Fluorescent proteins for live-cell imaging with super-resolution

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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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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