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

ABSTRACT: 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 the 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 intermittently. The same chip can be used to accommodate and immobilize arvae 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.

SUBMITTER: Chaudhury AR

PROVIDER: S-EPMC5559736 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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

Chaudhury Amrita Ray AR Insolera Ryan R Hwang Ran-Der RD Fridell Yih-Woei YW Collins Catherine C Chronis Nikos N

Lab on a chip 20170601 13

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 the 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 withou ...[more]

PMID: 28613308

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Project description:Importance:Targeting the early pathogenic steps in Stargardt disease type 1 (STGD1) is critical to advance our understanding of this condition and to develop potential therapies. Lipofuscin precursors may accumulate within photoreceptors, leading to photoreceptor damage and preceding retinal pigment epithelial (RPE) cell death. Fluorescence adaptive optics scanning light ophthalmoscopy can provide autofluorescence (AF) images in vivo with microscopic resolution to elucidate the cellular origin of AF abnormalities in STGD1. Objective:To study the spatial distribution of photoreceptor, RPE, and AF abnormalities in patients with STGD1 at a cellular level. Design, Setting, and Participants:Cross-sectional study using fluorescence adaptive optics scanning light ophthalmoscopy to compare the cones, rods, and RPE cells between 3 patients with STGD1 and 1 control individual. Imaging sessions were conducted at the University of Rochester. Further image analyses were performed at Beijing Tongren Eye Center and the University of Pittsburgh. Data were collected from August 2015 to February 2016, and analysis began in March 2016. Main Outcomes and Measures:Structural appearance of cones, rods, and AF structures at different retinal locations. Results:Two women and 1 man with macular atrophy phenotype of STGD1 and visual acuity loss ranging from 20/30 to 20/150 and 1 woman without STGD1 with 20/20 visual acuity were analyzed. Cone and rod spacing was increased in all 3 patients at all locations where photoreceptors were detectable; most cones had a dark appearance. Autofluorescence was low contrast but contained structures consistent with RPE cells in the periphery. In the transition zone peripheral to the foveal atrophic lesion, the structural pattern of AF was more consistent with photoreceptors than RPE cells. The microscopic AF was disrupted within areas of clinically detectable atrophy. Conclusions and Relevance:Adaptive optics high-resolution images of cones, rods, and RPE cells at the leading disease front of STGD1 macular atrophy show an AF pattern that appears to colocalize with photoreceptors or may result from a combination of AF signals from both RPE cells and photoreceptors. This in vivo observation is consistent with histologic reports of fluorescence arising from photoreceptors in STGD1. The detection of bisretinoid accumulation in the photoreceptors may represent an early pathologic step in STGD1 and can provide an in vivo imaging tool to act as a biomarker of disease progression.

Dataset Information

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

Publications

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

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