Logic Sensing of MicroRNA in Living Cells Using DNA-Programmed Nanoparticle Network with High Signal Gain

We have developed a novel TPE fluorescent probe (Q3CA-P), with a long Stokes shift and a high signal-to-background ratio, for hNQO1 detection and imaging.

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Molecular Beacons for Sensitive Gene Detection in Living Cells

Advances in Bioengineering ◽

10.1115/imece2003-42959 ◽

2003 ◽

Author(s):

Andrew Tsurkas ◽

Gang Bao

Keyword(s):

Gene Expression ◽

Real Time ◽

Molecular Beacon ◽

Point Mutations ◽

Living Cells ◽

Molecular Beacons ◽

High Signal ◽

Gene Detection ◽

Real Time Imaging ◽

Background Ratio

Real-time imaging of gene expression in living cells has the potential to significantly impact clinical and laboratory studies of cancer, including cancer diagnosis and analysis. Molecular beacons (MBs) provide a simple and promising tool for the detection of target mRNA as tumor markers due to their high signal-to-background ratio, and their improved specificity in detecting point mutations. However, the harsh intracellular environment does limit the sensitivity of MB-based gene detection. Specifically, MBs bound to target mRNAs cannot be distinguished from those degraded by nucleases, or opened due to non-specific interactions. To overcome this difficulty, we have developed a novel dual FRET molecular beacons approach in which a pair of molecular beacons, one with a donor fluorophore and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of false-positive events. We have performed systematic in-solution and cellular studies of dual FRET molecular beacon and demonstrated that this new approach allows for real-time imaging of gene expression in living cells.

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Room Temperature Bistability, Logic Gate Operation, Incoherent Switching and High Signal Gain with InSb Devices

Optical Bistability 2 ◽

10.1007/978-1-4684-4718-7_31 ◽

1984 ◽

pp. 215-222 ◽

Cited By ~ 1

Author(s):

S. D. Smith ◽

F. A. P. Tooley

Keyword(s):

Room Temperature ◽

Logic Gate ◽

High Signal ◽

Gate Operation ◽

Signal Gain

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A spinning disk confocal microscope system for rapid high resolution, multimode, fluorescence speckle microscopy and GFP imaging in living cells

Microscopy and Microanalysis ◽

10.1017/s1431927600026118 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 8-9

Author(s):

Paul Maddox ◽

Julie Canman ◽

Sonia Grego ◽

Wendy Salmon ◽

Clare Waterman-Storer ◽

...

Keyword(s):

High Resolution ◽

Fluorescent Protein ◽

Imaging System ◽

Ccd Camera ◽

High Sensitivity ◽

Time Lapse ◽

Living Cells ◽

High Signal ◽

High Quantum Efficiency ◽

Spinning Disk

High resolution fluorescent speckle microscopy (FSM) and green fluorescent protein (GFP) imaging in living cells can require image recording at low densities of fluorophores (10 or less/resolvable unit) with low light excitation to prevent photobleaching. This needs efficient optical components, a high quantum efficiency detector, and a digital image acquisition and display system for time-lapse recording of multiple channels. Recently, Shinya and Ted Inoue have described the advantages of the Yokogawa CSU-10 spinning-disk confocal scanning unit for obtaining high quality fluorescent images with brief exposures and low fluorescence bleaching. Based on their findings, we have combined the CSU-10 unit with a high sensitivity pan-chromatic CCD camera to facilitate high spatial and temporal resolution imaging of fluorescence in living cells. in addition, the high signal-to-noise in images obtained with this instrument provides the opportunity to obtain 3-D views of extraordinary resolution and image quality after iterative constrained de-convolution.Our imaging system is constructed around a Nikon TE300 inverted microscope equipped with either a 60X or 100X Plan Apochromat objective, and standard epi-fluorescence optics for visual inspection of the specimen to locate cells for recording.

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Stacking nonenzymatic circuits for high signal gain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1222807110 ◽

2013 ◽

Vol 110 (14) ◽

pp. 5386-5391 ◽

Cited By ~ 154

Author(s):

X. Chen ◽

N. Briggs ◽

J. R. McLain ◽

A. D. Ellington

Keyword(s):

High Signal ◽

Signal Gain

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Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

10.1101/2020.06.26.174102 ◽

2020 ◽

Author(s):

Sven A. Szilagyi ◽

Moritz Burmeister ◽

Q. Tyrell Davis ◽

Gero L. Hermsdorf ◽

Suman De ◽

...

Keyword(s):

Single Molecule ◽

Signal To Noise Ratio ◽

Numerical Aperture ◽

Light Sheet ◽

Living Cells ◽

High Signal ◽

Active Optics ◽

Single Molecule Level ◽

Light Sheet Microscopy ◽

Reflected Light

AbstractObservation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

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Bioluminescent Sensors for Ca++ Flux Imaging and the Introduction of a New Intensity-Based Ca++ Sensor

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.773353 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jie Yang ◽

Carl Hirschie Johnson

Keyword(s):

Dynamic Range ◽

Cultured Cells ◽

Signal To Noise Ratio ◽

Dynamic Change ◽

Living Cells ◽

High Signal ◽

Pros And Cons ◽

Rapid Kinetics

Sensitive detection of biological events is a goal for the design and characterization of sensors that can be used in vitro and in vivo. One important second messenger is Ca++ which has been a focus of using genetically encoded Ca++ indicators (GECIs) within living cells or intact organisms in vivo. An ideal GECI would exhibit high signal intensity, excellent signal-to-noise ratio (SNR), rapid kinetics, a large dynamic range within relevant physiological conditions, and red-shifted emission. Most available GECIs are based on fluorescence, but bioluminescent GECIs have potential advantages in terms of avoiding tissue autofluorescence, phototoxicity, photobleaching, and spectral overlap, as well as enhancing SNR. Here, we summarize current progress in the development of bioluminescent GECIs and introduce a new and previously unpublished biosensor. Because these biosensors require a substrate, we also describe the pros and cons of various substrates used with these sensors. The novel GECI that is introduced here is called CalBiT, and it is a Ca++ indicator based on the functional complementation of NanoBiT which shows a high dynamic change in response to Ca++ fluxes. Here, we use CalBiT for the detection of Ca++ fluctuations in cultured cells, including its ability for real-time imaging in living cells.

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Multiplexed live-cell visualization of endogenous proteins with nanometer precision by fluorobodies

10.1101/145698 ◽

2017 ◽

Author(s):

Alina Klein ◽

Susanne Hank ◽

Anika Raulf ◽

Felicitas Tissen ◽

Mike Heilemann ◽

...

Keyword(s):

High Throughput ◽

Protein Function ◽

Protein Targeting ◽

Fluorescent Proteins ◽

Specific Protein ◽

Living Cells ◽

Live Cell ◽

Cellular Structures ◽

High Signal ◽

Endogenous Proteins

AbstractThe visualization of endogenous proteins in living cells is a major challenge. A fundamental requirement for spatiotemporally precise imaging is a minimal disturbance of protein function at high signal-to-background ratio. Current approaches for visualization of native proteins in living cells are limited by dark emitting, bulky fluorescent proteins and uncontrollable expression levels. Here, we demonstrate the labeling of endogenous proteins using nanobodies with site-specifically engineered bright organic fluorophores, named fluorobodies. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing allowed for low background, low toxicity, and high-throughput. Multiplexed imaging of distinct cellular structures was facilitated by specific protein targeting, culminating in live-cell super-resolution imaging of protein networks. The high-throughput delivery of engineered nanobodies will open new avenues in visualizing native cellular structures with unprecedented accuracy in cell-based screens.

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