectories of diffusing molecules that interact with each other, considering a wide range of model parameters, and including resolution limitations and tracking errors. We found that FISIK has the sensitivity to determine association and dissociation rates, where its accuracy depends on the labeled fraction of molecules and the extent of molecule tracking errors. For cases where the labeled fraction is relatively low (e.g. to afford accurate tracking), combining dynamic but sparse single-molecule imaging data with almost whole-population oligomer distribution data improves FISIK’s performance. All in all, FISIK is a promising approach for the derivation of molecular interaction kinetics in their native environment from single-molecule imaging data.

scholarly journals High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization

2016 ◽  
Vol 113 (39) ◽  
pp. 11046-11051 ◽  
Author(s):  
Jeffrey R. Moffitt ◽  
Junjie Hao ◽  
Guiping Wang ◽  
Kok Hao Chen ◽  
Hazen P. Babcock ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cell ◽  
Single Molecule ◽  
Rna Profiling ◽  
Drastic Increase ◽  
Cell Gene Expression ◽  
A Cell ◽  
Labeling Approach

Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.

In-situ electrical-resistivity measurements in the high-voltage electron microscope

1978 ◽  
Vol 36 (1) ◽  
pp. 68-69
Author(s):  
W. E. King
Keyword(s):  
Electrical Resistivity ◽  
Electron Microscope ◽  
High Voltage ◽  
Resistivity Measurements ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
High Voltage Electron ◽  
Wide Range ◽  
Helium Gas

A side-entry type, helium-temperature specimen stage that has the capability of in-situ electrical-resistivity measurements has been designed and developed for use in the AEI-EM7 1200-kV electron microscope at Argonne National Laboratory. The electrical-resistivity measurements complement the high-voltage electron microscope (HVEM) to yield a unique opportunity to investigate defect production in metals by electron irradiation over a wide range of defect concentrations.A flow cryostat that uses helium gas as a coolant is employed to attain and maintain any specified temperature between 10 and 300 K. The helium gas coolant eliminates the vibrations that arise from boiling liquid helium and the temperature instabilities due to alternating heat-transfer mechanisms in the two-phase temperature regime (4.215 K). Figure 1 shows a schematic view of the liquid/gaseous helium transfer system. A liquid-gas mixture can be used for fast cooldown. The cold tip of the transfer tube is inserted coincident with the tilt axis of the specimen stage, and the end of the coolant flow tube is positioned without contact within the heat exchanger of the copper specimen block (Fig. 2).

Site specific agricultural soil management with the use of new technologies

2013 ◽  
Vol 16 (1) ◽  
pp. 59-67
Keyword(s):  
New Technologies ◽  
Digital Interface ◽  
Appropriate Treatment ◽  
Wide Range ◽  
Soil Variation ◽  
Soil Information ◽  
Remote Sensing Techniques ◽  
Agricultural Areas

<p>The Soil Science Institute of Thessaloniki produces new digitized Soil Maps that provide a useful electronic database for the spatial representation of the soil variation within a region, based on in situ soil sampling, laboratory analyses, GIS techniques and plant nutrition mathematical models, coupled with the local land cadastre. The novelty of these studies is that local agronomists have immediate access to a wide range of soil information by clicking on a field parcel shown in this digital interface and, therefore, can suggest an appropriate treatment (e.g. liming, manure incorporation, desalination, application of proper type and quantity of fertilizer) depending on the field conditions and cultivated crops. A specific case study is presented in the current work with regards to the construction of the digitized Soil Map of the regional unit of Kastoria. The potential of this map can easily be realized by the fact that the mapping of the physicochemical properties of the soils in this region provided delineation zones for differential fertilization management. An experiment was also conducted using remote sensing techniques for the enhancement of the fertilization advisory software database, which is a component of the digitized map, and the optimization of nitrogen management in agricultural areas.</p>

Sign in / Sign up

Export Citation Format

Share Document