This work was supported by the Lehigh University Start-Up Fund, the NIH (R00EB010071 to C.Z., R15EB019704 to C.Z. and A.L., R03AR063271 to A.L., and R01AG014713 and R01MH060009 to R.E.T.), the NSF (1455613 to C.Z. and A.L.), the Cure Alzheimer's Fund (to R.E.T.), and the Massachusetts General Hospital (Executive Committee on Research Award to A.L.). M.C. and Y.M. was supported by the National Key Basic Research Program of China (973 Program) under Grant No. 2014CB340404.

The authors declare no conflicts of interests related to the current study.

קצב פעימות לב המהיר של תסיסנית, עם HR מקסימום בסביבות 400 פעימות לדקה בשלבי זחל ובוגר, דורש מהירות גבוהה הדמיה כדי לפתור את הלב diastoles ו systoles (לא פחות מ -80 מסגרות / שנייה מבוסס על חוויות). בשל עובי דופן לב בקנה מידה קאמרי לב קטן בגודל מיקרון (5 - 10 מיקרומטר), ברזולוציה מרחב?...

מחקר אורך של הלב בחיות קטנות תורם להבנת מגוון רחב של מחלות לב וכלי דם אנושיות קשורות, כגון מומי לב מולד הקשורים גן 1,2. בעשורים האחרונים, במודלים של בעלי חיים שונים, כגון 3,4 עכבר, 5,6 Xenopus, 7,8 דג זברה, עופות 9, ו תסיסנית 10-16, שמש לנהל בלב-התפתחות האנושית הקשורים במחקר. מודל העכבר כבר בשימוש נרחב ללמוד התפתחות לב חריגה נורמליות פנוטיפים פגמו לב עקב הדמיון שלה עם 3,4 לב האנושי. עובר Xenopus שימושי במיוחד בחקר התפתחות לב עקב טיפולה הקל 5,6 שקיפות חלקית. השקיפות של עובר ואת הזחל המוקדם של מודל דג הזברה מאפשרת תצפית אופטית קלה של 7,8 פיתוח לב. מודל העופות הוא נושא משותף של מחקרי לב התפתחותי becausדואר בלב ניתן לגשת בקלות לאחר הסרת קליפות ביצים ואת הדמיון המורפולוגי של לבבות עופות לבני אדם 9. המודל תסיסנית יש מספר מאפיינים ייחודיים ההופכים אותו לאידיאלי עבור ביצוע מחקרים ארוכי טווח של הלב. ראשית, הצינור בלב תסיסנית הוא ~ 200 מיקרומטר מתחת לפני השטח הגבה, אשר מספק נוחות עבור גישה אופטית ותצפית על הלב. בנוסף, מנגנונים מולקולריים רבים מסלולים גנטיים שמורים בין תסיסנית ובעלי החוליות. Orthologs של למעלה מ -75% מגני מחל אנושיות נמצאו תסיסנית, אשר הפכה אותו בשימוש נרחב במחקרים מהונדסים 11,13. יתר על כן, יש לו מחזור חיים קצר ועלויות תחזוקה נמוכות, ויש בו נעשה שימוש נפוץ כמודל דגימה למחקר בביולוגיה התפתחותית 14-16. דיווחים קודמים תיאר את הפרוטוקולים לניטור תפקוד הלב תסיסנית כגון שהואArtBeat. עם זאת, תהליכים לנתיחה נדרשו 17,18. הדמיה אופטית מספקת דרך יעילה לחזות התפתחות לב בחיות בשל אופיו הלא-פולשנית. שיטות הדמיה אופטיות שונות יושמו במחקר לב חי ביצוע, כגון מיקרוסקופיה שני פוטונים 19, מיקרוסקופיה confocal 20,21, מיקרוסקופיה גיליון אור 22, ו טומוגרפיה קוהרנטיות אופטית (אוקטובר) 16,23-26. יחסית, אוק הוא מסוגל לספק עומק הדמיה גדול בלבבות חיה קטנים ללא שימוש בחומרי ניגוד תוך שמירה על רזולוציה גבוהה מהירות הדמית ultrahigh, אשר חשובה לבעלי חי הדמיה לחיות. בנוסף, העלות הנמוכה של פיתוח מערכת אוקטובר יש לפופולרי בטכניקה זו דימות אופטית של דגימות. אוקטובר שימש בהצלחה עבור מחקר אורך של דרוזופילה. באמצעות אוקטובר, הדמיה מורפולוגית ופונקציונלית לב שבוצעה כדי ללמוד את מבנה הלב, funcתפקידים תזונתיים של גנים, ואת המנגנונים של מומים קרדיווסקולרים מודלי מוטציה במהלך התפתחות לב. לדוגמא, ירידה בתפקוד לב תלוי גיל קבלה אוששה הקשורות אנזים למטה מוסדר ההמרה-אנגיוטנסין (ACER) גנים תסיסנית עם אוקטובר 27. Phenotyping של קרדיומיופתיה גנים הקשורים הודגם תסיסנית באמצעות אוקטובר 28-33. המחקר באמצעות אוקטובר חשף גם את התפקיד הפונקציונלי של גן SOX5 אדם בלב תסיסנית 34. לעומת אוקטובר, OCM משתמשת אובייקטיבית עם צמצם מספרי גבוה כדי לספק רזולוציה רוחבית טוב יותר. בעבר, התפקוד לקוי הלב נגרם על ידי השתקת גני יממת אדם ortholog dCry / dClock נחקר באמצעות מערכת OCM מנהג 15,16, כמו גם את ההשפעה של דיאטה עתירה שומן על cardiomyopathies תסיסנית להבין אדם מושרה שמן מחלות לב. 15 הנה, הפרוטוקול דואר ניסוי מסוכם עבור מחקר אורך של שינויים מורפולוגיים ופונקציונליים לב תסיסנית ב instar השני (L2), instar השלישי (L3), גולם יום 1 (PD1), גולם יום 2 (PD2), יום גולם 3 (PD3) גולם, יום 4 (PD4), גולם יום 5 (PD5), ומבוגרים (איור 1) באמצעות OCM להעמיק את הלימוד של מחלות לב מולדות אדם קשור. פרמטרים פונקציונלי לב, כגון משאבי אנוש CAP נותחו כמותית בשלבים התפתחותיים שונים כדי לחשוף את תכונות התפתחות הלב.

<table><tbody><tr><td>Custom OCM imaging system</td><td>Developed in our lab</td><td></td><td></td></tr><tr><td>my Temp Mini Digital Incubator</td><td>Benchmark</td><td>H2200-HC</td><td></td></tr><tr><td>Cover glass</td><td>AmScope</td><td>200PCS</td><td></td></tr><tr><td>Cotton Ball</td><td>RITE AID</td><td></td><td></td></tr><tr><td>Instant Drosophila Formula</td><td>CAROLINA</td><td>formula 4-24</td><td></td></tr><tr><td>Yeast</td><td>ActiveDry</td><td></td><td></td></tr><tr><td>Microscope</td><td>SONY</td><td>WILD M420</td><td></td></tr><tr><td>Brush</td><td>Loew-Cornell</td><td>245B</td><td>being used to move specimens</td></tr><tr><td>Labview software</td><td>National Instruments</td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td></td></tr><tr><td>Tweezer</td><td>Wiha</td><td>AA SA</td><td>to fix the fruit fly wings</td></tr><tr><td>FlyNap</td><td>Carolina Biological Supply Company</td><td>4,224,898</td><td></td></tr><tr><td>Scotch Permanent Double Sided Tape, 3 M</td><td>Scotch</td><td></td><td></td></tr><tr><td>Pipette</td><td>Fisherbrand</td><td>MU18837</td><td></td></tr><tr><td>Organic Extra Coconut Oil</td><td>Spring Valley</td><td>13183</td><td></td></tr><tr><td>Microscope Slide</td><td>CapitolBrand</td><td>M3504-E</td><td></td></tr><tr><td>&lt;i&gt;Drosophila&lt;/i&gt; Vials</td><td>SEOH</td><td>8401SS</td><td></td></tr><tr><td>All-trans-retinal</td><td>Sigma-Aldrich Co.</td><td>R2500</td><td></td></tr></tbody></table>

drosophila preparation and longitudinal imaging of heart function in vivo using optical coherence microscopy (ocm)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

הדמיה של הלב האורך נערך באמצעות זבובי פירות עם זן 24b-GAL4 / + בטמפרטורת החדר עם OCM. מדידות בוצעו L2, L3, ובאותו 8 במרווחים hr מ PD1 כדי PD4, ומבוגר יום 1 (AD1) כדי לעקוב אחר תהליך המטמורפוזה (טבלה 1). זחל, גולם מוקדם, גולם מאוחר זבובים בוגרים היו רכובים על שקופיות הזכוכית כפי שנ?...

Watch this Scientific Journal Video about Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM at JoVE.com

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

הכנת תסיסנית והדמיה אורכית תפקוד הלב in vivo באמצעות מיקרוסקופית קוהרנטיות אופטית (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

drosophila-preparation-longitudinal-imaging-heart-function-vivo-using

Research

JoVE Journal

Biology

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

10.8K Views.  Lehigh University. Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Video: Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

מיפוי אופטי של פוטנציאלים פעולה ו הארעית סידן בלב מאוס

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Bioengineering

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

אחד תסיסנית אומטידיום Dissection והדמיה

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Neuroscience

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

הדמיה לטווח ארוך בשידור חי של<em&gt; דרוזופילה</em&gt; דיסק העין

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Developmental Biology

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

לדמיין את הלב הפועם תסיסנית</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

חצי אוטומטיות Heartbeat ניתוח אופטי של לבבות קטנים

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

ניטור הלב לתפקד הזחל תסיסנית melanogaster ללימודים פיזיולוגיים

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Novel And Efficient Method for Drosophila Heart Fluorescence Staining with Cryosectioning

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila hearts before imaging is a meticulous time-intensive process that requires advanced training and motor skills. To address these challenges, we present an innovative protocol that utilizes cryosectioning for the fluorescence imaging of Drosophila heart tissue. The protocol has been demonstrated in imaging the adult Drosophila heart but could be adapted for developmental stages. The method enhances both the efficiency and accessibility of fluorescence staining while preserving the integrity of the tissue. This protocol simplifies the process without compromising the quality of imaging, thereby reducing the dependency on technicians with highly developed training and motor skills. Specifically, we replace complex techniques, such as capillary vacuum suction, with more straightforward methods like tissue embedding. This approach allows for the visualization of cardiac structures with greater ease and reproducibility. We demonstrate the utility of this protocol by effectively detecting key cardiac markers and&#160;achieving high-resolution fluorescence and immunostaining imaging that unveils intricate details of heart morphology and cellular organization. This method provides a robust and accessible tool for researchers exploring Drosophila cardiac biology, facilitating detailed analyses of heart development, function, and disease models.

The Drosophila heart sectioning and fluorescence imaging protocol simplifies studying heart structure and pathologies. This approach involves straightforward sectioning, staining, and imaging, bypassing the technical expertise needed for traditional dissection. It enhances accessibility, making Drosophila a more widely usable model for cardiac-related research within the broader scientific community.

שיטה חדשנית ויעילה לצביעה פלואורסצנטית של לב דרוזופילה עם הקפאה

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila ...

Genetics

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 ...

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.

חי הדמיה של חלבוני ה-GFP שהכותרת ב-<em&gt; דרוזופילה</em&gt; ביציות

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.

הכנת תסיסנית והדמיה אורכית תפקוד הלב in vivo באמצעות מיקרוסקופית קוהרנטיות אופטית (OCM)

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מיפוי אופטי של פוטנציאלים פעולה ו הארעית סידן בלב מאוס

אחד תסיסנית אומטידיום Dissection והדמיה

הדמיה לטווח ארוך בשידור חי של

לדמיין את הלב הפועם תסיסנית

חצי אוטומטיות Heartbeat ניתוח אופטי של לבבות קטנים

ניטור הלב לתפקד הזחל תסיסנית melanogaster ללימודים פיזיולוגיים

שיטה חדשנית ויעילה לצביעה פלואורסצנטית של לב דרוזופילה עם הקפאה

הכנת פיתוח למבוגרים תסיסנית מוח רשתיות עבור הדמיה חיה

חי הדמיה של חלבוני ה-GFP שהכותרת ב-

פיתוח מודלים דרוזופילה מלנוגסטר להדמיה ובקרה אופטוגנטית של תפקוד הלב