Hyperspectral Imaging Microscope and Scanner
At UTSW, we have been developing state-of-the-art hyperspectral imaging systems. This has included the ASTRAL DNA sequencer, the MAGNA slide array scanner and now the Hyperspectral Imaging Cytogenetics Microscope (patent applied for). This microscope is now in regular use at UTSW for research biological and medical research.
The Hyperspectral Imaging Cytogenetics project consists of three components: 1) the development of a hyperspectral imaging microscope, 2) the development of a unique set of fluorochrome labels for a variety of applications and 3) the application of these to clinical and biological research samples. The HIC microscope (patent pending) was co-invented by Skip Garner and Roger Schultz as a project in the Center for Biomedical Inventions. The first prototype was constructed by Tom Nielsen and Jeff Zavaleta, medical students at UTSW.
For complete details, please see: R. A. Schultz, T. Nielsen, J.R. Zavaleta, R. Ruch, R. Wyatt and H.R. Garner, “Hyperspectral Imaging: A Novel Approach For Microscopic Analysis,” Vol. 43, pgs. 239-247, Cytometry, 2001.
Summary
We have developed a prototype hyperspectral imaging microscope capable of collecting the complete fluorescent spectrum from a region of a microscope slide. This microscope correlates spatial and spectral information without requiring the use of optical filters, offering important advantages over a standard epi-fluorescence microscope.
Standard FISH protocols require the use of filters to view dyes with widely separated emission spectra, placing restrictions on the combinations and number of dyes used. By obtaining the entire spectrum emitted from a point on the slide, these limitations may be overcome.
At the center of our hyperspectral imaging system is a standard epi-fluorescence microscope equipped with a standard filter cubeset, multiple objectives, multiple excitation sources, and a motorized stage to allow precise movement. The side port of the microscope is optically coupled to an imaging spectrograph, usually set up to measure approximately 400-780 nm. The output of the spectrograph is recorded by a CCD camera with a resolution of 1536x1024 pixels.
Software developed in house controls all hardware for acquisition and contains many features for displaying the resultant XY images and spectral information. During a scan, individual pictures are taken in the l-y plane while the stage is moved in the x direction to build an image cube. The excitation energy is opticaly mapped to a single line of interest to maximize resolution and minimize washout of the dyes. The l-y images are received from the camera and loaded into memory to form an image cube which may be viewed in the XY plane. A graph of the spectrum for any pixel may be viewed by clicking on the cube. A standard linear curve fitting algorithm is used to determine the contribution of individual dyes to the measured spectrum. The software can also use a windowing technique which emulates standard filtering. An overlay feature allows the fluorochrome curve fitting results to be compared by displaying them in pseudocolor in a single image. The contribution of a dye may be emphasized by turning off other dyes or by scaling their values up or down. Overlapping colors may be added, averaged, or the maximum or minimum values may be viewed. The overlay feature also allows multiple scans to be performed on the same region of the slide and the results compared, allowing the use of multiple excitation sources to build one image.
The prototype has been tested using a large range of samples, and is now in regular use for cytogenetics, histology, FISH, cell fusion, microarray scanning and materials science. Testing with microspheres demonstrated a spatial resolution of approximately 0.2 um. Standard DAPI/FITC/Texas Red stained chromosomes have been resolved. The overlaping spectra of Texas Red and MitoTracker Orange were resolved using curve fitting.
