עבודה זו נתמכה על ידי תרומות של קרן וולש (I-1657) ו-NIH (RO1EY18884). PRH הוא חוקר יוג&#39;ין מק &#39;דרמוט במחקר ביו.

<ol>
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בדיקות ניאון משמש הדמיה כאן אנו מתארים את נקודות החוזק ואת המגבלות של הכנת תסיסנית מערכת הראייה עבור מבחר של בדיקות ניאון משלוש מחלקות: (1) בדיקות שיתווספו הכנה רקמת חיים exogenously; (2) חלבוני ניאון כי מקודדים גנטית הן לחיות הדמיה קבוע; (3) צבעי ניאון שיתווספו רקמות קבוע אימונוהיסטוכימיה. 1. אקסוגניים בדיקות: Lysotracker גרין DND-26 או אדום DND-99 (Invitrogen, Inc) אלה זמינים מסחרית פלורסנט קרום חדיר תרכובות להצטבר בתאים acidified חזק בתא, הבולט lysosomes, מאוחר endosomes, ואת autophagosomes. Lysotracker בריכוזים nanomolar (אנו משתמשים בדרך כלל 50 ננומטר) חודר דיסק L3 ו-P עין 20% באופן מלא פחות מדקה אחת. מאז Lysotracker ברציפות מצטבר ו מפעיל השפעה alkalizing, אנו התמונה פחות מ -5 דקות. בניגוד דיסקים העין, Lysotracker לא לחדור למוח ללא הפרעה של הרקמה. עם זאת, נזהר קריעת הקרום החיצוני מאפשר חדירה של קטרים ​​תא כמה באונה אופטיים. Lysosensor DND-189 (Invitrogen, Inc) בניגוד Lysotracker, Lysosensor לא לצבור בתאים acidified, אבל מפגין גדל הקרינה ב-pH חומצי. Lysosensor יש מאפיינים חדירה דומים מאוד Lysotracker. אנו משתמשים Lysosensor בריכוז של 1μM. 2. מקודד גנטית בדיקות: עבור רבים ניסויים הדמיה לחיות, אנחנו מבטאים גנים מתוייגים עם מולקולות ניאון שונים כמו ה-GFP, TagRFP 10 או ה-pH רגיש pHluorin 11 תחת שליטה של מערכת בינארית Gal4/UAS הביטוי 12. Gal4 קווים המבטאים ב photoreceptors בפיתוח כוללים GMR-Gal4 (כל photoreceptors) 8 ו-mDelta0.5 Gal4 (פיתוח R4 ו R7) 13. מספר תת ספציפיים Gal4 נהג קווים קיימים להשתמש יזמים שונים Rhodopsin 14. בדיקות ניאון שימושי במיוחד בתחום ההדמיה לחיות הם חלבונים photoconvertible. השתמשנו בהצלחה Dendra2, חלבון monomeric ירוק ל-אדום photoconvertable 15. שים לב Dendra2 נועד להיות photoconvertible באמצעות קו ארגון 488nm לייזר. עם זאת, לא נוכל להשיג באמצעות המרת אור כחול לעין שלנו באמצעות הגדרת במצב או קונבנציונלי או סריקת תהודה confocal. לעומת זאת, המרה עם לייזר UV 405nm יעיל. 3. אימונוהיסטוכימיה: עבור העין מבוגר קבוע, לעתים קרובות אנו לבחור phalloidin rhodamine (Invitrogen, Inc) או אנטי Chaoptin (נוגדן ספציפי photoreceptor 16) כדי להמחיש rhabdomeric מבנה (איור 1A). דרך טובה לנתח את מבנה מחסנית lamina היא לשלב אנטי chaoptin 16, סמן גליה אנטי הובנה 17, ואנטי sec6 18 (1B איור). כפי שמוצג באיור. 3, זה שילוב של נוגדנים המבדיל העיקרי presynaptic (chaoptin) ו postsynaptic (sec6) תהליכים, כמו גם גליה אפיתל (הובנה) ב lamina 19. הרכבת לשכת זלוף עבור הדמיה לחיות, אנחנו מעסיקים קאמרית זלוף המאפשר חילופי איטי של פתרונות מבלי להפריע הכנה רקמות. הפגנה של הרכבה קאמרית זלוף כלול וידאו ומוצגות סכמטי באיור 2. האסיפה של החדר זלוף מתוארת באיור. 2A. בגין על ידי הנחת אטם מעל הדגימה הראשונה, ולאחר מכן להמשיך עם הרכבה כפי שמוצג. מטרתו של אטם היא ליצור רווח בין הבסיס של החדר ואת המכסה, בתוך חלל שבו רקמה היא חתומה באמצעות פתרון אשר יזרמו. החלל בתוך אטם חייב לכלול את כניסת (פתרון נכנס כאן), הכנה רקמות, ולשקע שדרכו פתרון עוזב את החדר. שתי תכונות של אטם הם קריטיים: ראשית, אטם חייב להיות עבה מספיק בשביל המוח עדיין רזה מספיק כדי לאפשר הדמיה עם עדשה ברזולוציה גבוהה. עובי אטם אידיאלי תלויה הגדרת פרמטרים ספציפיים. שנית, חריץ אטם מספק בתא המאפשר בועה לעבור בלי קשר את הדגימה (איור 2B). הדמיה על המיקרוסקופ אנו משתמשים עדשה 63x (צמצם 1.3) טבילה גליצרין עבור החדר זלוף או עדשה 63x טבילה במים ישירות לתוך המדיום תרבות בשקופית. עדשות גליצרין לספק יותר עבודה מאשר המרחק עדשות שמן. אנו משתמשים מיקרוסקופ סורק תהודה confocal שסורקת בשיעורים הרבה יותר מהר מאשר סורק קונבנציונאלי (8000 הרץ לעומת 1400 הרץ, בהתאמה). סריקת תהודה מאפשר 3 מימדי ההקלטות לאורך זמן על מהירותאה מסגרת חליפין. בנוסף, מהירות סריקה מהירה יותר להפחית באופן משמעותי phototoxicity 6 לייזר אשר הוא החשש העיקרי עבור סריקות חוזרות ונשנות של רקמה חיה. איזון עוצמה לייזר מתח PMT (רווח) חייבת להיות מושגת על מנת למקסם את האות תוך מזעור רעש photobleaching. שיקול נוסף חשוב הוא כי כמות ההשפעה לייזר על איזור מסוים נקבעת על ידי החלטה וזום. לדוגמה, באזור הבחירה נסרק ברזולוציה באותו 256x256, זום 2x ליום 512x512, זום 1x, אך מהירות מקסימלית אפשרית הדמיה לחיות היא 4 פעמים מהר יותר ב 256x256, זום 2x. כדי להקליט את הפעילות של תאים תאיים נע במהירות, יש לנו נרשם בהצלחה עם ההגדרות הבאות: 5 frames per-z מחסנית בקצב של אחד Z-מחסנית ל 10 שניות עם מהירות סריקה של הרץ ו - 8000 בממוצע קו 2x. הפעלות הדמיה ארוך בהיקף של שעות, לעתים קרובות אנו להפחית את שיעור מסגרת אחת לכל כמה דקות ולבחור אזורים גדולים כמו הכנת סביר יותר משמרת. תסיסנית חזותית האנטומיה מערכת איור 3 מראה את האנטומיה של העין מבוגר תסיסנית (איור 3A), המוח הבוגר (איור 3B), ו 20% -25% מורכבים גלמי דיסק / עין המוח (איור 3 ג). איור 3A ממחיש את העין למבוגרים עם או בלי lamina ואת בתאי השומן המצורפת. במהלך דיסקציה העין מבוגרים, lamina, אשר נמצא במרכז העין השוכן בקערה שיצרו גופי התא photoreceptor (משמאל), יש להסיר מבלי לשבש את גופי התא מתחתיו (ימין). עבור לנתיחה המוח הבוגר, אחד צריך להיות מסוגל להבחין בין גופי התא photoreceptor, lamina, האונה אופטיים, המוח המרכזי, קנה הנשימה (איור 3B). Photoreceptors ואת קנה הנשימה צורך להסיר לנתיחה זה תוך השארת lamina המצורפת באונה אופטיים. כדי לסייע בזיהוי המורכב של 20-25% גלמי עין דיסק / המוח, תמונה חיה מוצג באיור 3C. <img src="/files/ftp_upload/1936/1936fig1.jpg" alt="איור 1" /> באיור 1. (א) במוח מבוגר. (ב) בוגר העין. (ג) 20% -25% מורכבים גלמי eye-disc/brain. <img src="/files/ftp_upload/1936/1936fig2.jpg" alt="איור 2" /> איור 2. (א) חדר זלוף הרכבה (ב) מיקום אטם יחסית הדגימה. <img src="/files/ftp_upload/1936/1936fig3.jpg" alt="איור 3" /> איור 3. (א) מכתים rhabdomere למבוגרים באמצעות אנטי chaoptin. (ב) lamina למבוגרים מכתים באמצעות אנטי chaoptin (ירוק, photoreceptors), אנטי הובנה (אדום, גליה), ו sec6 (כחול, interneurons).

ציוד מיוחד: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> היקף Dissection: Leica Mz12.5 עם מצלמה דיגיטלית </li><li style=";text-align:right;direction:rtl"> לייקה TCS SP5 Confocal טנדם-מיקרוסקופ סורק לסריקה קונבנציונאלי ומהדהד לחיות מיקרוסקופיה הדמיה, כולל כחול (488nm), ירוק (561nm), תפוזים (594nm) והרבה אדום (633nm) לייזרים וכן 63x מיוחד, 1.4 גליצרין ו 63x, 1.3 עדשות טבילה במים. </li><li style=";text-align:right;direction:rtl"> הרווארד מכשירים Confocal הדמיה לשכת RC-30 ו peristaltic משאבה דגם 720 </li><li style=";text-align:right;direction:rtl"> סאטר פיפטה חולץ P-97 </li><li style=";text-align:right;direction:rtl"> תוכנה: 5.2 עמירה, הדמיה הפרצוף </li></ol>

preparation of developing and adult drosophila brains and retinae for live imaging

<em&gt; תסיסנית</em&gt; המוח ומערכת הראייה מנוצלים נרחב במערכות מודל ללמוד פיתוח העצבית, פונקציה וניוון. כאן אנחנו מראים three ההכנות של המוח ומערכת הראייה כי לכסות את טווח ממתחם בפיתוח עין דיסק המוח המתפתח גלמים בעין הפרט לנתיחה המוח מפני זבובים בוגרים. כל הפרוטוקולים מותאמים לתרבות חיים של ההכנות. עם זאת, אנו מציגים גם את התנאים אימונוהיסטוכימיה רקמות קבוע במקומות המתאימים. לבסוף, אנו מראים התנאים הדמיה לחיות אלה ההכנות באמצעות קונבנציונאלי ומהדהד 4D הדמיה confocal לחיות בתא זלוף. יחד, פרוטוקולים אלה מספקים בסיס הדמיה לחיות על סולמות זמן שונים, החל מבחני תאיים פונקציונלי בקנה מידה של דקות תהליכים התפתחותיים או ניווניות בהיקף של שעות רבות.

Watch this Scientific Journal Video about Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging at JoVE.com

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

פרוטוקול זה מתאר שלוש<em&gt; תסיסנית</em&gt; ההכנות: 1) מוח לנתיחה למבוגרים, 2) ברשתית מבוגר לנתיחה ו 3) לפתח דיסק במוח עין לנתיחה קומפלקסים. הדגש מושם על טכניקות הכנה מיוחדת ותנאי הדמיה לחיות, למרות כל ההכנות ניתן להשתמש אימונוהיסטוכימיה רקמות קבוע.

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 ...

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Biology

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

35.2K Views.  University of Texas Southwestern Medical Center. פרוטוקול זה מתאר שלוש תסיסנית ההכנות: 1) מוח לנתיחה למבוגרים, 2) ברשתית מבוגר לנתיחה ו 3) לפתח דיסק במוח עין לנתיחה קומפלקסים. הדגש מושם על טכניקות הכנה מיוחדת ותנאי הדמיה לחיות, למרות כל ההכנות ניתן להשתמש אימונוהיסטוכימיה רקמות קבוע.

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

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

תרבויות עצבית ראשונית של המוח של Late Stage תסיסנית גלמים

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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 ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscopic Fluorescence טומוגרפיה עבור In-vivo הדמיה של פיתוח תסיסנית</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

מדורים פרפין-Embedded קפואים של תסיסנית סקס שרירים

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

הדמיה חיה של שחול Cell מן האפידרמיס של דג הזברה פיתוח

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

הכנת למבוגרים תסיסנית עיניים עבור חתך דק ניתוח מיקרוסקופי

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

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, ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

חי הדמיה של חיסול תאים אפופטוטיים במהלך<em&gt; דרוזופילה</em&gt; עובר

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

העגבניות / GFP-FLP / FRT שיטת ההדמיה חיה של פסיפס למבוגרים בלבד<em&gt; דרוזופילה</em&gt; תאי קולטי אור

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...