Hyperspectral imaging: a novel approach for microscopic analysis.
BACKGROUND
The usefulness of the light microscope has been dramatically enhanced by recent developments in hardware and software. However, current technologies lack the ability to capture and analyze a high-resolution image representing a broad diversity of spectral signatures in a single-pass view. We show that hyperspectral imaging offers such a technology. METHODS AND RESULTS We developed a prototype hyperspectral imaging microscope capable of collecting the complete emission spectrum from a microscope slide. A standard epifluorescence microscope was optically coupled to an imaging spectrograph, with output recorded by a CCD camera. Software was developed for image acquisition and computer display of resultant X--Y images with spectral information. Individual images were captured representing Y-wavelength planes, with the stage successively moved in the X direction, allowing an image cube to be constructed from the compilation of generated scan files. This prototype instrument was tested with samples relevant to cytogenetic, histologic, cell fusion, microarray scanning, and materials science applications.


CONCLUSIONS
Hyperspectral imaging microscopy permits the capture and identification of different spectral signatures present in an optical field during a single-pass evaluation, including molecules with overlapping but distinct emission spectra. This instrument can reduce dependence on custom optical filters and, in future imaging applications, should facilitate the use of new fluorophores or the simultaneous use of similar fluorophores.
McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-8591, USA.
R A
RA
Schultz
T
T
Nielsen
J R
JR
Zavaleta
R
R
Ruch
R
R
Wyatt
H R
HR
Garner
D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation* / Q000379:methods; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D008863:Microspheres; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods; D013194:Staining and Labeling