Real-Time Monitoring of Fluorescence in Situ Hybridization Kinetics

Ostromohov, Nadya; Huber, Deborah; Bercovici, Moran; Kaigala, Govind V.

doi:10.1021/acs.analchem.8b02630

Cited by 16 publications

(10 citation statements)

References 52 publications

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“…However, while most of these parameters can be optimized by controlling external conditions, reagent delivery is achieved locally on-chip and is particularly sensitive. For example, delays in bringing the smFISH probe into and out of contact with the embryo can lead to lower probe concentration or overexposure resulting in lower smFISH signal, uneven staining, and high background fluorescence. , Reagent exchange is challenging because it requires delivering chemicals to the embryo without losing the embryo in the process. To solve this problem, we added two design considerations to address sample loss from the trap entrance or the back-channel.…”

Section: Results and Discussionmentioning

confidence: 99%

High-Temporal-Resolution smFISH Method for Gene Expression Studies in Caenorhabditis elegans Embryos

et al. 2020

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Recent development in fluorescence-based molecular tools has contributed significantly to developmental studies, including embryogenesis. Many of these tools rely on multiple steps of sample manipulation, so obtaining large sample sizes presents a major challenge as it can be labor-intensive and time-consuming. However, large sample sizes are required to uncover critical aspects of embryogenesis, for example, subtle phenotypic differences or gene expression dynamics. This problem is particularly relevant for single-molecule fluorescence in situ hybridization (smFISH) studies in Caenorhabditis elegans embryogenesis. Microfluidics can help address this issue by allowing a large number of samples and parallelization of experiments. However, performing efficient reagent exchange on chip for large numbers of embryos remains a bottleneck. Here, we present a microfluidic pipeline for large-scale smFISH imaging of C. elegans embryos with minimized labor. We designed embryo traps and engineered a protocol allowing for efficient chemical exchange for hundreds of C. elegans embryos simultaneously. Furthermore, the device design and small footprint optimize imaging throughput by facilitating spatial registration and enabling minimal user input. We conducted the smFISH protocol on chip and demonstrated that image quality is preserved. With one device replacing the equivalent of 10 glass slides of embryos mounted manually, our microfluidic approach greatly increases throughput. Finally, to highlight the capability of our platform to perform longitudinal studies with high temporal resolution, we conducted a temporal analysis of par-1 gene expression in early C. elegans embryos. The method demonstrated here paves the way for systematic high-temporal-resolution studies that will benefit large-scale RNAi and drug screens and in systems beyond C. elegans embryos.

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Section: Results and Discussionmentioning

confidence: 99%

High-Temporal-Resolution smFISH Method for Gene Expression Studies in Caenorhabditis elegans Embryos

et al. 2020

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“…Additional control of the confinement size and shape can be achieved through different channel geometries, for example the use of two aspiration channels or a coaxial channel configuration to achieve symmetric or circular confinements. Multiple injection and aspiration channels could also be considered in the future to create more complex confinement geometries, such as quadrupoles or nested confinements, as well as addition of on‐chip structures for injection of different processing solutions, similar to previously demonstrated geometries in pressure driven probes …”

Section: Discussionmentioning

confidence: 99%

Electrokinetic Scanning Probe

Ostromohov

Rofman

Bercovici

et al. 2019

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The theoretical analysis and experimental demonstration of a new concept are presented for a non‐contact scanning probe, in which transport of fluid and molecules is controlled by electric fields. The electrokinetic scanning probe (ESP) enables local chemical and biochemical interactions with surfaces in liquid environments. The physical mechanism and design criteria for such a probe are presented, and its compatibility with a wide range of processing solutions and pH values are demonstrated. The applicability of the probe is shown for surface patterning in conjunction with localized heating and 250‐fold analyte stacking.

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“…Probes could also be shunted over the tissue to reduce their required volume per test to around 100 nL. Indeed, with the probe hybridization so fast, the team was also able to study the kinetics of probe hybridization [77].…”

Section: Vertical Microfluidic Probe For Cell Layers and Tissue Slicesmentioning

confidence: 99%

“…Reproduced with permission from Ref. [77] investigation varied, some using only 50-100 cells [41,52,58] or on the order of 1000 cells [57,75], others at tens of thousands [56,59].…”

Section: Comparison Of Microfluidic Fish Platformsmentioning

confidence: 99%

FISH and chips: a review of microfluidic platforms for FISH analysis

Rodriguez-Mateos

Azevedo

Almeida

et al. 2020

Med Microbiol Immunol

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Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory. Keywords Microfluidics-assisted FISH • µFISH • Microfluidics • Lab-on-a-Chip (LOC) • Fluorescence in situ hybridization (FISH) Edited by Volkhard A. J. Kempf.

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Real-Time Monitoring of Fluorescence in Situ Hybridization Kinetics

Cited by 16 publications

References 52 publications

High-Temporal-Resolution smFISH Method for Gene Expression Studies in Caenorhabditis elegans Embryos

High-Temporal-Resolution smFISH Method for Gene Expression Studies in Caenorhabditis elegans Embryos

Electrokinetic Scanning Probe

FISH and chips: a review of microfluidic platforms for FISH analysis

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