scholarly journals Rapid Sequential in Situ Multiplexing With DNA-Exchange-Imaging

2017 ◽  
Author(s):  
Yu Wang ◽  
Johannes B. Woehrstein ◽  
Noah Donoghue ◽  
Mingjie Dai ◽  
Maier S. Avendaño ◽  
...  
Keyword(s):  
Single Channel ◽  
Super Resolution ◽  
Clinical Pathology ◽  
Single Step ◽  
Tissue Sections ◽  
Fresh Frozen ◽  
Biological Environment ◽  
New Applications ◽  
20 Nm

AbstractTo decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 °C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

scholarly journals Demonstration of Apoptotic Cells in Tissue Sections by In Situ Hybridization Using Digoxigenin-labeled Poly(A) Oligonucleotide Probes to Detect Thymidine-rich DNA Sequences

1997 ◽  
Vol 45 (1) ◽  
pp. 13-20 ◽  
Author(s):  
David A Hilton ◽  
Seth Love ◽  
Rachel Barber
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Early Stage ◽  
Nuclear Genome ◽  
Apoptotic Cells ◽  
Oligonucleotide Probes ◽  
Tissue Sections ◽  
Fresh Frozen

The recognition of apoptotic cells by morphological appearance alone may be difficult. We have investigated the use of in situ hybridization (ISH) with digoxigenin-labeled poly(A) probes to detect apoptotic cells in tissue sections. This method was compared to conventional morphologic assessment and in situ end-labeling (ISEL) in a range of tissues in which apoptosis is known to occur. ISH with poly(A) probes detected apoptotic nuclei in all tissues in which there was evidence of apoptosis as judged by conventional histology. ISH and, to a lesser extent, ISEL preferentially labeled shrunken but still intact nuclei with margination of chromatin, presumably at an early stage of apoptosis. The poly(A) hybridization was abolished by pretreatment of tissue sections with DNAse. After denaturation of DNA, poly(A) hybridized to nuclei in all cells. No convincing hybridization signal was detected in alcohol-fixed or fresh-frozen sections. Both ISEL and ISH labeled some of the nuclei in ischemic tissues. ISH with poly(A) oligonucleotide probes offers a simple alternative to ISEL for detection of cells in early stages of apoptosis. These probes hybridize to thymidine-rich sequences of DNA in the highly repeated Alu sequences within the nuclear genome. These sequences are believed to become available for hybridization after formalin fixation and paraffin embedding as a result of the apoptosis-related increase in the susceptibility of nuclear DNA to denaturation.

FC 017DEEP-LEARNING ENABLED QUANTIFICATION OF SINGLE-CELL SINGLE-MRNA TRANSCRIPTS AND CORRELATIVE SUPER-RESOLVED PODOCYTE FOOT PROCESS MORPHOMETRY IN ROUTINE KIDNEY BIOPSY SPECIMEN

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Florian Siegerist ◽  
Eleonora Hay ◽  
Julien Dang ◽  
Nassim Mahtal ◽  
Pierre-Louis Tharaux ◽  
...  
Keyword(s):  
Deep Learning ◽  
Single Cell ◽  
Kidney Biopsy ◽  
Super Resolution ◽  
Single Step ◽  
Cell Nuclei ◽  
Tissue Sections ◽  
Mrna Transcripts ◽  
Foot Process ◽  
Normfinder Algorithm

Abstract Background and Aims Although high-throughput single-cell transcriptomic analysis, super-resolution light microscopy and deep-learning methods are broadly used, the gold-standard to evaluate kidney biopsies is still the histologic assessment of formalin-fixed and paraffin embedded (FFPE) samples with parallel ultrastructural evaluation. Recently, we and others have shown that super-resolution fluorescence microscopy can be used to study glomerular ultrastructure in human biopsy samples. Additionally, in the last years mRNA in situ hybridization techniques have been improved to increase specificity and sensitivity to enable transcriptomic analysis with single-mRNA resolution (smFISH). Method For smFISH, we used the fluorescent multiplex RNAscope kit with probes targeting ACE2, WT1, PPIB, UBC and POLR2A. To find an on-slide reference gene, the normfinder algorithm was used. The smFISH protocol was combined with a single-step anti-podocin immunofluorescence enabled by VHH nanobodies. Podocytes were labeled by tyramide-signal amplified immunofluorescence using recombinant anti-WT1 antibodies. Slides were imaged using confocal laser scanning, as well as 3D structured illumination microscopy. Deep-learning networks to segment glomeruli and cell nuclei (UNet and StarDist) were trained using the ZeroCostDL4Mic approach. Scripts to automate analysis were developed in the ImageJ1 macro language. Results First, we show robust functionality of threeplex smFISH in archived routine FFPE kidney biopsy samples with single-mRNA resolution. As variations in sample preparation can negatively influence mRNA-abundance, we established PPIB as an ideal on-slide reference gene to account for different RNA-integrities present in biopsy samples. PPIB was chosen for its most stable expression in microarray dataset of various glomerular diseases determined by the Normfinder algorithm as well as its smFISH performance. To segment glomeruli and to label glomerular and tubulointerstitial cell subsets, we established a combination of smFISH and immunofluorescence. As smFISH requires intense tissue digestion to liberate cross-linked RNAs, immunofluorescence protocols had to be adapted: For podocin, a small-sized single-step label approach enabled by small nanobodies and for WT1, tyramide signal amplification was used. For enhanced segmentation performance, we used deep learning: First, a network was customized to recognize DAPI+ cell nuclei and WT1/DAPI+ podocyte nuclei. Second, a UNet was trained to segment glomeruli in podocin-stained tissue sections. Using these segmentation masks, we could annotate PPIB-normalized single mRNA transcripts to individual cells. We established an ImageJ script to automatize transcript quantification. As a proof-of-principle, we demonstrate inverse expression of WT1 and ACE2 in glomerular vs. tubulointerstitial single cells. Furthermore, in the podocyte subset, WT1 highly clustered whereas no significant ACE2 expression was found under baseline conditions. Additionally, when imaged with super-resolution microscopy, podocyte filtration slit morphology could be visualized The optical resolution was around 125 nm and therefore small enough to resolve individual foot processes. The filtration slit density as a podocyte-integrity marker did not differ significantly from undigested tissue sections proving the suitability for correlative podocyte foot process morphometry with single-podocyte transcript analysis. Conclusion Here we present a modular toolbox which combines algorithms for multiplexed, normalized single-cell gene expression with single mRNA resolution in cellular subsets (glomerular, tubulointerstitial and podocytes). Additionally, this approach enables correlation with podocyte filtration slit ultrastructure and gross glomerular morphometry.

scholarly journals Quantitative Comparison of Pretreatment Regimens Used to Sensitize In Situ Hybridization Using Oligonucleotide Probes on Paraffin-embedded Brain Tissue

1997 ◽  
Vol 45 (12) ◽  
pp. 1707-1713 ◽  
Author(s):  
Kevin R. Oliver ◽  
Robert P. Heavens ◽  
Dalip J. S. Sirinathsinghji
Keyword(s):  
In Situ Hybridization ◽  
Quantitative Comparison ◽  
Oligonucleotide Probes ◽  
Frozen Sections ◽  
Double Labeling ◽  
Tissue Sections ◽  
Fresh Frozen ◽  
Rat Tissue ◽  
Made In

Paraffin embedding of tissue is generally perceived to dramatically reduce RNA detectability. As a consequence, in situ hybridization on paraffin-embedded tissue is largely confined to detection of high-copy RNA species (e.g., viral RNA) and/or to detection using typically more sensitive cDNA probes or riboprobes. In this study, several procedures for in situ hybridization on paraffin-embedded rat tissue using oligonucleotide probes complementary to cellular transcripts were developed and quantitatively compared. Certain pretreatments showed marked increases in sensitivity compared to untreated sections. Furthermore, through quantitative assessment using image analysis, sensitivity of optimal pretreatments was equal to that of routinely used fresh-frozen, postfixed tissue sections. The development of such techniques permitting in situ hybridization to be carried out on paraffin-embedded tissue allows a comparison of protein and mRNA distribution to be made in adjacent sections and provides the potential for double labeling by in situ hybridization and immunohistochemistry which may not be possible on post-fixed frozen sections.

Flat embeddment of monolayer cells, tissue sections, and other cellular materials attached onto glass substrate

1990 ◽  
Vol 48 (3) ◽  
pp. 766-767
Author(s):  
Jeffrey P. Chang ◽  
Jaang J. Wang
Keyword(s):  
Present Report ◽  
Cellular Materials ◽  
Cover Glass ◽  
Tissue Sections ◽  
Embedding Technique ◽  
Exfoliated Cells ◽  
Black Paper ◽  
Cell Concentrations ◽  
Flat Embedding

Flat embeddment of certain specimens for electron microscopy is necessary for three classes of biological materials: namely monolayer cells, tissue sections of paraffin or plastics, as well as cell concentrations, exfoliated cells, and cell smears. The present report concerns a flat-embedding technique which can be applied to all these three classes of materials and which is a modified and improved version of Chang's original methodology.Preparation of coverglasses and microslides. Chemically cleaned coverglasses, 11 × 22 mm or other sizes, are laid in rows on black paper. Ink-mark one coner for identifying the spray-side of the glass for growing cells. Lightly spray with Teflon monomer (Heddy/Contact Inductries, Paterson, NO 07524, U.S.A.) from a pressurized can. Bake the sprayed glasses at 500°F for 45 min on Cover-Glass Ceramic Racks (A. Thomas Co. Philadelphia), for Teflon to polymerize.Monolayer Cells. After sterilization, the Teflon-treated coverglasses, with cells attached, are treated or fixed in situ in Columbia staining dishes (A. Thomas Co., Philadelphia) for subsequent processing.

In-Situ Observation of Clay Minerals Hydrated and Intercalated With Liquid Chemicals Using Film-Sealed Environmental Cell

1980 ◽  
Vol 38 ◽  
pp. 208-209 ◽  
Author(s):  
K. Fukushima ◽  
N. Kohyama ◽  
A. Fukami
Keyword(s):  
Carbon Film ◽  
Resolving Power ◽  
In Situ Observation ◽  
Interlayer Water ◽  
Saturated Water ◽  
Structure Changes ◽  
Environmental Cell ◽  
Gas Layer ◽  
20 Nm

A film-sealed high resolution environmental cell(E.C) for observing hydrated materials had been developed by us(l). Main specification of the E.C. is as follows: 1) Accelerated voltage; 100 kV. 2) Gas in the E.C.; saturated water vapour with carrier gas of 50 Torr. 3) Thickness of gas layer; 50 μm. 4) Sealing film; evaporated carbon film(20 nm thick) with plastic microgrid. 5) Resolving power; 1 nm. 6) Transmittance of electron beam; 60% at 100 kV. The E.C. had been successfully applied to the study of hydrated halloysite(2) (3). Kaolin minerals have no interlayer water and are basically non-expandable but form intercalation compounds with some specific chemicals such as hydrazine, formamide and etc. Because of these compounds being mostly changed in vacuum, we tried to reveal the structure changes between in wet air and in vacuum of kaolin minerals intercalated with hydrazine and of hydrated state of montmori1lonite using the E.C. developed by us.

scholarly journals Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

2021 ◽  
Vol 10 (2) ◽  
pp. 319
Author(s):  
Hee Cheol Yang ◽  
Won Jong Rhee
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Molecular Beacon ◽  
Disease Diagnosis ◽  
Single Step ◽  
Flow Cytometer ◽  
Surface Proteins ◽  
Biomarker Detection ◽  
In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

scholarly journals Computational and experimental investigations of one-step conversion of poly(carbonate)s into value-added poly(aryl ether sulfone)s

2016 ◽  
Vol 113 (28) ◽  
pp. 7722-7726 ◽  
Author(s):  
Gavin O. Jones ◽  
Alexander Yuen ◽  
Rudy J. Wojtecki ◽  
James L. Hedrick ◽  
Jeannette M. García
Keyword(s):  
High Performance ◽  
Density Functional ◽  
Value Added ◽  
Single Step ◽  
Nucleophilic Attack ◽  
Experimental Investigations ◽  
Aryl Ether ◽  
One Step ◽  
Poly Carbonate

It is estimated that ∼2.7 million tons poly(carbonate)s (PCs) are produced annually worldwide. In 2008, retailers pulled products from store shelves after reports of bisphenol A (BPA) leaching from baby bottles, reusable drink bottles, and other retail products. Since PCs are not typically recycled, a need for the repurposing of the PC waste has arisen. We report the one-step synthesis of poly(aryl ether sulfone)s (PSUs) from the depolymerization of PCs and in situ polycondensation with bis(aryl fluorides) in the presence of carbonate salts. PSUs are high-performance engineering thermoplastics that are commonly used for reverse osmosis and water purification membranes, medical equipment, as well as high temperature applications. PSUs generated through this cascade approach were isolated in high purity and yield with the expected thermal properties and represent a procedure for direct conversion of one class of polymer to another in a single step. Computational investigations performed with density functional theory predict that the carbonate salt plays two important catalytic roles in this reaction: it decomposes the PCs by nucleophilic attack, and in the subsequent polyether formation process, it promotes the reaction of phenolate dimers formed in situ with the aryl fluorides present. We envision repurposing poly(BPA carbonate) for the production of value-added polymers.

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