Using Microfluidics Chips for Live Imaging and Study of Injury Responses in &lt;em&gt;Drosophila&lt;/em&gt; Larvae

Mishra, Bibhudatta; Ghannad-Rezaie, Mostafa; Li, Jiaxing; Wang, Xin; Hao, Yan; Yan, Bing; Chronis, Nikos; Collins, Catherine A.

doi:10.3791/50998

Cited by 24 publications

(25 citation statements)

References 34 publications

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“…Recently, microfluidic devices and Micro-Electrical-Mechanical Systems (MEMS) have been successfully used in many Drosophila assays, [38][39][40][41][42][43][44][45][46] mainly because of their capability to provide scalable and controllable environments for quantitative embryonic [38][39][40][41][42][43] and larval [44][45][46] investigations in flies. These miniaturized devices that are made primarily from the elastomer polydimethylsiloxane (PDMS) have significantly enhanced the throughput, accuracy, and reproducibility of these assays.…”

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confidence: 99%

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

et al. 2015

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Drosophila melanogaster (fruit fly) is a model organism and its behaviours including oviposition (egg-laying) on agar substrates have been widely used for assessment of a variety of biological processes in flies. Physical and chemical properties of the substrate are the dominant factors affecting Drosophila's oviposition, but they have not been investigated precisely and parametrically with the existing manual approaches. As a result, many behavioral questions about Drosophila oviposition, such as the combined effects of the aforementioned substrate properties (e.g., exposure area, sugar content, and stiffness) on oviposition and viability, and their threshold values, are yet to be answered. In this paper, we have devised a simple, easily implementable, and novel methodology that allows for modification of physical and chemical composition of agar substrates in order to quantitatively study survival and oviposition of adult fruit flies in an accurate and repeatable manner. Agar substrates have been modified by surface patterning using single and hexagonally arrayed through-hole polydimethylsiloxane (PDMS) membranes with various diameters and interspacing, as well as by substrate stiffness and sugar content modification via alteration of chemical components. While pure PDMS substrates showed a significant lethal effect on flies, a 0.5 mm diameter through-hole access to agar was found to abruptly increase the survival of adult flies to more than 93%. Flies avoided ovipositing on pure PDMS and on top of substrates with 0.5 mm diameter agar exposure areas. At a hole diameter of 2 mm (i.e., 0.25% exposure area) or larger, eggs were observed to be laid predominately inside the through-holes and along the edges of the PDMS-agar interface, showing a trending increase in site selection with 4 mm (i.e., 1% exposure area threshold) demonstrating natural oviposition rates similar to pure agar. The surfacemodified agar-PDMS hybrid devices and the threshold values reported for the substrate physical and chemical conditions affecting oviposition are novel; therefore, we advocate their use for future in-depth studies of oviposition behaviour in Drosophila melanogaster with accuracy and repeatability. The technique is also useful for development of novel assays for learning and decision-making studies as well as miniaturized devices for self-assembly of eggs and embryonic developmental investigations. V C 2015 AIP Publishing LLC. [http://dx

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confidence: 99%

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

et al. 2015

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“…# H8898 Sigma Aldrich Inc.), put on a glass coverslip (#12-544E, Fisherbrand), and then covered by placing the transparent chip on top using manual alignment under a stereo dissection microscope. A tight seal between the PDMS chip, oil and coverslip was created by applying vacuum pressure (600 mTorr) using a 20 cc syringe to the vacuum port of the microfluidic network in ‘Layer 1’ [47]. …”

Section: Experimental Methodsmentioning

confidence: 99%

On chip cryo-anesthesia of Drosophila larvae for high resolution in vivo imaging applications

et al. 2017

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We present a microfluidic chip for immobilizing Drosophila melanogaster larvae for high resolution, in vivo imaging. The chip creates a low-temperature micro-environment that anaesthetizes and immobilizes the larva in under 3 minutes. We characterized the temperature distribution within chip and analyzed the resulting larval body movement using high resolution fluorescence imaging. Our results indicate that the proposed method minimizes submicron movements of internal organs and tissue without affecting the larva physiology. It can be used to continuously immobilize larvae for short periods of time (minutes) or for longer periods (several hours) if used intermittingly. The same chip can be used to accommodate and immobilize larvae across all developmental stages (1st instar to late 3rd instar), and loading larvae onto the chip does not require any specialized skills. To demonstrate the usability of the chip, we observed mitochondrial trafficking in neurons from the cell bodies to the axon terminals along with mitochondrial fusion and neuro-synaptic growth through time in intact larvae. Besides studying sub-cellular processes and cellular development, we envision the use of on chip cryo-anesthesia in a wide variety of biological in vivo imaging applications, including observing organ development of the salivary glands, fat bodies and body-wall muscles.

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“…We tracked endogenous Rpl13a protein production in single cells in vivo by measuring red and blue fluorescence intensities in the nucleus over time scales of seconds to days (Methods). To avoid the effects of anesthesia on protein translation, we imaged awake animals immobilized using a suction microfluidics chamber (Mishra et al 2014). Images were acquired at varying time intervals to verify that the time course of fluorescence signals were not due to changes in animal positioning, movement, imaging depth, photobleaching, or changes in the nucleus and nucleolus (Figure 1C, Methods).…”

Section: Measuring Protein Synthesis Dynamics In Single Cells In the mentioning

confidence: 99%

Parental Allele-Specific Protein Expression in Single CellsIn Vivo

Chen

2018

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Allelic expression from each parent-of-origin is important as a backup and to ensure that enough protein products of a gene are produced. Thus far, it is not known how each cell throughout a tissue differs in parental allele expression at the level of protein synthesis. Here, we measure the expression of the Ribosomal protein L13a (Rpl13a) from both parental alleles simultaneously in single cells in the living animal. We use genome-edited Drosophila that have a quantitative reporter of protein synthesis inserted into the endogenous Rpl13a locus. We find that individual cells can have large (>10-fold) differences in protein expression between the two parental alleles. Cells can produce protein from only one allele oftentimes, and time-lapse imaging of protein production from each parental allele in each cell showed that the imbalance in expression from one parental allele over the other can invert over time. One sentence summaryParental allele-specific protein expression varies widely across cells and over time. Highlights1. We used genome editing to insert a quantifiable protein translation reporter into the endogenous Ribosomal protein L13a gene and thus track the protein expression of both parental alleles simultaneously in every single cell in the awake animal.2. Cells can have a large difference in protein expression for one parental allele over the other, and this can invert over time, and can occur in clusters of cells within a tissue.3. We demonstrate the highly variable nature of heterozygous and homozygous definitions across single cells, and over time.4. Our study demonstrates a new paradigm that can be used to examine inherited epigenetic control of expression from a specific parent-of-origin allele across single clonal cells from a common progenitor in vivo.

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Using Microfluidics Chips for Live Imaging and Study of Injury Responses in <em>Drosophila</em> Larvae

Cited by 24 publications

References 34 publications

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

On chip cryo-anesthesia of Drosophila larvae for high resolution in vivo imaging applications

Parental Allele-Specific Protein Expression in Single CellsIn Vivo

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