Простой микрофлюидных устройство было разработано для выполнения анестетика бесплатно<em> В естественных условиях</em> Визуализации<em> C. Элеганс</em>, Неповрежденные<em> Drosophila</em> Личинок и рыбок данио личинки. Устройство используется деформируемый мембраны PDMS, чтобы обездвижить этих модельных организмов в целях выполнения промежуток времени визуализации многочисленных процессов, таких как сердцебиение, деление клеток и субклеточных нейронов транспорта. Мы демонстрируем использование этого устройства и приведены примеры различных типов данных, собранных из различных модельных системах.
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.
PDMS микрофлюидных устройств оптически прозрачным, следовательно, может быть использована для высокого разрешения в естественных изображений любых прозрачных / полупрозрачных моделей организма. Наш дизайн подходит для больших увеличениях пространственно-временных изображений к…
The authors have nothing to disclose.
Мы благодарим доктора Krishanu Ray для акций Drosophila, Tarjani Agarwal для поддержания дрозофилы клетки, Петр Juo для nuIs25 и CGC для C. Элеганс штаммов. SPK сделал jsIs609 в лаборатории Майкла Нонет в. Мы благодарим Arpan Агнихотри (BITS Pilani) за помощь в покадровой визуализации митохондрий перевозки jsIs609 животных в микрофлюидных устройств. Мы благодарны д-р ватсала Thirumalai и Сурия Пракаш за предоставление нам эмбрионов рыбок данио. Мы благодарим доктора Кришны и CIFF в НКО для использования вращающийся диск конфокальной микроскопии при поддержке Департамента Науки и Технологии-центр нанотехнологий (№ SR/55/NM-36-2005). Мы также благодарим Kaustubh Рау, В. Венкатраман и Четана Sachidanand для обсуждения. Эта работа финансировалась DBT пост-докторские стипендии (SM), DST ускоренной схеме (SM) и DBT гранта (SPK). SA была поддержана DST и CSIR гранты SPK.
Name of the reagent | Company | Catalogue number | Comments (optional) |
Silicon wafers | University wafer | 150 mm (100) Mech Grade SSP Si | |
Clewin Software | WieWeb software | Version 2.90 | |
Laser plotter | Fine Line Imaging | 65,024 DPI | |
HMDS | Sigma-Aldrich | 440191-100ML | |
SU8 | Microchem | SU8-2025, SU8-2050 | |
Developer | Microchem | SU8 Developer | |
Silane | Sigma-Aldrich | 448931-10G | |
PDMS | Dow corning | Sylgard 184 | |
UV lamp | Oriel | 66943 | 200W Hg Oriel Light |
Hot air oven | Ultra Instruments | Custom made | Set at 50 °C |
Hot plate | IKA Laboratory Equipment | 3810000 | http://www.ika.com |
Plasma cleaner | Harrick Plasma | PDC-32G | |
Spinner | Semiconductor Production Systems | SPIN150-NPP | www.SPS-Europe.com |
Glass cover slip | Gold Seal | 22 X 22 mm, No. 1 thickness | |
C. elegans | Caenorhabditis Genetics Center (CGC) | e1265, ayIs4 | |
Drosophila | Bloomington | P{chaGAL4}/cyo, UAS-syt.eGFP | |
Zebrafish | Indian wild type | Wild type | |
Tygon tube | Sigma | Z279803 | |
Micro needle | Sigma | Z118044 | Cut into 1 cm pieces |
3-way stopcock | Sigma | S7521 | |
Harris puncher | Sigma | Z708631 | |
Compressed nitrogen gas | Local Gas supplier | Use a regulator to control the pressure | |
Stereo microscope | Nikon | SMZ645 | |
Confocal microscope | Andor & Olympus | Yokogawa spinning disc confocal microscope | |
ImageJ | National Institutes of Health | www.rsbweb.nih.gov/ij | Java based image processing program |