Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish
Neurobiology, NCBS-TIFR.Journal of Visualized Experiments (Impact Factor: 1.33). 09/2012; DOI: 10.3791/3780
Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques (1-3). Microfluidic devices have been used for sub-cellular imaging (4,5), in vivo laser microsurgery (2,6) and cellular imaging (4,7). In vivo imaging requires immobilization of organisms. This has been achieved using suction (5,8), tapered channels (6,7,9), deformable membranes (2-4,10), suction with additional cooling (5), anesthetic gas (11), temperature sensitive gels (12), cyanoacrylate glue (13) and anesthetics such as levamisole (14,15). Commonly used anesthetics influence synaptic transmission (16,17) and are known to have detrimental effects on sub-cellular neuronal transport (4). In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 μm to ~800 μm for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min. We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F (4)). Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.
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ABSTRACT: Microfluidics is an emerging field of in vitro science which is generating many new patents. Microfluidics employs small ‘biochips’ of glass, plastic or other materials, which contain an internal array of wells and channels, often with valves and other embedded devices. Cells, tissues or embryos can be implanted in the wells of a sterilized biochip. Then, by connecting the biochip to a pump, culture medium can be circulated through the wells, thereby providing a constant flow-through of nutrients and removal of metabolites. This flow-through culture environment may be closer in some respects to physiological (in vivo) conditions than conventional static replacement cultures. For these and other reasons, discussed in this review, microfluidics has found important applications in the field of regenerative medicine, in which the culture of complex tissues in physiological conditions is a crucial goal. Recent patents cover various modifications of chip architecture that allow the three-dimensional culture of cells, tissues and organs. Microfluidic devices, several of them patented, have been developed for culturing a wide range of different cell types, including primary endothelial cells, interstitial cells, mammalian adherent cell lines, embryonic stem cells, fibroblasts, tumor cells and neurons. Devices have also been described, and some of them patented, in which artificial capillary networks can be grown from endothelial cells. Other devices allow different tissues to be co-cultured in a way that mimics the functions of an organ. Examples of these ‘organs-on-a-chip’ include lungs, heart, kidneys, gastrointestinal tract and brain culture models. Microfluidic devices for the culture and manipulation of whole embryos of zebrafish, the nematode Caenorhabditis elegans and mouse, have also been described and/or patented. These and other microfluidic culture systems are also finding various biomedical applications, such as safety and efficacy testing of drugs, and several patents have been published for these applications. In this review, we summarize recent scientific advances and patents in the field of microfluidics that have special relevance to regenerative medicine.
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ABSTRACT: A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.
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ABSTRACT: Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3(rd) instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.
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