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We have developed a new, high performance, hyperspectral microscope for biological and other applications. For each voxel within a three-dimensional specimen, the microscope simultaneously records the emission spectrum from 500 nm to 800 nm, with better than 3 nm spectral resolution. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical sectioning. Optical throughput and detection efficiency are maximized through the use of a custom prism spectrometer and a backside thinned electron multiplying charge coupled device (EMCCD) array. A custom readout mode and synchronization scheme enable 512-point spectra to be recorded at a rate of 8300 spectra per second. In addition, the EMCCD readout mode eliminates curvature and keystone artifacts that often plague spectral imaging systems. The architecture of the new microscope is described in detail, and hyperspectral images from several specimens are presented. OCIS codes: 180.0180, 180.1790, 180.2520.

We describe the design, construction, and operation of a hyperspectral microarray scanner for functional genomic research. The hyperspectral instrument operates with spatial resolutions ranging from 3 to 30 [micro]m and records the emission spectrum between 490 and 900 nm with a spectral resolution of 3 nm for each pixel of the microarray. This spectral information, when coupled with multivariate data analysis techniques, allows for identification and elimination of unwanted artifacts and greatly improves the accuracy of microarray experiments. Microarray results presented in this study clearly demonstrate the separation of fluorescent label emission from the spectrally overlapping emission due to the underlying glass substrate. We also demonstrate separation of the emission due to green fluorescent protein expressed by yeast cells from the spectrally overlapping autofluorescence of the yeast cells and the growth media. OCIS codes: 170.3880, 170.2520.

Raman spectroscopic markers have been determined for fatigue-related microdarnage in bovine bone. Microdamage was induced using a cyclic fatigue loading regime. After loading, the specimens were stained en-bloc with basic fuchsin to facilitate damage visualization and differentiate fatigue-induced damage from cracks generated during subsequent histological sectioning. Bone tissue specimens were examined by light microscopy and hyperspectral near-infrared Raman imaging microscopy. Three regions were defined - tissue with no visible damage, tissue with microcracks, and tissue with diffuse damage. Raman transects, lines of 150-200 Raman spectra, were used for initial tissue surveys. Exploratory factor analysis of the transect Raman spectra has identified spectroscopically distinct chemical microstmctures of the bone specimens that correlate with damage. In selected regions of damage, full hyperspectral Raman images were obtained with 1.4-(mu)m spatial resolution. In regions of undamaged tissue, the phosphate (nu)(sub 1) band is found at 957 cm(super -1), as expected for the carbonated hydroxyapatic bone mineral. However, in regions of visible microdamage, an additional phosphate (nu)(sub 1) band is observed at 963 cm(super -1) and interpreted as a more stoichiometric, less carbonated mineral species. Raman imaging confirms the qualitative relationship between the Rarnan spectral signature of bone mineral and the type of microdamage in bovine bone. Two tentative explanations for the presence of less carbonated phosphate in damaged regions are proposed.