scholarly journals Graphene-enabled, spatially controlled electroporation of adherent cells for live-cell super-resolution microscopy

2019 ◽  
Author(s):  
Seonah Moon ◽  
Wan Li ◽  
Ke Xu
Keyword(s):  
Mammalian Cells ◽  
Single Layer ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Biological Research ◽  
Super Resolution Microscopy ◽  
Affinity Probes ◽  
Intracellular Targets

AbstractThe incorporation of exogenous molecules into live cells is essential for both biological research and therapeutic applications. In particular, for the emerging field of super-resolution microscopy of live mammalian cells, reliable fluorescent labeling of intracellular targets remains a challenge. Here, utilizing the unique mechanical, electrical, and optical properties of graphene, a single layer of bonded carbon atoms, we report a facile approach that enables both high-throughput delivery of fluorescent probes into adherent live cells and in situ super-resolution microscopy on the same device. ∼90% delivery efficiencies are achieved for free dyes and dye-tagged affinity probes, short peptides, and whole antibodies, thus enabling high-quality super-resolution microscopy. Moreover, we demonstrate excellent spatiotemporal controls, which, in combination with the ready patternablity of graphene, allow for the spatially selective delivery of two different probes for cells at different locations on the same substrate. We thus open up a new pathway to the microscopic manipulation and visualization of live cells.

A Platinum(II) Based Correlative Probe for Bacteria

2019 ◽  
Author(s):  
Anna Maria Ranieri ◽  
Kathryn Leslie ◽  
Song Huang ◽  
Stefano Stagni ◽  
Denis Jacquemin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Super Resolution ◽  
Molecular Probes ◽  
Live Cells ◽  
Structured Illumination ◽  
Luminescent Probe ◽  
Structured Illumination Microscopy ◽  
Cellular Localisation ◽  
Super Resolution Microscopy ◽  
Nanosims Analysis

There is a lack of molecular probes for imaging bacteria, in comparison to the array of such tools available for the imaging of mammalian cells. This is especially so for correlative probes, which are proving to be powerful tools for enhancing the imaging of live cells. In this work a platinum(II)-naphthalimide molecule has been developed to extend small molecule correlative probes to bacterial imaging. The probe was designed to exploit the naphthalimide moiety as a luminescent probe for super-resolution microscopy, with the platinum(II) centre enabling visualisation of the complex with ion nanoscopy. Photophysical characterisation and theoretical studies confirmed that the emission properties of the naphthalimide are not altered by the platinum(II) centre. Structured illumination microscopy (SIM) imaging on live <i>Bacillus cereus</i>revealed that the platinum(II) centre does not change the sub-cellular localisation of the naphthalimide, and confirmed the suitability of the probe for super-resolution microscopy. NanoSIMS analysis of the sample was used to monitor the uptake of the platinum(II) complex within the bacteria and proved the correlative action of the probe. The successful combination of these two probe moieties with no perturbation of their individual detection introduces a platform for a versatile range of new correlative probes for bacteria.

scholarly journals Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy

2020 ◽  
Vol 6 (22) ◽  
pp. eaba4542 ◽  
Author(s):  
Chenyi Mao ◽  
Min Yen Lee ◽  
Jing-Ru Jhan ◽  
Aaron R. Halpern ◽  
Marcus A. Woodworth ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Processing Conditions ◽  
Sample Processing ◽  
Tissue Fluorescence ◽  
Uniform Labeling ◽  
Tissue Microscopy

Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.

scholarly journals Fringe optimization for structured illumination super-resolution microscope with digital micromirror device

2019 ◽  
Vol 12 (03) ◽  
pp. 1950014 ◽  
Author(s):  
Xibin Yang ◽  
Qian Zhu ◽  
Zhenglong Sun ◽  
Gang Wen ◽  
Xin Jin ◽  
...  
Keyword(s):  
Modulation Depth ◽  
Low Cost ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Digital Micromirror Device ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Wide Range ◽  
Fringe Patterns

Structured illumination microscopy (SIM) is a promising super-resolution technique for imaging subcellular structures and dynamics due to its compatibility with most commonly used fluorescent labeling methods. Structured illumination can be obtained by either laser interference or projection of fringe patterns. Here, we proposed a fringe projector composed of a compact multi-wavelength LEDs module and a digital micromirror device (DMD) which can be directly attached to most commercial inverted fluorescent microscopes and update it into a SIM system. The effects of the period and duty cycle of fringe patterns on the modulation depth of the structured light field were studied. With the optimized fringe pattern, [Formula: see text] resolution improvement could be obtained with high-end oil objectives. Multicolor imaging and dynamics of subcellular organelles in live cells were also demonstrated. Our method provides a low-cost solution for SIM setup to expand its wide range of applications to most research labs in the field of life science and medicine.

scholarly journals Spatial separation of ribosomes and DNA in Asgard archaeal cells

2021 ◽  
Author(s):  
Burak Avcı ◽  
Jakob Brandt ◽  
Dikla Nachmias ◽  
Natalie Elia ◽  
Mads Albertsen ◽  
...  
Keyword(s):  
Super Resolution ◽  
Spatial Separation ◽  
Eukaryotic Cell ◽  
Catalyzed Reporter Deposition ◽  
Card Fish ◽  
Open Question ◽  
Super Resolution Microscopy ◽  
Rrna Sequences ◽  
Genomic Material

AbstractThe origin of the eukaryotic cell is a major open question in biology. Asgard archaea are the closest known prokaryotic relatives of eukaryotes, and their genomes encode various eukaryotic signature proteins, indicating some elements of cellular complexity prior to the emergence of the first eukaryotic cell. Yet, microscopic evidence to demonstrate the cellular structure of uncultivated Asgard archaea in the environment is thus far lacking. We used primer-free sequencing to retrieve 715 almost full-length Loki- and Heimdallarchaeota 16S rRNA sequences and designed novel oligonucleotide probes to visualize their cells in marine sediments (Aarhus Bay, Denmark) using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Super-resolution microscopy revealed 1–2 µm large, coccoid cells, sometimes occurring as aggregates. Remarkably, the DNA staining was spatially separated from ribosome-originated FISH signals by 50–280 nm. This suggests that the genomic material is condensed and spatially distinct in a particular location and could indicate compartmentalization or membrane invagination in Asgard archaeal cells.

scholarly journals STED super-resolution microscopy in Drosophila tissue and in mammalian cells

2011 ◽  
Author(s):  
Lana Lau ◽  
Yin Loon Lee ◽  
Maja Matis ◽  
Jeff Axelrod ◽  
Tim Stearns ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Influenza A virus surface proteins are organized to help penetrate host mucus

2019 ◽  
Author(s):  
Michael D. Vahey ◽  
Daniel A. Fletcher
Keyword(s):  
Sialic Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Spatial Organization ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Viral Envelope ◽  
Surface Proteins ◽  
Virus Surface ◽  
Super Resolution Microscopy

AbstractInfluenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating an organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus’s conflicting needs to both penetrate mucus and stably attach to the underlying cells, and could contribute to the prevalence of the filamentous phenotype in clinical isolates of IAV.

scholarly journals Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

2021 ◽  
Author(s):  
Yasunori Horikoshi ◽  
Hiroki Shima ◽  
Wataru Kobayashi ◽  
Jiying Sun ◽  
Volker J Schmid ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Super Resolution ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Sister Chromatids ◽  
Super Resolution Microscopy ◽  
Damaged Dna

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

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