Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.

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Low-Power Two-Color Stimulated Emission Depletion Microscopy for Live Cell Imaging

Biosensors ◽

10.3390/bios11090330 ◽

2021 ◽

Vol 11 (9) ◽

pp. 330

Author(s):

Jia Zhang ◽

Xinwei Gao ◽

Luwei Wang ◽

Yong Guo ◽

Yinru Zhu ◽

...

Keyword(s):

Low Power ◽

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Stimulated Emission ◽

Live Cells ◽

Sted Microscopy ◽

Resolution Imaging ◽

Stimulated Emission Depletion

Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.

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Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging

Molecular Cell ◽

10.1016/j.molcel.2017.06.018 ◽

2017 ◽

Vol 67 (2) ◽

pp. 282-293.e7 ◽

Cited By ~ 169

Author(s):

Tadasu Nozaki ◽

Ryosuke Imai ◽

Mai Tanbo ◽

Ryosuke Nagashima ◽

Sachiko Tamura ◽

...

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Chromatin Domains ◽

Dynamic Organization

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Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

Frontiers in Immunology ◽

10.3389/fimmu.2017.01030 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 42

Author(s):

Bjoern Traenkle ◽

Ulrich Rothbauer

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Single Domain ◽

Single Domain Antibodies ◽

Super Resolution Microscopy

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

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.

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CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

10.1101/2020.03.03.974923 ◽

2020 ◽

Author(s):

Patricia A. Clow ◽

Nathaniel Jillette ◽

Jacqueline J. Zhu ◽

Albert W. Cheng

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Repetitive Sequences ◽

Binary Sequence ◽

Three Dimensional ◽

Live Cell ◽

Live Cells ◽

Imaging Method ◽

Genomic Locus ◽

Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

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C. elegans neurons have functional dendritic spines

eLife ◽

10.7554/elife.47918 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

Author(s):

Andrea Cuentas-Condori ◽

Ben Mulcahy ◽

Siwei He ◽

Sierra Palumbos ◽

Mei Zhen ◽

...

Keyword(s):

Motor Neurons ◽

Dendritic Spines ◽

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Model Organisms ◽

Spine Density ◽

C Elegans ◽

Spine Function

Dendritic spines are specialized postsynaptic structures that transduce presynaptic signals, are regulated by neural activity and correlated with learning and memory. Most studies of spine function have focused on the mammalian nervous system. However, spine-like protrusions have been reported in C. elegans (Philbrook et al., 2018), suggesting that the experimental advantages of smaller model organisms could be exploited to study the biology of dendritic spines. Here, we used super-resolution microscopy, electron microscopy, live-cell imaging and genetics to show that C. elegans motor neurons have functional dendritic spines that: (1) are structurally defined by a dynamic actin cytoskeleton; (2) appose presynaptic dense projections; (3) localize ER and ribosomes; (4) display calcium transients triggered by presynaptic activity and propagated by internal Ca++ stores; (5) respond to activity-dependent signals that regulate spine density. These studies provide a solid foundation for a new experimental paradigm that exploits the power of C. elegans genetics and live-cell imaging for fundamental studies of dendritic spine morphogenesis and function.

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Malic Acid Carbon Dots: From Super-resolution Live-Cell Imaging to Highly Efficient Separation

ACS Nano ◽

10.1021/acsnano.8b01619 ◽

2018 ◽

Vol 12 (6) ◽

pp. 5741-5752 ◽

Cited By ~ 62

Author(s):

Bo Zhi ◽

Yi Cui ◽

Shengyang Wang ◽

Benjamin P. Frank ◽

Denise N. Williams ◽

...

Keyword(s):

Malic Acid ◽

Carbon Dots ◽

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Efficient Separation ◽

Highly Efficient

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Customizable live-cell imaging chambers for multimodal and multiplex fluorescence microscopy

Biochemistry and Cell Biology ◽

10.1139/bcb-2020-0064 ◽

2020 ◽

Vol 98 (5) ◽

pp. 612-623

Author(s):

Adam Tepperman ◽

David Jiao Zheng ◽

Maria Abou Taka ◽

Angela Vrieze ◽

Austin Le Lam ◽

...

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Imaging Modalities ◽

Experimental Conditions ◽

Microscopy Imaging ◽

Glass Coverslip ◽

Multiple Imaging ◽

Microscopy Techniques

Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires the provision of appropriate experimental conditions in a format that meets the optical requirements of the microscope. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized, live-cell imaging chambers that are compatible with a range of imaging modalities, including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and that supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting microfluidic devices made of polydimethylsiloxane. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach for producing imaging chambers that are compatible with modern microscopy techniques.

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