Name: an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images
Uploaded: 2012-08-31T13:00:00
Description: Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

אנו מודים הביולוגית הדמית מרכז הפיתוח (BIDC) מאוניברסיטת קליפורניה, סן פרנסיסקו לשימוש בImaris, Imaris XT ו Matlab. אנו מודים V. Kharazia לסיוע הטכני ועל הנרי, LK פלורן, L. ךייטש על תרומתם לעריכה של כתב היד. עבודה זו נתמכה על ידי מימון ממדינת קליפורניה מחקר רפואי על אלכוהול התעללות וסמים דרך קליפורניה בסן פרנסיסקו לSEB, המכונים הלאומיים לבריאות: 1R21DA029966-01 ופרס NIH מסלול המהיר להקרין את אוסף MLSMR לSEB, קליפורניה בסן פרנסיסקו בית הספר לרוקחות ( משרד הדיקן ורוקחות קליניות) ובית הספר לרפואה (פרמקולוגיה קלינית &amp; Therapeutics ניסיוני) לCLHK.

1. ניתוח מורפומטריות תלת ממדי של שינויים פנוטיפי תא בודד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> (HEK293) תאי כליה עובריים אנושיים היו transfected עם hemagglutinin (HA) מתויג-corticotropin משחרר factor receptor-2 (CRF-R2), הקולטן G-חלבון בשילוב (GPCR) כפי שתואר לעיל 4, 5. </li><li style=";text-align:right;direct...

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אנחנו הראינו כי טיפול CRF מושרה שינוי משמעותי במורפולוגיה ומיקום של CRF-R2. השינוי בCRF-R2 נבלם על ידי טיפול אנטגוניסט סלקטיבי. אנחנו הראינו כי שינויי הקולטן הם לא אותרו ולא ניתן למדוד באמצעות טכניקות multispectral 2D הסטנדרטיות. היכולת ללמוד תמונות 3D מורכבות היא קריטית כדי לשלב א?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות (אופציונלי) </td></tr><tr><td> הכליה עוברית אנושית (HEK293) </td><td> אוסף תרבות סוג אמריקאי </td><td> CRL-1573 </td><td></td></tr><tr><td> של Dulbecco שינוי הנשר הבינוני (DMEM) </td><td> Invitrogen </td><td> 11965118 </td><td></td></tr><tr><td> עוברי בסרום שור (FBS) </td><td> Invitrogen </td><td> SH30070.03 </td><td></td></tr><tr><td> AlexaFluor-488 (IgG2b) </td><td> Invitrogen </td><td> -11001 </td><td></td></tr><tr><td> חד שבטי אנטי HA.11 (1 IgG) </td><td> Covance </td><td> 16B12 </td><td></td></tr><tr><td> DAPI </td><td> מעבדות וקטור </td><td> H-1200 </td><td>; </td></tr><tr><td> CRF </td><td> סיגמא </td><td> C2917 </td><td></td></tr><tr><td> Antisauvagine-30 (AS-30) </td><td> סיגמא </td><td> A4727 </td><td></td></tr></tbody></table>

an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

פתחנו פלטפורמת תוכנה שמנצלת Imaris Neuroscience, ImarisXT וMATLAB כדי למדוד את השינויים במורפולוגיה של צורה מוגדרת נלקחה מפלואורסצנטי confocal תלת ממדי של תאים בודדים. זו גישה חדשנית יכולה לשמש כדי לכמת את השינויים בצורת התא בא הפעלה קולטנית ולכן מייצגת כלי נוסף אפשרי לגילוי סמים.

כלי אנליטי שמכמת שינויי מורפולוגיה תאיים מתמונות פלואורסצנציה תלת ממדים

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

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