Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

Mapping Intimacies ◽

10.1101/840082 ◽

2019 ◽

Author(s):

Adam D. Cawte ◽

Peter J. Unrau ◽

David S. Rueda

Keyword(s):

Single Molecule ◽

Signal To Noise Ratio ◽

Super Resolution ◽

Live Cell ◽

Rna Aptamers ◽

Live Cells ◽

Structured Illumination ◽

Rna Molecules ◽

Cellular Processes ◽

Rna Imaging

AbstractRNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to fluorophore exchange. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

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Extending resolution of structured illumination microscopy with sparse deconvolution

10.21203/rs.3.rs-279271/v1 ◽

2021 ◽

Author(s):

Weisong Zhao ◽

Shiqun Zhao ◽

Liuju Li ◽

Xiaoshuai Huang ◽

Shijia Xing ◽

...

Keyword(s):

Signal To Noise Ratio ◽

Three Dimensional ◽

Super Resolution ◽

Live Cell ◽

Frame Rate ◽

Live Cells ◽

Photon Flux ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Spatiotemporal Resolution

Abstract The spatial resolutions of live-cell super-resolution microscopes are limited by the maximum collected photon flux. Taking advantage of a priori knowledge of the sparsity and continuity of biological structures, we develop a deconvolution algorithm that further extends the resolution of super-resolution microscopes under the same photon budgets by nearly twofold. As a result, sparse structured illumination microscopy (Sparse-SIM) achieves ~60 nm resolution at a 564 Hz frame rate, allowing it to resolve intricate structural intermediates, including small vesicular fusion pores, ring-shaped nuclear pores formed by different nucleoporins, and relative movements between the inner and outer membranes of mitochondria in live cells. Likewise, sparse deconvolution can be used to increase the three-dimensional resolution and contrast of spinning-disc confocal-based SIM (SD-SIM), and operates under conditions with the insufficient signal-to-noise-ratio, all of which allows routine four-color, three-dimensional, ~90 nm resolution live-cell super-resolution imaging. Overall, sparse deconvolution may be a general tool to push the spatiotemporal resolution limits of live-cell fluorescence microscopy.

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Seeing Is Believing: Visualizing Circular RNAs

Non-Coding RNA ◽

10.3390/ncrna6040045 ◽

2020 ◽

Vol 6 (4) ◽

pp. 45

Author(s):

Pruthvi Raj Bejugam ◽

Aniruddha Das ◽

Amaresh Chandra Panda

Keyword(s):

Single Molecule ◽

Rna Binding ◽

Rna Binding Proteins ◽

Imaging Techniques ◽

Sequence Similarity ◽

Rna Aptamers ◽

Circular Rnas ◽

Cellular Processes ◽

Imaging Probes ◽

Rna Imaging

Advancement in the RNA sequencing techniques has discovered hundreds of thousands of circular RNAs (circRNAs) in humans. However, the physiological function of most of the identified circRNAs remains unexplored. Recent studies have established that spliceosomal machinery and RNA-binding proteins modulate circRNA biogenesis. Furthermore, circRNAs have been implicated in regulating crucial cellular processes by interacting with various proteins and microRNAs. However, there are several challenges in understanding the mechanism of circRNA biogenesis, transport, and their interaction with cellular factors to regulate cellular events because of their low abundance and sequence similarity with linear RNA. Addressing these challenges requires systematic studies that directly visualize the circRNAs in cells at single-molecule resolution along with the molecular regulators. In this review, we present the design, benefits, and weaknesses of RNA imaging techniques such as single-molecule RNA fluorescence in situ hybridization and BaseScope in fixed cells and fluorescent RNA aptamers in live-cell imaging of circRNAs. Furthermore, we propose the potential use of molecular beacons, multiply labeled tetravalent RNA imaging probes, and Cas-derived systems to visualize circRNAs.

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3D super-resolved imaging in live cells using sub-diffractive plasmonic localization of hybrid nanopillar arrays

Nanophotonics ◽

10.1515/nanoph-2020-0105 ◽

2020 ◽

Vol 9 (9) ◽

pp. 2847-2859

Author(s):

Soojung Kim ◽

Hyerin Song ◽

Heesang Ahn ◽

Seung Won Jun ◽

Seungchul Kim ◽

...

Keyword(s):

Signal To Noise Ratio ◽

Imaging Techniques ◽

Optical Loss ◽

Super Resolution ◽

Living Cells ◽

Live Cells ◽

Complex Biological System ◽

Observation Volume ◽

Resolution Imaging ◽

Nanopillar Arrays

AbstractAnalysing dynamics of a single biomolecule using high-resolution imaging techniques has been had significant attentions to understand complex biological system. Among the many approaches, vertical nanopillar arrays in contact with the inside of cells have been reported as a one of useful imaging applications since an observation volume can be confined down to few-tens nanometre theoretically. However, the nanopillars experimentally are not able to obtain super-resolution imaging because their evanescent waves generate a high optical loss and a low signal-to-noise ratio. Also, conventional nanopillars have a limitation to yield 3D information because they do not concern field localization in z-axis. Here, we developed novel hybrid nanopillar arrays (HNPs) that consist of SiO2 nanopillars terminated with gold nanodisks, allowing extreme light localization. The electromagnetic field profiles of HNPs are obtained through simulations and imaging resolution of cell membrane and biomolecules in living cells are tested using one-photon and 3D multiphoton fluorescence microscopy, respectively. Consequently, HNPs present approximately 25 times enhanced intensity compared to controls and obtained an axial and lateral resolution of 110 and 210 nm of the intensities of fluorophores conjugated with biomolecules transported in living cells. These structures can be a great platform to analyse complex intracellular environment.

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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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A millisecond structured illumination microscope for super-resolution live cell imaging

Applied Physics Express ◽

10.35848/1882-0786/ab7cef ◽

2020 ◽

Vol 13 (4) ◽

pp. 045002

Author(s):

Tomu Suzuki ◽

Shinji Kajimoto ◽

Narufumi Kitamura ◽

Mayumi Takano-Kasuya ◽

Naoko Furusawa ◽

...

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Structured Illumination

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Live cell single molecule-guided Bayesian localization super resolution microscopy

Cell Research ◽

10.1038/cr.2016.160 ◽

2016 ◽

Vol 27 (5) ◽

pp. 713-716 ◽

Cited By ~ 10

Author(s):

Fan Xu ◽

Mingshu Zhang ◽

Wenting He ◽

Renmin Han ◽

Fudong Xue ◽

...

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Live Cell ◽

Super Resolution Microscopy

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3D tracking of extracellular vesicles by holographic fluorescence imaging

Science Advances ◽

10.1126/sciadv.abc2508 ◽

2020 ◽

Vol 6 (45) ◽

pp. eabc2508

Author(s):

Matz Liebel ◽

Jaime Ortega Arroyo ◽

Vanesa Sanz Beltrán ◽

Johann Osmond ◽

Ala Jo ◽

...

Keyword(s):

Extracellular Vesicles ◽

Single Molecule ◽

Three Dimensional ◽

Super Resolution ◽

Fluorescent Detection ◽

Live Cells ◽

Fluorescent Light ◽

3D Tracking ◽

Anisotropic Motion ◽

Lag Times

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Here, we report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. We expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, we image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), we resolve near-isotropic 3D diffusion and directional transport. For longer lag times, we observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.

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

Journal of Innovative Optical Health Sciences ◽

10.1142/s1793545819500147 ◽

2019 ◽

Vol 12 (03) ◽

pp. 1950014 ◽

Cited By ~ 2

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.

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