Microscope system renders 3-D images
At the Biotechnology Research Department of the Orange County Water District (Orange, CA), researchers have built a digitally based confocal microscope system capable of rendering three-dimensional (3-D) images. In operation, the images are obtained using a light microscope equipped with oil-immersion ultraviolet (UV) and illumination optics from Olympus (Melville, NY). Rather than use a continuous-wave mercury lamp as the source of excitation light, a short-arc xenon flash from EG&G Optoelectronics (Santa Clara, CA) minimizes the photobleaching of cells, thereby allowing simultaneous quantitative determination of fluorescence.
Projection eyepieces ranging from 1.67X to 6.7X are used in the microscope head to convey images to a modified image intensifier from B.E. Meyers (Redmond, WA). Images are captured using a Model 4815-5000 2/3-in. CCD camera from Cohu (San Diego, CA), which uses an RS170 link to transfer data to a modified frame grabber from Imagraph (Chelmsford, MA).
Digital image size is 512 ¥ 480 pixels, which, with a 2.5X projection eyepiece, yields a spatial resolution of 9.227 mm in the x axis and 0.191 mm in the y axis. Each image in the 8-bit image stack is first processed to filter noise using ImageProPlus from Media Cybernetics (Silver Spring, MD) and then digitally deconvolved using MicroTome AT deconvolution software from VayTek (Fairfield, IA) to remove defocused information.
After an image stack is processed, it is transmitted via an Ethernet link to an Iris Indigo Elan workstation from Silicon Graphics (Mountain View, CA). The image stack is assembled into a volume using VoxBlast UNIX software from VayTek. Data are resampled, and then interpolation is used to produce a voxel volume that represents the¥axis of the original specimen.
Voxel volume data are typically rendered in 24-bit false color to accentuate contrast of the original gray-level image, permitting both manual and machine recognition of features within the rendered volume. The resulting virtual reconstruction can be manipulated by the microscopist in all three axes, allowing stereoptic display, virtual dissections, polygon extraction of areas of interest, and both two- and three-dimensional tomography. Lastly, the images are either digitally archived to tape or printed using a ColorEase dye-sublimation printer from Eastman Kodak Co. (Rochester, NY) onto either a paper or a transparency medium. ©
