עבודה זו נתמכה על ידי אחוות Wellcome Trust מחקר בכיר א דייויס.

<ol>
	<li>Arias, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+and+the+development+of+biology+in+the+20th+century.">Drosophila melanogaster and the development of biology in the 20th century.</a> Methods Mol. Biol. 420, 1-25 (2008).</li><li>Parton, R. M., Valles, A. -. M., Dobbie, I. D., Davis, I., Goldman, R. D., Swedlow, J. R., Spector, D. 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Cell Biol. 2, 247-256 (2001).</li><li>Van De Bor, V., Hartswood, E., Jones, C., Finnegan, D., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gurken+and+the+I+factor+retrotransposon+RNAs+share+common+localization+signals+and+machinery.">gurken and the I factor retrotransposon RNAs share common localization signals and machinery.</a> Dev. Cell. 9 (1), 51-62 (2005).</li><li>Delanoue, R., Herpers, B., Soetaert, J., Davis, I., Rabouille, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Squid&#47;hnRNP+helps+Dynein+switch+from+a+gurken+mRNA+transport+motor+to+an+ultrastructural+static+anchor+in+sponge+bodies.">Drosophila Squid&#47;hnRNP helps Dynein switch from a gurken mRNA transport motor to an ultrastructural static anchor in sponge bodies.</a> Dev. Cell. 13 (4), 523-538 (2007).</li><li>Jaramillo, A. M., Weil, T. T., Goodhouse, J., Gavis, E. R., Schupbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+fluorescently+labeled+endogenous+gurken+mRNA+in+Drosophila.">The dynamics of fluorescently labeled endogenous gurken mRNA in Drosophila.</a> J. Cell. Sci. 121 (Pt. 6), 887-894 (2008).</li><li>Forrest, K. M., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+endogenous+mRNA+reveals+a+diffusion+and+entrapment+mechanism+for+nanos+mRNA+localization+in+Drosophila.">Live imaging of endogenous mRNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila.</a> Curr. Biol. 13, 1159-1168 (2003).</li><li>Bertrand, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+ASH1+mRNA+particles+in+living+yeast.">Localization of ASH1 mRNA particles in living yeast.</a> Mol. Cell. 2 (4), 437-445 (1998).</li><li>Spradling, A. C., Bate, M., Martinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+oogenesis.">Developmental genetics of oogenesis.</a> The Development of Drosophila melanogaster. 1, 1-70 (1993).</li><li>MacDougall, N., Clark, A., MacDougall, E., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+gurken+(TGFalpha)+mRNA+localizes+as+particles+that+move+within+the+oocyte+in+two+dynein-dependent+steps.">Drosophila gurken (TGFalpha) mRNA localizes as particles that move within the oocyte in two dynein-dependent steps.</a> Dev. Cell. 4 (3), 307-319 (2003).</li><li>Tekotte, H., Tollervey, D., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+migrating+border+cell+cluster+in+living+Drosophila+egg+chambers.">Imaging the migrating border cell cluster in living Drosophila egg chambers.</a> Dev. Dyn. 236 (10), 2818-2824 (2007).</li><li>Weil, T. T., Parton, R., Davis, I., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+bicoid+mRNA+anchoring+highlight+conserved+mechanisms+during+the+oocyte-to-embryo+transition.">Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition.</a> Curr. Biol. 18, 1055-1061 (2008).</li><li>Prasad, M., Jang, A. C., Starz-Gaiano, M., Melani, M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+culturing+Drosophila+melanogaster+stage+9+egg+chambers+for+live+imaging.">A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging.</a> Nat. Protoc. 2 (10), 2467-2473 (2007).</li><li>Morris, L. X., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live+imaging+provides+new+insight+into+stem+cell+regulation+and+germline-soma+coordination+in+the+Drosophila+ovary.">Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary.</a> Development. 138 (11), 2207-2215 (2011).</li></ol>

אין לנו מה למסור.

<html> הדמיה תא חי הוא assay עוצמה לבחינת תהליכים תאיים בזמן אמת. בנוסף תצפית פשוטה בתחום בהיר, הוספת תוויות ניאון כדי חלבונים RNAs של עניין יש להוביל לפריצות דרך רבות. כאן אנו התוויתי פרוטוקול ביציות הדמיה חיים בודדים שניתן לנצלם בשילוב עם מבחני גנטיים ביוכימיים. בפרוטוקול זה, אנחנו גם מסבירים כיצד לתפעל באופן ניסיוני ביציות החיים בזריקה. קיימות אפשרויות רבות עבור חומר להזריק, כולל חוץ גופית ב-RNA מסונתז שכותרתו ניאון assay את היכולת של המבנה משני לוקליזציה RNA ישירה (כדור ו דייוויס, לא פורסם) וכן נוגדנים המעכבים את הפונקציה של חלבונים 6. העבודה העתידית צפויה לראות את המבוא של רכיבים אחרים תיוג ומכונות הסלולר ביציות חיים המאפשרים המנגנונים המולקולריים להיבדק. t &quot;&gt; שמירה על יכולת הקיום והבריאות של רקמה חיונית בעבודה עם תאים חיים. בפרוטוקול זה, אנו מציינים מספר צעדים שיכולים להוביל ללחץ של תא הביצה. לדוגמה, על אף היותו שמן מעולה עבור הדמיה, תרבות המורחבת בשמן יכול להוביל ללחץ על תא הביצה. זה ניתן לפקח בקלות על ידי בחינת המורפולוגיה עמדה גרעינית, קרום הביצית אשר לעוות ו בועה תחת לחץ (איור להשוות 7E (מוטעמות) כדי Figure7F תחת חשיפה לשדות בהיר ( הדגיש)). שלב 9 בתאי ביצה להראות לוקליזציה RNA של התא הגבול ההגירה מעל 12 שעות מרובות. עם זאת, בשלב 7/8 תא הביצה תתחיל להציג תופעות לוואי בשמן פחמן הילה לאחר כ 40 דקות של השחלה סולקו מן נשים. מאוחרת ביצה הבמה חדרי, בשלב 11-14, יכול להתפתח בדרך כלל בשמן או בתקשורת מימית בגלל הפרשת מעטפת הביצית מתאי זקיק בכל השלבים האלה 13. זה כבר reported כי תוספת של אינסולין עד בינוני חרק מימית יכול לשמור 9 ביציות הבמה עד 6 שעות 14 ו germaria עד 14 שעות 15. בכל המקרים, תמרון אגרסיבי של תא הביצה יש להימנע, כפי שהוא מדגיש את ביציות ומפחית את יכולת הקיום שלה. הדמיה פחות בתאי ביצה ולדאוג לטיפול בכל בעדינות היא הדרך הטובה ביותר להבטיח את יכולת הקיום האופטימלי.

כלים המשמשים לנתיחה צריך להיות נקי אבל לא צריך להיות autoclaved. נגב כלים עם ETOH ולאפשר להם להתייבש לפני ההתחלה. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> בסדר צבע היטה מברשת (אמנים סייבל, עגול, בגודל 00 או 0) </td><td> זמין מ רוב חנויות ציוד אמנות </td><td></td><td> מברשת איכותית היא חשובה </td></tr><tr><td> Halocarbon מוצרי החברה, סדרה 95 </td><td> Halocarbon מוצרים Corporation, 887 Kinderkamack כביש, נהר Edge NJ 07661 </td><td></td><td> אירופה המפיץ: Solvadis (GmbH) הקפד על ידי חמצן מבעבע אוויר דרך השמן. </td></tr><tr><td> מכסים, לא להחליק. 1.5, 22 x 50 מ&quot;מ </td><td> הבינלאומי-No1-VWR cat = 631-0137-22-50mm </td><td> מנצל-גלזר-22-40mm MNJ-400-070T </td><td></td></tr><tr><td> דומון מס &#39;5 - Dumostar </td><td> מדע כלים עדינים </td><td> 11253-20 </td><td> &quot;Biologie&quot; עצה טובה גם אבל שברירי יותר </td></tr><tr><td> לנתח בדיקה </td><td> מדע כלים עדינים </td><td> 10140-01 </td><td></td></tr><tr><td> לנתח מיקרוסקופ </td><td> אולימפוס </td><td> SZ61 </td><td></td></tr><tr><td> CO2 כרית </td><td> זמין דרך <a href="http://www.flystuff.com">http://www.flystuff.com</a> / (חלוקת Genesee מדעי) </td><td> 59-114 (789,060 Dutscher) </td><td> אירופה המפיץ: www. dutscherscientific. com</a> / זה גם קל יחסית מותאם אישית לבנות רפידות משלך זבוב </td></tr><tr><td> KimCare </td><td> Kimbery קלארק, KimCare רפואיות מגבונים </td><td> KLEW3020 </td><td></td></tr><tr><td> מיקרוסקופ-DeltaVision </td><td> יישומי Precision </td><td> DC-CORE </td><td></td></tr><tr><td> Micromanipulator </td><td> Burleigh Micromanipulators מ Lumen הדינמיקה הקבוצתית Inc. 2260 Argentia כביש, Mississauga, אונטריו, קנדה L5N 6H7 </td><td> Burleigh PCS-5000 הסדרה, TS-5000-300 </td><td> <a href="http://www.ldgi-burleigh.com">http://www.ldgi-burleigh.com</a> / (לשעבר Exfo) </td></tr><tr><td> זריקת המכשיר </td><td> Tritech Research, Inc 2961 ותיק Ave לוס אנג&#39;לס, קליפורניה 90064 </td><td> MINJ-1 </td><td> שונה עם בעל לקחת Eppendorf Femptotips </td></tr><tr><td> הזרקת מחט </td><td> Eppendorf סטרילי Femtotips I ו-II </td><td> 930000043 </td><td> טיפים שונים טובים יותר עבור יישומים שונים </td></tr><tr><td> טוען טיפים 20μl </td><td> Eppendorf </td><td> 5242956.003 </td><td></td></tr><tr><td> שמרים יבשים פעילים </td><td> פליישמן של שמרים </td><td> # 2192 </td><td></td></tr></tbody></table>

preparing individual drosophila egg chambers for live imaging

הדמיה תא חי הוא טכניקה חשוב ליישם מספר של רקמות תסיסנית המשמשים מודלים לחקור נושאים כגון מפרט ציר, התמיינות תאים ו organogenesis 1. הכנה נכונה של דגימות הניסוי הוא מכריע, לעיתים קרובות מוזנחים, צעד. מטרת ההכנה היא להבטיח התאמה פיזיולוגית ולקבוע תנאים אופטימליים הדמיה. כדי לשמור על הכדאיות רקמות, חיוני כדי למנוע התייבשות, הידרדרות היפוקסיה, התחממות יתר או בינוני 2. תא הביצה תסיסנית היא מערכת מבוססת היטב לבחינת שאלות הנוגעות, אך לא רק, על דפוסים הגוף, לוקליזציה mRNA וארגון cytoskeletal 3,4. עבור בתאי ביצה המוקדמות ו באמצע הבמה, גובר בשמן halocarbon טוב להישרדות כי היא מאפשרת דיפוזיה ללא חמצן, מונע התייבשות היפוקסיה ויש לו תכונות אופטיות מעולה במיקרוסקופ. Imההזדקנות של חלבונים ניאון ניתן באמצעות הכנסת transgenes לתוך תא ביצית או הזרקת הפיזי של רנ&quot;א, חלבונים שכותרתו או נוגדנים 5-7. לדוגמה, תוספת של MS2 בונה על הגנום של בעלי חיים המאפשרת תצפית בזמן אמת של mRNAs ב הביצית 8. מבנים אלו מאפשרים על תווית vivo של ה-mRNA באמצעות ניצול האינטראקציה bacteriophage MS2 RNA גזע לולאה עם חלבון המעיל שלה 9. כאן, אנו מציגים פרוטוקול להוצאת השחלות, כמו גם בידוד ovarioles בודדים ותאי ביצה מ תסיסנית הנשי. לתיאור מפורט של תסיסנית oogenesis הכס אלן ג Spradling (1993, נדפס 2009) 10.

Watch this Scientific Journal Video about Preparing Individual Drosophila Egg Chambers for Live Imaging at JoVE.com

Preparing Individual Drosophila Egg Chambers for Live Imaging

<em&gt; תסיסנית</em&gt; ביצה קאמרית הוא מודל מצוין לחקר המנגנונים של לוקליזציה mRNA. על מנת ללכוד את האירועים דינמיים ביסודה של תהליכי לוקליזציה, ברזולוציה גבוהה הדמיה מהירה של רקמה חיה נדרש. כאן, אנו מציגים פרוטוקול לנתיחה והדמיה של דגימות חי עם הפרעה מזערית.

הכנת הפרט תסיסנית ביצה צ&#39;יימברס הדמיה חיים

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

preparing-individual-drosophila-egg-chambers-for-live-imaging

Research

JoVE Journal

Biology

Preparing Individual Drosophila Egg Chambers for Live Imaging

19.1K Views.  University of Oxford.  תסיסנית ביצה קאמרית הוא מודל מצוין לחקר המנגנונים של לוקליזציה mRNA. על מנת ללכוד את האירועים דינמיים ביסודה של תהליכי לוקליזציה, ברזולוציה גבוהה הדמיה מהירה של רקמה חיה נדרש. כאן, אנו מציגים פרוטוקול לנתיחה והדמיה של דגימות חי עם הפרעה מזערית.

Video: Preparing Individual Drosophila Egg Chambers for Live Imaging

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

הדמיה חיה של נדידת תאים גליה את הדיסק העין דמותי תסיסנית

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

הכנת תסיסנית עוברים Live-הדמיה באמצעות פרוטוקול Drop Hanging

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

חיים הדמיה של המוח על ידי עוברי דג הזברה מיקרוסקופיה confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

מסיק והכנת תסיסנית עוברים הקלטה אלקטרו ונהלים אחרים

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

הדמיה חיה של תסיסנית melanogaster Hemocyte נדידות עובריים

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

הדמיה חיה של תנועתיות התא ואת cytoskeleton אקטין של נוירונים בודדים לבין תאים קרסט עצבית בעוברים דג הזברה

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

הכנת פיתוח למבוגרים תסיסנית מוח רשתיות עבור הדמיה חיה

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

אלקטרונים ספין תהודה מיקרו הדמיה המינים Live עבור מיפוי חמצן

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

הדמיה של זקופה תסיסנית עוברים

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.