אנו מודים לד&quot;ר Krishanu ריי למניות תסיסנית, Tarjani Agarwal לשמירת תסיסנית כלוב, פיטר Juo לnuIs25 וCGC לג זני elegans. SPK עשה jsIs609 במעבדתו של מייקל התשיעייה. אנו מודים Arpan Agnihotri (Pilani BITS) על עזרתו בזמן לשגות הדמיה של תחבורת מיטוכונדריה של jsIs609 בעלי חיים בהתקני microfluidic. אנו מודים לד&quot;ר Vatsala Thirumalai וסורייה פראקאש שספק לנו עוברי דג זברה. אנו מודים לד&quot;ר קרישנה וCIFF בבנק המרכזי לאומי לשימוש במיקרוסקופ confocal הדיסק מסתובב נתמך על ידי משרד מדע וטכנולוגיה, המרכז לננוטכנולוגיה (מס SR/55/NM-36-2005). אנו מודים גם לKaustubh ראה, ו &#39;Venkatraman וChetana Sachidanand לדיונים. עבודה זו מומנה על ידי מלגת DBT פוסט הדוקטורט (SM), ערכת מסלול מהיר DST (SM) וDBT מענק ל( SPK). SA נתמך על ידי מענקי DST וCSIR לSPK.

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אין ניגודי האינטרסים הכריזו.

התקני microfluidic PDMS הם שקופים אופטי ולכן ניתן להשתמש בו לרזולוציה גבוהה בתחום הדמית vivo של כל אורגניזם מודל שקוף / שקוף. התכנון שלנו הוא מתאים להדמית הגדלה גבוהה זמנית spatio של אירועים הסלולר ומשנה הסלולר בבעלי חיים שלמים. Microfabrication באמצעות טכניקות ליתוגרפיה רכות מ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות (אופציונלי) </td></tr><tr><td> פרוסות סיליקון </td><td> רקיק אוניברסיטה </td><td> 150 מ&quot;מ (100) Mech כיתת SSP סי </td><td></td></tr><tr><td> Clewin תוכנה </td><td> WieWeb תוכנה </td><td> גרסה 2.90 </td><td></td></tr><tr><td> פלוטר ליזר </td><td> הדמית קו דקה </td><td> 65024 DPI </td><td></td></tr><tr><td> HMDS </td><td> סיגמה אולדריץ </td><td> 440191-100 מ&quot;ל </td><td></td></tr><tr><td> SU8 </td><td> Microchem </td><td> SU8-2025, SU8-2050 </td><td></td></tr><tr><td> מפתחים </td><td> Microchem </td><td> SU8 מפתחים </td><td></td></tr><tr><td> Silane </td><td> סיגמה אולדריץ </td><td> 448931-10G </td><td></td></tr><tr><td> PDMS </td><td> Dow Corning </td><td> Sylgard 184 </td><td></td></tr><tr><td> מנורת UV </td><td> אוריאל </td><td> 66943 </td><td> 200W הכספי אוריאל אור </td></tr><tr><td> תנור אוויר חם </td><td> מכשירי Ultra </td><td> Custom made </td><td> הגדר ב 50 מעלות צלזיוס </td></tr><tr><td> פלטה </td><td> ציוד מעבדת איקא </td><td> 3810000 </td><td> <a href="http://www.ika.com" target="_blank">http://www.ika.com</a> </td></tr><tr><td> שואב פלזמה </td><td> Harrick פלזמה </td><td> PDC-32G </td><td></td></tr><tr><td> טווה </td><td> מערכות ייצור מוליכים למחצה </td><td> SPIN150-NPP </td><td> <a href="http://www.SPS-Europe.com" target="_blank">www.SPS-Europe.com</a> </td></tr><tr><td> תלוש כיסוי זכוכית </td><td> זהב חותם </td><td> 22 22 מ&quot;מ X, עובי מס &#39;1 </td><td></td></tr><tr><td>סי אלגנס </td><td> Caenorhabditis גנטיקת המרכז (CGC) </td><td> e1265, ayIs4 </td><td></td></tr><tr><td> דרוזופילה </td><td> בלומינגטון </td><td> P {chaGAL4} / ארגון נוער קתולי, כטב&quot;מ-syt.eGFP </td><td></td></tr><tr><td> דג זברה </td><td> סוג פראי הודי </td><td> סוג פרוע </td><td></td></tr><tr><td> צינור Tygon </td><td> סיגמא </td><td> Z279803 </td><td></td></tr><tr><td> מחט מייקרו </td><td> סיגמא </td><td> Z118044 </td><td> חותך לחתיכות 1 סנטימטר </td></tr><tr><td> 3-way ברזלים </td><td> סיגמא </td><td> S7521 </td><td></td></tr><tr><td> אגרופן אריס </td><td> סיגמא </td><td> Z708631 </td><td></td></tr><tr><td> גז חנקן דחוס </td><td> ספק גז מקומי </td><td></td><td> השתמש בוסת, כדי לשלוט בלחץ </td></tr><tr><td> מיקרוסקופ סטריאו &lt;/ Td&gt; <td> ניקון </td><td> SMZ645 </td><td></td></tr><tr><td> מיקרוסקופ confocal </td><td> תידור ואולימפוס </td><td> Yokogawa ספינינג דיסק confocal מיקרוסקופ </td><td></td></tr><tr><td> ImageJ </td><td> המכונים הלאומיים לבריאות </td><td> <a href="http://www.rsbweb.nih.gov/ij" target="_blank">www.rsbweb.nih.gov / ij</a> </td><td> Java תכנית עיבוד תמונה מבוססת </td></tr></tbody></table>

simple microfluidic devices for in vivo imaging of c. elegans, drosophila and zebrafish

מכשירי fluidic מייקר מפוברק לספק סביבת מייקרו נגישה למחקרי vivo על אורגניזמים קטנים. תהליכי ייצור פשוטים זמינים עבור התקני microfluidic באמצעות טכניקות ליתוגרפיה רכות 1-3. התקני microfluidic שמשו במשך הדמית משנה הסלולר 4,5, in vivo ליזר microsurgery 2,6 וסלולרית הדמיה 4,7. בחי ההדמיה דורשת קיבוע של אורגניזמים. זו הושגה באמצעות שאיבה 5,8, ערוצים מחודדים 6,7,9, ממברנות deformable 2-4,10, יניקה עם קירור נוסף 5, הרדמת גז 11, ג&#39;לים רגיש לטמפרטורה 12, דבק cyanoacrylate 13 והרדמה כגון levamisole 14 , בן 15. הרדמה נפוצה להשפיע על הולכה סינפטית 16,17 וידועים יש השפעות מזיקות על 4 הובלה עצביות משנה הסלולר. ברחוב זהג&#39;ודי אנחנו מדגימים קרום בסיס פולי דימתיל-siloxane מכשיר (PDMS) המאפשר קיבוע ללא הרדמה של אורגניזמים מודל גנטיים תקינים כגון elegans Caenorhabditis (סי אלגנס), זחלי דרוזופילה וזחלי דג זברה. אורגניזמים מודל אלו מתאימים למחקרי vivo במכשירי microfluidic בגלל קוטרם הקטן והגופים שקופים אופטי או שקופים. קטרי גוף לנוע בין ~ 10 מיקרומטר ל ~ 800 מיקרומטר לשלבים מוקדמים של זחל ג זחלי elegans ודג זברה ודורש התקני microfluidic בגדלים שונים כדי להשיג קיבוע מלא ל הדמיה ברזולוציה גבוהה זמן לשגות. אורגניזמים אלה משותקים באמצעות לחץ המופעל על ידי גז חנקן דחוס דרך עמודת נוזל וצלם באמצעות מיקרוסקופ הפוך. בעלי חיים ששוחררו מהמלכודת לחזור לתנועה רגילה בתוך 10 דקות. אנחנו מדגימים ארבעה יישומים של הדמית זמן לשגות בג elegansכלומר, תחבורת הדמית המיטוכונדריה בתאי עצב, תחבורת שלפוחית ​​מראש הסינפטי בתחבורת תחבורה-פגומת מוטציה, קולטן הגלוטמט וחלוקת תא neuroblast ש. נתונים שהתקבלו מסרטים כאלה מראים שקיבוע microfluidic הוא אמצעי שימושי ומדויק של רכישה בנתוני vivo של אירועים הסלולר ומשנה הסלולר בהשוואה לחיות מורדמות (האיור 1J ו3C-F 4). מידות מכשיר שונו כדי לאפשר הדמית זמן לשגות בשלבים שונים של ג elegans, תחילה זחלי instar דרוזופילה וזחלי דג זברה. הובלה של שלפוחית ​​מסומנים synaptotagmin מתויג עם GFP (syt.eGFP) בנוירונים חושיים תערוכות תנועה מכוונת של סמני שלפוחית ​​סינפטיים לידי ביטוי בתאי עצב תחושתיים כולינרגיות בזחלי תסיסנית שלמים 1 instar. מכשיר דומה כבר נעשה שימוש כדי לבצע הדמית זמן לשגות של פעימות לב ב~ הפרית הודעת שעה 30 (hpf)דג זברת זחלים. נתונים אלו מראים כי מכשירים הפשוטים שפתחנו ניתן ליישם במגוון רחב של מערכות מודל ללמוד כמה תאי תופעות ביולוגיות וההתפתחותי בגוף חי.

מכשיר הקיבוע הוא בלוק bilayer PDMS המפוברק על ידי שתי שכבות מליטות: שכבת זרימה (שכבה 1) ושכבת שליטה (שכבה 2) כפי שמוצג באיור 1. המלכודת הראשית מחוברת לבלון גז חנקן באמצעות רגולטור וזין תחנה 3-דרך ליישם את הלחץ דרוש (3-14 psi) על גבי הקרום דרך עמודת נוזל (איור 1 א). הק...

Watch this Scientific Journal Video about Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish at JoVE.com

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

מכשיר microfluidic פשוט פותח כדי לבצע הרדמה חופשיה<em&gt; In vivo</em&gt; הדמיה של<em&gt; ג elegans</em&gt;, שלם<em&gt; דרוזופילה</em&gt; זחלים וזחלי דג זברה. המכשיר מנצל PDMS קרום deformable לשתק אורגניזמים המודל הבא על מנת לבצע זמן לשגות הדמיה של תהליכים רבים כגון קצב לב, חלוקת תא והובלה עצבית משנה הסלולר. אנו מדגימים את השימוש במכשיר זה ולהראות דוגמאות לסוגים שונים של נתונים שנאספו ממערכות מודל שונות.

התקני microfluidic פשוטים עבור<em&gt; In vivo</em&gt; הדמיה של<em&gt; ג elegans</em&gt;,<em&gt; דרוזופילה</em&gt; ודג זברה

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

simple-microfluidic-devices-for-vivo-imaging-c-elegans-drosophila

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

17.0K Views.  NCBS-TIFR. מכשיר microfluidic פשוט פותח כדי לבצע הרדמה חופשיה In vivo הדמיה של ג elegans, שלם דרוזופילה זחלים וזחלי דג זברה. המכשיר מנצל PDMS קרום deformable לשתק אורגניזמים המודל הבא על מנת לבצע זמן לשגות הדמיה של תהליכים רבים כגון קצב לב, חלוקת תא והובלה עצבית משנה הס?...

Video: Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

חרוזים ועל הפרדת תאים ריבוי ערוצים מכשירים microfluidic שימוש dielectrophoresis ו זרימה למינרית

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

חלקיקים נגיפיים עבור<em&gt; בחי</em&gt; הדמית גידול

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

טכניקת Microfluidic לחקור deformability הסלולרי

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Microstructured מכשירי Microinjection אופטימיזציה והדימות של הזחלים דג זברה

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Developmental Biology

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

התקן הדמיה חיה לטווח ארוך עבור משופרת מניפולציה ניסויית של הזחלים דג זברה

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

פלטפורמה Microfluidic הדמיה האורך ב Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

באמצעות שבב חוש הריח של Microfluidic מותאם עבור ההדמיה של פעילות. עצבית בתגובה פרומונים בנוירונים הראש הגברי C. Elegans

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Neuroscience

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

שימוש בהתקן מיקרופלואידיקה גירוי מכני, רזולוציה גבוהה הדמיה של C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

שבב מיקרופלואידי פשוט לגדילה והדמיה ארוכת טווח של Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging

Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune cell migration. Microfluidic devices are used for entrapment to support long-term imaging of multicellular organisms, including zebrafish. However, the fabrication of these devices using soft lithography requires specialized facilities and competency in 3D printing, which may not be accessible to every lab. Our adaptation of a previously developed low-cost polyethylene terephthalate lamination method for constructing microfluidic devices increases accessibility by enabling design fabrication and iteration for a fraction of the technical investment of conventional techniques. We use a device made with this method, the Rotational Assistant for Danio Imaging of Subsequent Healing (RADISH), to accommodate drug treatment, manual wounding, and long-term imaging of up to four embryos in the same field of view. With this new design, we successfully capture gross morphological characteristics of the calcium&#160;signal around laser ablation and manual transection wounds for multiple embryos in the 2 h immediately following injury, as well as neutrophil recruitment to the wound edge for 24 h.

An innovative method for fabricating microfluidic devices using polyethylene terephthalate (PET) lamination significantly reduces the cost and complexity of entrapping and imaging multiple live zebrafish embryos.

Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune ...

התקני microfluidic פשוטים עבור

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