scholarly journals Development and Applications of Bioluminescent and Chemiluminescent Reporters and Biosensors

2019 ◽  
Vol 12 (1) ◽  
pp. 129-150 ◽  
Author(s):  
Hsien-Wei Yeh ◽  
Hui-Wang Ai
Keyword(s):  
Excited State ◽  
External Excitation ◽  
Excitation Light ◽  
Fluorescent Reporters ◽  
Fluorescence Measurements ◽  
Chemiluminescent Sensors ◽  
Biomedical Fields ◽  
Bioluminescent Reporters

Although fluorescent reporters and biosensors have become indispensable tools in biological and biomedical fields, fluorescence measurements require external excitation light, thereby limiting their use in thick tissues and live animals. Bioluminescent reporters and biosensors may potentially overcome this hurdle because they use enzyme-catalyzed exothermic biochemical reactions to generate excited-state emitters. This review first introduces the development of bioluminescent reporters, and next, their applications in sensing biological changes in vitro and in vivo as biosensors. Lastly, we discuss chemiluminescent sensors that produce photons in the absence of luciferases. This review aims to explore fundamentals and experimental insights and to emphasize the yet-to-be-reached potential of next-generation luminescent reporters and biosensors.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

A multipurpose instrument for quantitative intravital microscopy

1992 ◽  
Vol 73 (1) ◽  
pp. 296-306 ◽  
Author(s):  
A. Toth ◽  
M. E. Tischler ◽  
M. Pal ◽  
A. Koller ◽  
P. C. Johnson
Keyword(s):  
Parenchymal Cell ◽  
Fluorescence Emission ◽  
Repeated Measurements ◽  
Open Architecture ◽  
In Vitro Tests ◽  
Fluorescence Measurements ◽  
Tissue Area ◽  
Microcirculatory Function

An in vivo microscope system has been developed that can measure fluorescence emission and/or light absorption at up to five wavelengths in a tissue area of 18–30 microns diam while imaging adjacent microcirculatory vessels with a video system. The system also incorporates a computer-controlled stage and data acquisition system for rapid and repeated measurements from a number of tissue sites. The tissue area monitored for fluorescence or absorption can be defined further by a confocal arrangement of the microscope optics. Tests of the system for NADH fluorescence measurements show good agreement between the fluorescence at 450 nm and NADH concentration in vitro and in skeletal muscle. The instrument can also be used simultaneously for spectrophotometric determination of O2 saturation and hematocrit in microcirculatory vessels. In vitro tests indicate suitable accuracy for such measurements. The open architecture and modular arrangement of the instrument facilitates its use for a variety of simultaneous measurements of parenchymal cell and microcirculatory function.

Structural effects in chemistry and biology

2000 ◽  
Vol 04 (04) ◽  
pp. 382-385 ◽  
Author(s):  
JACK FAJER
Keyword(s):  
Excited State ◽  
Physicochemical Properties ◽  
Crystal Structures ◽  
Protein Complexes ◽  
Structural Effects ◽  
Conformational Variations ◽  
Prosthetic Groups ◽  
Skeletal Deformations

Conformationally designed, non-planar porphyrins afford new classes of structurally distinct chromophores with significantly altered optical, redox, magnetic, radical and excited state properties. The synthetic, non-planar porphyrins model and illustrate the consequences of the skeletal deformations and plasticity increasingly observed in crystal structures of protein complexes comprising porphyrinic chromophores and prosthetic groups. Conformational variations thus offer attractively simple mechanisms for modulating the physicochemical properties of porphyrins in vivo and in vitro.

The Development of Non-Radiative Probes for In Vivo Applications

2003 ◽  
Author(s):  
Andrew Tsourkas ◽  
Lee Josephson ◽  
Ralph Weissleder
Keyword(s):  
Relaxation Time ◽  
Magnetic Relaxation ◽  
Magnetic Particles ◽  
Superparamagnetic Iron Oxide ◽  
Great Promise ◽  
T2 Relaxation Time ◽  
T2 Relaxation ◽  
Fluorescence Measurements

Recently, the field of activatable probes has been extended from fluorescence to magnetic resonance (MR). Magnetic relaxation switches take advantage of the change in T2 relaxation time that occurs upon binding of multiple bioconjugated superparamagnetic iron-oxide nanoparticles to a target. Here, we use a model system to detect biotinilated BSA using switchable magnetic particles. The presence of the biotinilated BSA results in the aggregation of nanoparticles and leas to a substantial decrease in the T2 relaxation time. Magnetic relaxation switches show great promise for clinical in vitro diagnostics and in vivo imaging since changes in T2 are independent of the sample medium. Tests can be performed in turbid solutions without loss of sensitivity, unlike with fluorescence measurements.

scholarly journals MiniFAST: A sensitive and fast miniaturized microscope for in vivo neural recording

2020 ◽  
Author(s):  
Jill Juneau ◽  
Guillaume Duret ◽  
Joshua P. Chu ◽  
Alexander V. Rodriguez ◽  
Savva Morozov ◽  
...  
Keyword(s):  
Open Source ◽  
High Speed ◽  
High Sensitivity ◽  
High Gain ◽  
Image Sensor ◽  
High Speed Imaging ◽  
Light Power ◽  
Excitation Light

AbstractObserving the activity of large populations of neurons in vivo is critical for understanding brain function and dysfunction. The use of fluorescent genetically-encoded calcium indicators (GECIs) in conjunction with miniaturized microscopes is an exciting emerging toolset for recording neural activity in unrestrained animals. Despite their potential, current miniaturized microscope designs are limited by using image sensors with low frame rates, sensitivity, and resolution. Beyond GECIs, there are many neuroscience applications which would benefit from the use of other emerging neural indicators, such as fluorescent genetically-encoded voltage indicators (GEVIs) that have faster temporal resolution to match neuron spiking, yet, require imaging at high speeds to properly sample the activity-dependent signals. We integrated an advanced CMOS image sensor into a popular open-source miniaturized microscope platform. MiniFAST is a fast and sensitive miniaturized microscope capable of 1080p video, 1.5 µm resolution, frame rates up to 500 Hz and high gain ability (up to 70 dB) to image in extremely low light conditions. We report results of high speed 500 Hz in vitro imaging of a GEVI and ∼300 Hz in vivo imaging of transgenic Thy1-GCaMP6f mice. Finally, we show the potential for a reduction in photobleaching by using high gain imaging with ultra-low excitation light power (0.05 mW) at 60 Hz frame rates while still resolving Ca2+ spiking activity. Our results extend miniaturized microscope capabilities in high-speed imaging, high sensitivity and increased resolution opening the door for the open-source community to use fast and dim neural indicators.

scholarly journals Optical Imaging of Pancreatic Innervation

2021 ◽  
Vol 12 ◽  
Author(s):  
Madina Makhmutova ◽  
Alejandro Caicedo
Keyword(s):  
Central Nervous System ◽  
Imaging Techniques ◽  
Fluorescent Reporters ◽  
Technological Advances ◽  
Specific Manner ◽  
The Central Nervous System ◽  
Visceral Innervation ◽  
Stimulation Of

At the time of Ivan Pavlov, pancreatic innervation was studied by looking at pancreas secretions in response to electrical stimulation of nerves. Nowadays we have ways to visualize neuronal activity in real time thanks to advances in fluorescent reporters and imaging techniques. We also have very precise optogenetic and pharmacogenetic approaches that allow neuronal manipulations in a very specific manner. These technological advances have been extensively employed for studying the central nervous system and are just beginning to be incorporated for studying visceral innervation. Pancreatic innervation is complex, and the role it plays in physiology and pathophysiology of the organ is still not fully understood. In this review we highlight anatomical aspects of pancreatic innervation, techniques for pancreatic neuronal labeling, and approaches for imaging pancreatic innervation in vitro and in vivo.

scholarly journals Rapid multicomponent bioluminescence imaging via substrate unmixing

2019 ◽  
Author(s):  
Colin M. Rathbun ◽  
Anastasia A. Ionkina ◽  
Zi Yao ◽  
Krysten A. Jones ◽  
William B. Porterfield ◽  
...  
Keyword(s):  
Time Scale ◽  
Bioluminescence Imaging ◽  
Wide Range ◽  
Multiplexed Imaging ◽  
Sequential Addition ◽  
Bioluminescent Reporters

ABSTRACTEngineered luciferases and luciferins have dramatically expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Here we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale, a vast improvement over conventional protocols.

scholarly journals Novel tool to quantify with single-cell resolution the number of incoming AAV genomes co-expressed in the mouse nervous system

Gene Therapy ◽  
2021 ◽  
Author(s):  
Carola J. Maturana ◽  
Jessica L. Verpeut ◽  
Mahdi Kooshkbaghi ◽  
Esteban A. Engel
Keyword(s):  
Nervous System ◽  
Single Cell ◽  
Brain Connectivity ◽  
Genetic Diseases ◽  
Brain Regions ◽  
Anterior Cingulate ◽  
Fluorescent Reporters ◽  
Cargo Capacity

AbstractAdeno-associated viral (AAV) vectors are an established and safe gene delivery tool to target the nervous system. However, the payload capacity of <4.9 kb limits the transfer of large or multiple genes. Oversized payloads could be delivered by fragmenting the transgenes into separate AAV capsids that are then mixed. This strategy could increase the AAV cargo capacity to treat monogenic, polygenic diseases and comorbidities only if controlled co-expression of multiple AAV capsids is achieved on each transduced cell. We developed a tool to quantify the number of incoming AAV genomes that are co-expressed in the nervous system with single-cell resolution. By using an isogenic mix of three AAVs each expressing single fluorescent reporters, we determined that expression of much greater than 31 AAV genomes per neuron in vitro and 20 genomes per neuron in vivo is obtained across different brain regions including anterior cingulate, prefrontal, somatomotor and somatosensory cortex areas, and cerebellar lobule VI. Our results demonstrate that multiple AAV vectors containing different transgenes or transgene fragments, can efficiently co-express in the same neuron. This tool can be used to design and improve AAV-based interrogation of neuronal circuits, map brain connectivity, and treat genetic diseases affecting the nervous system.

scholarly journals In vitro teratogenicity testing using a 3D, embryo-like gastruloid system

2021 ◽  
Author(s):  
Veronika Mantziou ◽  
Peter Baillie-Benson ◽  
Manuela Jaklin ◽  
Stefan Kustermann ◽  
Alfonso Martinez Arias ◽  
...  
Keyword(s):  
Axial Patterning ◽  
Fluorescent Reporters ◽  
Lead Compounds ◽  
Lineage Differentiation ◽  
Aberrant Gene Expression ◽  
Species Specific ◽  
Aberrant Gene ◽  
Selection Of

AbstractPharmaceuticals that are intended for use in patients of childbearing potential need to be tested for teratogenicity before marketing. Several pharmaceutical companies use animal-free in vitro models which allow a more rapid selection of lead compounds and contribute to 3Rs principles (‘replace, reduce and refine’) by streamlining the selection of promising compounds that are submitted to further regulatory studies in animals. Currently available in vitro models typically rely on adherent monolayer cultures or disorganized 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo. A newly developed 3D ‘gastruloid’ model has the potential to achieve a more reliable prediction of teratogenicity by providing a robust recapitulation of gastrulation-like events alongside morphological coordination at relatively high-throughput. In this first proof-of-concept study, we used both mouse and human gastruloids to examine a panel of seven reference compounds, with associated in vivo data and known teratogenic risk, to quantitatively assess in vitro teratogenicity. We observed several gross morphological effects, including significantly reduced elongation or decreased size of the gastruloids, upon exposure to several of the reference compounds. We also observed aberrant gene expression using fluorescent reporters, including SOX2, BRA, and SOX17, suggestive of multi-lineage differentiation defects and disrupted axial patterning. Finally, we saw that gastruloids recapitulated some of the known in vivo species-specific susceptibilities between their mouse and human counterparts. We therefore suggest that gastruloids represent a powerful tool for teratogenicity assessment by enabling relevant physiological recapitulation of early embryonic development, demonstrating their use as a novel in vitro teratogenic model system.

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