High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators

Hofmann, Urs; Fabritius, Arne; Rebling, Johannes; Estrada, Héctor; Deán‐Ben, Xosé Luís; Griesbeck, Oliver; Razansky, Daniel

doi:10.1016/j.isci.2019.11.034

Cited by 5 publications

(5 citation statements)

References 44 publications

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“…The historical approach for optimizing GECIs makes use of high-throughput options that allow screening of recombinant candidate proteins devised by targeted genetic mutations. The most common way of doing so is to develop platforms that facilitate high-throughput screening of a large number of E. coli bacterial colonies which express GECI proteins [168,169]. Bacterial colony screening offers a cost-and time-effective way to screen large numbers of biosensor variants which exploit various energy transfer mechanisms [168].…”

Section: High-throughput Methods For Improving Geci Optimizationmentioning

confidence: 99%

“…Bacterial colony screening offers a cost-and time-effective way to screen large numbers of biosensor variants which exploit various energy transfer mechanisms [168]. First conducted in bacteria, a majority of the screening for optimal variants have been tailored towards fluorescence imaging performance, but expanding these protein screening platforms can include optimization for absorption-based imaging as well [169]. Screening can also be conducted directly in eukaryotic cells, such as the structure-guided mutagenesis and neuron-based screening for optimizing GCaMP6 with various modes of in vivo imaging [114,127].…”

Section: High-throughput Methods For Improving Geci Optimizationmentioning

confidence: 99%

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Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

Saha

2021

Biomolecules

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Since the 1970s, the emergence and expansion of novel methods for calcium ion (Ca2+) detection have found diverse applications in vitro and in vivo across a series of model animal systems. Matched with advances in fluorescence imaging techniques, the improvements in the functional range and stability of various calcium indicators have significantly enhanced more accurate study of intracellular Ca2+ dynamics and its effects on cell signaling, growth, differentiation, and regulation. Nonetheless, the current limitations broadly presented by organic calcium dyes, genetically encoded calcium indicators, and calcium-responsive nanoparticles suggest a potential path toward more rapid optimization by taking advantage of a synthetic biology approach. This engineering-oriented discipline applies principles of modularity and standardization to redesign and interrogate endogenous biological systems. This review will elucidate how novel synthetic biology technologies constructed for eukaryotic systems can offer a promising toolkit for interfacing with calcium signaling and overcoming barriers in order to accelerate the process of Ca2+ detection optimization.

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Section: High-throughput Methods For Improving Geci Optimizationmentioning

confidence: 99%

Section: High-throughput Methods For Improving Geci Optimizationmentioning

confidence: 99%

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

Saha

2021

Biomolecules

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“…A helpful step toward developing such a sensor would include the standardization of PA imaging setups and protocols for high-throughput screening of biosensor candidates (Hofmann et al, 2019).…”

Section: Dynamic Sensorsmentioning

confidence: 99%

Photoacoustic Neuroimaging - Perspectives on a Maturing Imaging Technique and its Applications in Neuroscience

Bodea

Westmeyer

2021

Front. Neurosci.

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A prominent goal of neuroscience is to improve our understanding of how brain structure and activity interact to produce perception, emotion, behavior, and cognition. The brain’s network activity is inherently organized in distinct spatiotemporal patterns that span scales from nanometer-sized synapses to meter-long nerve fibers and millisecond intervals between electrical signals to decades of memory storage. There is currently no single imaging method that alone can provide all the relevant information, but intelligent combinations of complementary techniques can be effective. Here, we thus present the latest advances in biomedical and biological engineering on photoacoustic neuroimaging in the context of complementary imaging techniques. A particular focus is placed on recent advances in whole-brain photoacoustic imaging in rodent models and its influential role in bridging the gap between fluorescence microscopy and more non-invasive techniques such as magnetic resonance imaging (MRI). We consider current strategies to address persistent challenges, particularly in developing molecular contrast agents, and conclude with an overview of potential future directions for photoacoustic neuroimaging to provide deeper insights into healthy and pathological brain processes.

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Section: Introductionmentioning

confidence: 99%

“…This intrinsic contrast is often insufficient to study tissue morphology and function, including metabolic and neuronal processes, which has driven research into transgene OA labels [1][2][3][4] . However, current methods of developing and screening new transgene OA labels are inefficient because they are based on measuring OA signals from E. coli colonies on growth plates 5,6 , which limits throughput and the use of mammalian cells for library expression. Optoacoustic flow cytometry (OA-FCM) could allow for high-throughput screening of variants of transgene OA labels expressed in bacterial or mammalian cells, enabling the use of directed-evolution strategies for rapid label development, analogous to the methods used to design fluorescent labels 7,8 .…”

mentioning

confidence: 99%

In vitro optoacoustic flow cytometry with light scattering referencing

Seeger

Stiel

Ntziachristos

2021

Sci Rep

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Morphological and functional optoacoustic imaging is enhanced by dedicated transgene reporters, in analogy to fluorescence methods. The development of optoacoustic reporters using protein engineering and directed evolution would be accelerated by high-throughput in-flow screening for intracellular, genetically encoded, optoacoustic contrast. However, accurate characterization of such contrast is impeded because the optoacoustic signals depend on the cell’s size and position in the flow chamber. We report herein an optoacoustic flow cytometer (OA-FCM) capable of precise measurement of intracellular optoacoustic signals of genetically-encoded chromoproteins in flow. The novel system records light-scattering as a reference for the detected optoacoustic signals in order to account for cell size and position, as well as excitation light flux in the focal volume, which we use to reference the detected optoacoustic signals to enhance the system’s precision. The OA-FCM was calibrated using micrometer-sized particles to showcase the ability to assess in-flow objects in the size range of single-cells. We demonstrate the capabilities of our OA-FCM to identify sub-populations in a mixture of two E. coli stocks expressing different reporter-proteins with a precision of over 90%. High-throughput screening of optoacoustic labels could pave the way for identifying genetically encoded optoacoustic reporters by transferring working concepts of the fluorescence field such as directed evolution and activated cell sorting.

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High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators

Cited by 5 publications

References 44 publications

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

Photoacoustic Neuroimaging - Perspectives on a Maturing Imaging Technique and its Applications in Neuroscience

In vitro optoacoustic flow cytometry with light scattering referencing

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