एक विश्लेषणात्मक उपकरण है कि तीन आयामी प्रतिदीप्ति छवियों से सेलुलर आकृति विज्ञान परिवर्तन quantifies
Summary
हम Imaris तंत्रिका विज्ञान, ImarisXT और MATLAB एक अपरिभाषित आकार के तीन आयामी एकल कक्षों के confocal प्रतिदीप्ति से लिया morphology में बदलाव को मापने का इस्तेमाल एक सॉफ्टवेयर प्लेटफार्म विकसित किया है. इस उपन्यास दृष्टिकोण सेल आकार में रिसेप्टर सक्रियण के बाद बदलाव यों इस्तेमाल किया जा सकता है और इसलिए दवाओं की खोज के लिए एक संभव अतिरिक्त उपकरण का प्रतिनिधित्व करता है.
Abstract
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 Eidgenössische 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.
Protocol
Discussion
हम पता चला है कि सीआरएफ उपचार आकारिकी और सीआरएफ-R2 के स्थान में एक महत्वपूर्ण परिवर्तन प्रेरित किया. चयनात्मक प्रतिपक्षी उपचार से हिचकते सीआरएफ-R2 में परिवर्तन किया गया था. हम पता चला है कि रिसेप्टर संशो?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम Imaris, Imaris XT और Matlab के इस्तेमाल के लिए जैव इमेजिंग विकास केंद्र (BIDC) कैलिफोर्निया विश्वविद्यालय, सैन फ्रांसिस्को धन्यवाद. हम तकनीकी सहायता के लिए और हेनरी, लालकृष्ण Floren, पांडुलिपि का संपादन करने के लिए उनके योगदान के लिए एल Daitch वी. Kharazia धन्यवाद. : 1R21DA029966-01 और NIH फास्ट ट्रैक एसईबी फार्मेसी, UCSF स्कूल MLSMR संग्रह स्क्रीन पुरस्कार (यह काम शराब और मादक द्रव्यों के सेवन पर एसईबी, स्वास्थ्य के राष्ट्रीय संस्थानों के माध्यम से UCSF कैलिफोर्निया मेडिकल रिसर्च के राज्य से धन के द्वारा समर्थित किया गया डीन कार्यालय और क्लीनिकल फार्मेसी) और स्कूल ऑफ मेडिसिन CLHK (नैदानिक भेषजगुण और प्रायोगिक चिकित्सा).
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Human Embryonic Kidney (HEK293) | American Type Culture Collection | CRL-1573 | |
| Dulbecco’s Modified Eagle Medium (DMEM) | Invitrogen | 11965118 | |
| Fetal Bovine Serum (FBS) | Invitrogen | SH30070.03 | |
| AlexaFluor-488 (IgG2b) | Invitrogen | A-11001 | |
| monoclonal anti-HA.11 (IgG1) | Covance | 16B12 | |
| DAPI | Vector Laboratories | H-1200 | |
| CRF | Sigma | C2917 | |
| Antisauvagine-30 (AS-30) | Sigma | A4727 |
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