Center for Microscopy and Image Analysis.
Three Dimentional Image Analysis
In resent years, thanks to the support of MRDDRC at CNMC, CMIA has expanded its capabilities for 3D image analysis. Two workstations equipped with Image Pro Plus and 3D constructor are available. Investigators should not hesitate to ask the CMIA director about the existing capabilities, and how they can be integrated in their specific project. 3D image analysis provides valuable complement to the 2D analysis, for efficient modeling of experimental systems. In addition to qualitative observations, the software also offeres options for 3D data aquisition. Carefully considered experimental desighn for 3D data collection could provide extreemely valuable results, particularly in anatomically complex systems.
Series of MRI images were used for creation of this movie. The MRI images were encoded with spectral lookup table encoding density, and opened with 3D constructor of Image Pro Plus software available at the CMIA. The highest densities were further displayed as bgowinish iso-surface. Blue spectra represent low density and green moderate density. A threshold was applied to eliminate non-significant image information.
In this example, the vestibular primary afferents were llabeled with biocytin (green) and synaptic vesicle protein, synaptotagmin was used as a marker for synaptic terminals (red). Volumes of co-localization represent synaptic terminals of primary vestibular origin. Co-localization was represented as blue iso-surface. The image on the right side demonstrates co-localization volumes. Left arrow, pints on digitally magnified fields to model a synaptic terminal.
In this experiment a potassium channel subunit expression (Kv1.1) was detected by immmunolabeling (Kv1.1, Alexa Fluor 594, red) simultaneous with a marker for neurons (MAP2, Alexa Fluor 488, green). A series of confocal images were collected with 60x, 1.4 objective. Further, the confocal stack was imported to Image Pro Plus, 3D constructor (A). Kv1.1 labeling at the initial segemnt was difficult to select based on conventional 2D image analysis approach. The initial segment was present in composite image (A) in the boxed in region, and in B, with MAP2 labeling digitally removed. 3D analysis of the region of interest, revealed several imunolabled clusters (C), which were further merged to represent the total labeling of this potassium channel subunit at this sub-cellular compartment. Total volume, integrated optical brightness were extracted.
The Center for Microscopy and Image Analysis, The George Washington University Medical Center, Ross Hall 406, 2300 I St NW, Washington DC 20037 Ph. 202.994.2881 Fax. 202.994.8885, anaasp@gwumc.edu
This movie was created from series of confocal images. Vestibular fibers were labeled with biocytin (red) and neurons were labeled with MAP2 (green). At the end of the movie MAP2 labeling was removed to enable visualization of the complex synaptic terminals around the neuronal cell body.
Two planes of view of a 3D reconstructions from series of confocal images representing neurons labeled with MAP2 (red) and connexin 43 labeling mainly at the vicinity of the neurons (green).
