We thank O. Subach for providing the original H2B-PSmOrange plasmid and our fish facility team for fish care. We are grateful to the Nikon Imaging Center at the University of Heidelberg for access to microscopy equipment and analysis software. We acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the CBTM (DFG INST 91027/10-1 FUGG). This work was supported by the Excellenzcluster CellNetworks, EcTop Spatio-temporal coordination of signaling processes (EcTop 2), University of Heidelberg to C.A.B. and the Medical Faculty Mannheim of the University Heidelberg and the DFG (FOR 1036/2, 298/3-1 and 298/6-1) to M.C.

1. תמלול mRNA H2B-PSmOrange במבחנה טיהור mRNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Linearize את PSmOrange H2B המכיל pCS2 + פלסמיד באמצעות אנזים הגבלה NotI פי הוראות היצרן. הערה: בצע את השלבים הבאים באמצעות הגנות מתאימות כגון כפפות וחלוק מעבדה על מנת למנוע זיהום והשפלה mRNA. </li><li style=";text-align:right;direction:rtl"> לטהר את ה- DNA לינארית באמצעות שיטות המבוססות ערכת טיהור PCR או פנול, כלורופורם על פי פרוטוקולים של היצרנים. </li><li style=";text-align:right;direction:rtl"> השתמש 1 מיקרוגרם של תבנית ה- DNA לינארית עבור שעתוק SP6-mRNA על פי הוראות היצרן. </li><li style=";text-align:right;direction:rtl"> הסר DNA על ידי הוספת 1.0 U RNase חינם DNaseI במשך 10-20 דק &#39;ב 37 ° C. </li><li style=";text-align:right;direction:rtl"> לנקות את ה- mRNA באמצעות RNA לנקות ערכה על פי פרוטוקול של היצרן. </li><li style=";text-align:right;direction:rtl"> כדי להגדיל טוהר mRNA וריכוז, לזרז את mRNA על ידי הוספת 6 נתרן אצטט μl (3.0 M, pH 5.2) ו 150 μl 96% אתנול. דגירה מפתרון ixed ב -20 מעלות צלזיוס במשך 30 דקות לפחות עד 24 שעות. </li><li style=";text-align:right;direction:rtl"> אסוף את ה- mRNA על ידי ספינינג הפתרון ב 18.407 XG במשך 45 דקות ב 4 ° C. מחק את הנוזל ו resuspend mRNA ב 150 μl 70% אתנול. מערבבים צנטריפוגות הפתרון עבור 15 דקות נוספות ב 4 ° C. מחק את הנוזל, לייבש את הגלולה במשך 5 דקות על קרח ו resuspend mRNA ב 20 מים חינם μl ultrapure RNase. </li><li style=";text-align:right;direction:rtl"> להעריך את ריכוז והטוהר של mRNA על ידי מדידה photometric של דילול 1: mRNA 100 (mRNA 1 μl ב 99 μl מים חינם ultrapure RNase). </li><li style=";text-align:right;direction:rtl"> עבור אחסון לטווח קצר, לשמור על פתרון mRNA ב -20 ° C. עבור אחסון לטווח ארוך, לשמור על mRNA ב -80 מעלות צלזיוס. </li></ol> 2. H2B-PSmOrange mRNA Microinjection לעוברי דג הזברה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדרת ההזדווגות זוגות בלילה לפני הזריקה באמצעות TG (foxD3: GFP); TG (FLH: GFP) דג מהונדס כפול (או כל דג מהונדס GFP אחר). השאירו את הדגים מופרדים על ידי הצבת spacerביניהם כדי לקבוע את הזמן של הזדווגות. </li><li style=";text-align:right;direction:rtl"> הסר את spacer בבוקר ההזרקה ולתת זוג דגים למשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> במהלך ההזדווגות זמן, לדלל את ה- mRNA H2B-PSmOrange לריכוז סופי של 130 pg / NL (במים חינם RNase) פתרון הזרקת μl העברת 6 לתוך נימי microinjection באמצעות קצה microloader. בדוק את עוצמת זריקה עם שקופית זכוכית מכוילת. התאם את לחץ ההזרקה להזריק 2 פתרון mRNA NL (260 pg). </li><li style=";text-align:right;direction:rtl"> אסוף את הביצים לתוך צלחת פטרי פלסטיק מעוקר המכילים עובר 1x בינוני (E3). </li><li style=";text-align:right;direction:rtl"> עבר 20 עד 30 עובר למנת הזרקה בעזרת פיפטה פסטר מפלסטיק. </li><li style=";text-align:right;direction:rtl"> להזריק 2 mRNA NL המכיל פתרון לתוך התא או סתם מתחת לתא בחלמון בשלב אחד התא 11. </li><li style=";text-align:right;direction:rtl"> העברת עוברים מוזרק לתוך צלחת פטרי שמכילה בינוני 1x E3, להסיר בדרך כלל מופרית או לא עוברים לפתח ולגדל אותם על 28 מעלות צלזיוס. הערה: o הגנת אורf עובר אינו נדרש לאורך הניסוי. </li><li style=";text-align:right;direction:rtl"> תרים עוברי דג הזברה במדיום 1x E3 ולהוסיף 0.2 מ&quot;מ PTU (1-פניל-2 thiourea) לאחר gastrulation לעכב פיגמנטציה. שינוי המדיום פעמים ביום כדי למזער את הסכנה של זיהומים חיידקיים. </li></ol> 3. עובר והטבעה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בחר את ה- GFP (ירוק) ו PSmOrange (כתום / אדום) עובר להביע שיתוף באמצעות מיקרוסקופ משקפת מצוידת מנורת קרינה ומסנני פליטה מתאימות. מעביר את העוברים החיוביים לתוך פטרי צלחת סטרילי המכילה בינוני 1x E3. </li><li style=";text-align:right;direction:rtl"> עוברים dechorionate תחת סטראו באמצעות מלקחיים 12. </li><li style=";text-align:right;direction:rtl"> העברת עובר אחד בתוך שפופרת 1.5 מ&quot;ל המכיל 1.0 מ&quot;ל של מחומם מראש agarose התכה נמוכה 1.0% (אה&quot;ע) שהוכנו מים ultrapure באמצעות פיפטה לחתוך קצה (P200). הערה: מומלץ לחמם את אה&quot;ע 80 ° C ו להתקרר במשך 3-5 דקות ב RT לפני הטבעה העובר. </li><li style=";text-align:right;direction:rtl"> מעבירים את העובר ב -150 אה&quot;ע μl לתוךתאיים coverglass ולהתאים את האוריינטציה של העובר, בצד הגבי למשל למטה בניסוי שתואר על A1R מיקרוסקופ סורק לייזר confocal הפוכה + (או כל מיקרוסקופ מתאימה אחרת), באמצעות קצה פלסטיק משובח. כאשר אה&quot;ע הוא polymerized, למלא את התא במים דגים המכיל 0.2 מ&quot;מ PTU ו 0.02% אתיל 3-aminobenzoate methansulfonate (Tricaine) עבור הרדמה. </li><li style=";text-align:right;direction:rtl"> לפני הדמיה המדגם, לחכות 15 דקות כדי לקבוע במדויק את השפעת Tricaine. ההרדמה מונעת תנועות עוברות, אשר ניתן לנטר באמצעות סטראו מצויד תאורת brightfield. הערה: התאים ניתן לעשות שימוש חוזר פעמים. </li></ol> 4. PSmOrange photoconversion <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את המדגם תחת מיקרוסקופ confocal ולסרוק את הדגימה כדי לזהות את האזור photoconvert באמצעות 488 ננומטר ו 561 nm לייזרים הדמיה GFP ו PSmOrange בהתאמה. הערה: השתמש A1R מיקרוסקופ סורק לייזר confocal הפוכה + על sta הפוכהge TiEcontrolled ידי תוכנת הדמיה מיקרוסקופ ומצוידים 488 ננומטר, 561 ננומטר ו 640 nm לייזרים עבור photoconversion והדמיה. עם זאת, היישום הוא בהחלט לא מוגבל מיקרוסקופ זה כל עוד קווי לייזר לגיור הדמיה זמינים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שים את המטרה אוויר 20X למקומו (NA: 0.75; WD: 1.0 מ&quot;מ; FOV: 0.64 x 0.64 מ&quot;מ). </li><li style=";text-align:right;direction:rtl"> השתמש בהגדרות מיקרוסקופ הבא כדי לזהות את החלבון PSmOrange לפני photoconversion: 561 ננומטר לייזר, 0.74 mW הנמדדים המטוס מוקד מעל המטרה. הערה: זה מקביל ל -40% במערכת זו (מדידה במישור המוקד הוא הדרך היחידה לקבוע את כוח יעיל). התאם את עוצמת הלייזר בהתאם לצרכים ניסיוניים כמו יעילות של זריקות H2B-PSmOrange יכול להשתנות. </li><li style=";text-align:right;direction:rtl"> להוסיף גורמי זום כדי להדגיש את תחומי העניין. בהגדלות גדולות להפחית את זמן רכישת תמונה ולכן phototoxicity. </li></ol></li><li style=";text-align:right;direction:rtl"> לרכוש Z- מחסנית כיסוי המבנההים של עניין באמצעות 488 ננומטר ו 561 nm לייזרים במצב רציף (מצב קו 1-&gt; 4). תקן ה- z צעד בין 1.0 ו -2.0 מיקרומטר. ממוצע קו ותדיר סריקה ניתן להשתמש כדי לייעל את איכות תמונה. </li><li style=";text-align:right;direction:rtl"> סרוק את המדגם לבחור את האזור של כלי עניין (ROI) כדי להדגיש את האזור photoconvert. תקן את ההחזר על ההשקעה כאזור גירוי. בחר את מודול הפעלת תמונה / הלבנה, להפעיל את הלייזר 488 ננומטר על ידי סימון התיבה המתאימה ולהגדיר את לייזר 488 ננומטר עד 80% (כוח לייזר 1 mW הנמדדים המטרה). הגדר את מהירות הסריקה ל -0.5 שניות / מסגרת. </li><li style=";text-align:right;direction:rtl"> פתח את כלי העזר photoconversion (ND גירוי) וציין את ההגדרות photoconversion. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הקש על הפקודה &quot;הוסף&quot; כדי לציין את פרוטוקול photoconversion. </li><li style=";text-align:right;direction:rtl"> גדר &quot;שלב&quot; 1 בתפריט &quot;רכישה / הגירוי&quot; ל &quot;הרכישה&quot; ומציין את מספר התמונות להירכש לפני photoconversion בתפריט &quot;הלולאות&quot;. </li><li style=";text-align:right;direction:rtl"> גדר &quot;שלב&quot; 2 עד &quot;Stimulation &quot;והזן את מספר אירועי גירוי להתבצע. </li><li style=";text-align:right;direction:rtl"> התאם &quot;שלב&quot; 3 כמתואר עבור &quot;שלב&quot; 1. לחלופין, כנס המתנה &quot;שלב&quot; אחרי &quot;שלב&quot; 2 על ידי הזנת הזמן לחכות לפני הסיבוב האחרון של רכישת תמונה. </li><li style=";text-align:right;direction:rtl"> לאחר כל הפרמטרים נקבעים, להחיל את ההגדרה גירוי ולהפעיל את photoconversion. הערה: כוח לייזר, תדירות סריקה ומספר חזרות עבור כל סיבוב של photoconversion יכול להשתנות נבדלים העובר העובר. הגדרה להתחיל עם מוצג בטבלה 1. </li></ol></li><li style=";text-align:right;direction:rtl"> לרכוש Z- מחסנית הסופי באמצעות 488 ננומטר, 561 ננומטר ו 640 nm לייזרים החלת הגדרות כנ&quot;ל. הגדר את לייזר 640 ננומטר כדי להמחיש את PSmOrange המרה באמצעות כוח לייזר גבוהה (עד 4.5 mW). </li></ol> 5. העובר לרדת מעל אה&quot;ע <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר את העובר המכיל קאמרית מן מיקרוסקופ. מוציאים בזהירות את העובר מן u agaroseלשיר מלקחיים. </li><li style=";text-align:right;direction:rtl"> מעבירים את העובר לתוך צלחת פטרי-פלסטיק חדש מעוקר או לתוך צלחת 6-היטב מעוקר המכיל E3 1x ו -0.2 מ&quot;מ PTU. דגירה העובר על 28 מעלות צלזיוס עד לשלב של פיתוח הרצוי. </li></ol> 6. ניתוח הגורל של תאים המבטאים חלבון photoconverted PSmOrange <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Re-להטביע את העובר כפי שמתואר תחת נקודות 3.3 ו -3.4. </li><li style=";text-align:right;direction:rtl"> מניחים את המדגם תחת מיקרוסקופ confocal ולסרוק את הדגימה כדי לזהות תאים photoconverted (640 ננומטר) במבנה מהונדס GFP עניין (488 ננומטר). </li><li style=";text-align:right;direction:rtl"> לרכוש Z- מחסנית מכסה את המבנים של העניין באמצעות 488 ננומטר, 561 ננומטר ו 640 nm לייזרים במצב רציף (מצב קו 1-&gt; 4). תקן ה- z צעד בין 1.0 ו -2.0 מיקרומטר. ממוצע קו ותדיר סריקה ניתן להשתמש כדי לייעל את איכות תמונה. </li><li style=";text-align:right;direction:rtl"> השתמש באחת מהשיטות הבאות כדי לזהות תאים, אשר GFP שיתוף מפורש ואת חלבון photoconverted. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מותאם ללקוח תמונת פיג&#39;י אוטומטית3 J מאקרו <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שזירת ערימת מסמכי המקור באמצעות תוסף &quot;GaussianBlur&quot; (→ תהליך → מסנן → GaussianBlur) ולחסר את הערימות החלקות מהמקור באמצעות תוסף &quot;מחשבון תמונה&quot; (Image מחשבון → תהליך →). את הצעד הזה כדי לחזות מבנים של עניין ולהפחית את הזמן חישובית לניתוח. </li><li style=";text-align:right;direction:rtl"> החל סף ספציפי הערוצים הירוקים ומרחיקים בצבע האדום על מנת להדגיש את תאי photoconverted (→ תמונה → התאם → סף). השתמש באפשרות &quot;לנתח חלקיקים&quot; כדי לזהות את תחומי סף עם הגדרה מתאימה (→ לנתח → לנתח חלקיקים). </li><li style=";text-align:right;direction:rtl"> פתח את תוסף &quot;מחשבון תמונה&quot; ולהציג את ROIs חופף בערוץ ירוק ומרחיקות האדומים בצהוב (→ מחשבון תמונת → תהליך). </li></ol></li><li style=";text-align:right;direction:rtl"> תוכנת הדמיה מיקרוסקופ להערכת נתוני 3D <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הצג את המחסנית3D באמצעות אפשרות הצגת נפח הצגה (→ צפה נפח צג → תפריט ויזואליזציה 3D). השתמש בממשק הגרפי כדי לבחור את הערוצים הירוקים, אדום ומרחיקות האדומים, להתאים את הבהירות הניגודיות לחתוך את מחסנית 3D כדי להדגיש את אזור photoconverted (איור 1). הערה: שיטות דומות כדי לנתח את הנתונים זמינים ברוב התוכנות הנפוצות לניתוח תמונה. </li></ol></li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+intracellular+protein+movements+using+photoswitchable+fluorescent+proteins+PS-CFP2+and+Dendra2.">Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.</a> Nature Protocols. 2, 2024-2032 (2007).</li><li>Subach, F. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+mCherry+for+high-resolution+two-color+fluorescence+microscopy.">Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.</a> Nature Methods. 6, 153-159 (2009).</li><li>Patterson, G. H., Lippincott-Schwartz, 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+photoactivatable+GFP+for+selective+photolabeling+of+proteins+and+cells.">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> Science. 297, 1873-1877 (2002).</li><li>Subach, F. V., Patterson, G. H., Renz, M., Lippincott-Schwartz, J., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+monomeric+photoactivatable+red+fluorescent+protein+for+two-color+super-resolution+sptPALM+of+live+cells.">Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.</a> J Am Chem Soc. 132, 6481-6491 (2010).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), e1115 (2009).</li><li>Concha, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+tissue+interactions+across+the+dorsal+midline+of+the+forebrain+establish+CNS+laterality.">Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality.</a> Neuron. 39, 423-438 (2003).</li><li>Auer, T. O., Duroure, K., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+conversion+of+eGFP-+into+Gal4-transgenic+lines+in+zebrafish.">CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish.</a> Nature Protocols. 9, 2823-2840 (2014).</li><li>Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+CRISPR/Cas9-mediated+knock-in+in+zebrafish+by+homology-independent+DNA+repair.">Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.</a> Genome Research. 24, 142-153 (2014).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+zebrafish+early+embryonic+development+by+scanned+light+sheet+microscopy.">Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.</a> Science. 322, 1065-1069 (2008).</li></ol>

The authors have nothing to disclose.

עובר מהונדסים נושאת כתבי ניאון סייע ביסודו להבין התפתחות עוברית. עם זאת, יש עדיין הצורך החיוני עבור יזמים כדי להקל על ויזואליזציה הספציפי של מבנים בפרט. בהיעדרם, החוקרים מסתמכים על טכניקות כגון photoconversion של חלבוני ניאון כדי לברר על מוצאם והתפתחותם של מבנה האינטרס שלהם...

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days3. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.
Fluorescent photoconvertible proteins are well suited for cell tracking during embryonic development. The prerequisites for the application of such proteins are a long-lived nature, a well-separated emission range upon conversion and bright fluorescence. Available photoconvertible proteins comprise those that change their emission range upon conversion such as Kaede4, KiGR5, mEos26, PS-CFP2 or Dendra27 and others which are only fluorescent when photoactivated (PAmCherry8, PAGFP9 or PATagRFP10). Their applications to track cells in existing FP-transgenic animals are however limited as they often involve a green fluorescent state or do not fulfill all of the above criteria. Only recently, Subach and colleagues reported the PSmOrange protein, which changes its emission from orange/red to far red upon photoconversion and was successfully applied in cells in culture and cultured cells injected into mice2.
To investigate the protein's suitability for cell tracking in a living embryo, we generated an expression construct for the microinjection of nuclear-tagged H2B-PSmOrange into zebrafish embryos. We find that the protein fulfills all prerequisites for successful cell tracking in GFP transgenic backgrounds during the first 4 (and possibly more) days of zebrafish embryonic development. During this time, most of the cell migratory events are completed in fish making the PSmOrange system an excellent addition to the zebrafish toolkit.

<table><tbody><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>mMESSAGE mMACHINE SP6 Transcription kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>RNeasy MiniElute Cleanup kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>Plastic Pasteur</td><td>alpha laboratories</td><td>LW4000</td><td></td></tr><tr><td>Original H2B-PSmOrange Plasmid</td><td>Addgene</td><td>31920</td><td>The plasmid described in the paper is available in the Carl lab</td></tr><tr><td>FemtoJet Microinjector</td><td>Eppendorf</td><td>5247 000.013</td><td></td></tr><tr><td>Forceps (5 Inox)</td><td>NeoLab</td><td>2-1633</td><td></td></tr><tr><td>Lab-Tek II Chambered #1.5 German Coverglass System&amp;nbsp;</td><td>Nunc</td><td>155382</td><td></td></tr><tr><td>Nikon A1R+</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>Nikon PLAN Apo &amp;lambda; 20X air objective&amp;nbsp;</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>NIS Elements AR Software (v. 4.30.02)</td><td>Nikon GmbH Germany/Laboratory Imaging</td><td>No Number</td><td></td></tr></tbody></table>

tracking cells in gfp-transgenic zebrafish using the photoconvertible psmorange system

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

איור 1 מדגים דוגמה של מערכת photoconversion PSmOrange. מתחם האצטרובל הוא מבנה משומר ב diencephalon הגבה חוליות. כמו חוליות רבות אחרות, מורכבים זה מורכב של איבר האצטרובל במרכז של diencephalon ותאי parapineal צדדי-השמאל. ניסויים משחררים רפרוף אלגנטיים אבל גוזל זמן הראו כי...

Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

מעקב תאים דג הזברה GFP מהונדס שימוש במערכת photoconvertible PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

tracking-cells-gfp-transgenic-zebrafish-using-photoconvertible

Research

JoVE Journal

Developmental Biology

8.5K Views.  Heidelberg University. We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Video: Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Photoconversion תא בודד בהמחיה שלם דג זברה

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

הפרקים הבאים רקמה Endocardial ויה Photoconversion תאים של העובר דג זברה

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

שני הפוטונים מבוסס Photoactivation עוברי דג הזברה לחיות

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Biology

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Photoconversion של חלבונים מטוהרים פלורסנט ו Dual-בדיקה אופטי סימון בתאים חיים

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

ססגוניות זמן לשגות הדמיה של דג הזברה טראנסגנטי: חזותי בתאי גזע רשתית הופעל על ידי אבלציה ממוקד תאים עצביים

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

תא בודד הגורל מיפוי של דג הזברה

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

מעקב נייד באמצעות חלבוני Photoconvertible במהלך פיתוח דג זברה

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

דור של תרביות תאים המפוזרים Presomitic המזודרם להדמיה של דג הזברה פילוח השעון בתאים בודדים

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

ROS הדמיית תאים חיים במהלך התפתחות עצבית

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

Neuroscience

Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

Source: Asakawa, K. et al. <a href="https://www.jove.com/v/62932">Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae.</a> J. Vis. Exp. (2022)
In this video, we describe a phase separation assay, wherein intracellular proteins comprising intrinsically disordered regions fused to a photosensitive oligomerization domain are induced via blue-light exposure to self-associate into membrane-less liquid-like condensates.

בדיקת הפרדת פאזות חלבון: שיטה אופטוגנטית להפרדת שלב חלבון קושר RNA מוטנטי בנוירונים מוטוריים בעמוד השדרה של זחלי דגי זברה

Source: Asakawa, K. et al. Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae. J. Vis. Exp. (2022)
In this video, we ...