A microfluidic device and computational platform for high throughput live imaging of gene expression

1] Department of Biology, Duke University, Durham, North Carolina, USA. [2] Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria. [3] Duke Center for Systems Biology, Duke University, Durham, North Carolina, USA. [4].
Nature Methods (Impact Factor: 32.07). 09/2012; 9(11). DOI: 10.1038/nmeth.2185
Source: PubMed


To fully describe gene expression dynamics requires the ability to quantitatively capture expression in individual cells over time. Automated systems for acquiring and analyzing real-time images are needed to obtain unbiased data across many samples and conditions. We developed a microfluidics device, the RootArray, in which 64 Arabidopsis thaliana seedlings can be grown and their roots imaged by confocal microscopy over several days without manual intervention. To achieve high throughput, we decoupled acquisition from analysis. In the acquisition phase, we obtain images at low resolution and segment to identify regions of interest. Coordinates are communicated to the microscope to record the regions of interest at high resolution. In the analysis phase, we reconstruct three-dimensional objects from stitched high-resolution images and extract quantitative measurements from a virtual medial section of the root. We tracked hundreds of roots to capture detailed expression patterns of 12 transgenic reporter lines under different conditions.

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    • "The ability to identify unique cell types from 3D images creates the possibility to perform virtual cell-type-specific analyses. This can be accomplished through the introduction of distinct cell lineage markers (Long et al., 2009; Federici et al., 2012) or the creation of user-defined cellular reference lookup (Busch et al., 2012; Schmidt et al., 2014). The ability to extend these approaches to different organs and contexts relies upon additional user input to define either novel reference atlases or to introduce lineage markers into additional genetic backgrounds. "
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    ABSTRACT: Diverse molecular networks underlying plant growth and development are rapidly being uncovered. Integrating these data into the spatial and temporal context of dynamic organ growth remains a technical challenge. We developed 3DCellAtlas, an integrative computational pipeline that semiautomatically identifies cell types and quantifies both 3D cellular anisotropy and reporter abundance at single-cell resolution across whole plant organs. Cell identification is no less than 97.8% accurate and does not require transgenic lineage markers or reference atlases. Cell positions within organs are defined using an internal indexing system generating cellular level organ atlases where data from multiple samples can be integrated. Using this approach, we quantified the organ-wide cell-type-specific 3D cellular anisotropy driving Arabidopsis thaliana hypocotyl elongation. The impact ethylene has on hypocotyl 3D cell anisotropy identified the preferential growth of endodermis in response to this hormone. The spatiotemporal dynamics of the endogenous DELLA protein RGA, expansin gene EXPA3, and cell expansion was quantified within distinct cell types of Arabidopsis roots. A significant regulatory relationship between RGA, EXPA3, and growth was present in the epidermis and endodermis. The use of single-cell analyses of plant development enables the dynamics of diverse regulatory networks to be integrated with 3D organ growth. © 2015 American Society of Plant Biologists. All rights reserved.
    The Plant Cell 04/2015; 27(4). DOI:10.1105/tpc.15.00175 · 9.34 Impact Factor
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    • "Confocal laser microscopy has been used to assess dynamic gene expression of root initiation and cell growth within the root tissues (Busch et al., 2012; Vermeer et al., 2014). Linked studies of gene regulation, growth regulators, intercellular communication and tissue development have led to advances in mechanistic multiscale modeling that can be used to predict root phenotypes (Band et al., 2012). "
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    ABSTRACT: I. II. III. IV. References Summary There is wide breadth of root function within ecosystems that should be considered when modeling the terrestrial biosphere. Root structure and function are closely associated with control of plant water and nutrient uptake from the soil, plant carbon (C) assimilation, partitioning and release to the soils, and control of biogeochemical cycles through interactions within the rhizosphere. Root function is extremely dynamic and dependent on internal plant signals, root traits and morphology, and the physical, chemical and biotic soil environment. While plant roots have significant structural and functional plasticity to changing environmental conditions, their dynamics are noticeably absent from the land component of process-based Earth system models used to simulate global biogeochemical cycling. Their dynamic representation in large-scale models should improve model veracity. Here, we describe current root inclusion in models across scales, ranging from mechanistic processes of single roots to parameterized root processes operating at the landscape scale. With this foundation we discuss how existing and future root functional knowledge, new data compilation efforts, and novel modeling platforms can be leveraged to enhance root functionality in large-scale terrestrial biosphere models by improving parameterization within models, and introducing new components such as dynamic root distribution and root functional traits linked to resource extraction.
    New Phytologist 09/2014; DOI:10.1111/nph.13034 · 7.67 Impact Factor
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    • "A second microfluidics device, the RootArray, uses confocal microscopy to image up to 64 roots in three dimensions and at high resolution. However, long acquisition times in this system limit the temporal resolution; imaging even a subset of the roots at high resolution takes several hours [6]. While extremely powerful, both of these systems require plants to be grown in the microfluidic device so that the roots grow close to a cover slip which enables high resolution imaging. "
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    ABSTRACT: High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots. The promoter of Hsp17.6 was used to build a Hsp17.6p:GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p:GFP response dynamics. Hsp17.6p:GFP signal increased with temperature increases from 28[degree sign]C to 37[degree sign]C. At 40[degree sign]C the kinetics and localization of the response are markedly different from those at 37[degree sign]C. This suggests that different mechanisms mediate heat shock responses above and below 37[degree sign]C. Finally, we demonstrate that Hsp17.6p:GFP expression exhibits wave like dynamics in growing roots. The RootScope system is a simple and powerful platform for investigating the heat shock response in plants.
    BMC Plant Biology 10/2013; 13(1):158. DOI:10.1186/1471-2229-13-158 · 3.81 Impact Factor
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