HYPERSPECTRAL SLIDE SCANNING: POTENTIAL FOR RAPID WHOLE BRAIN GENE IMAGING

V.L. Sutherland¹*; J.A. Timlin²; M.B. Sinclair²;
M.K. Chawla¹; B.L. McNaughton¹; P.F. Worley³;
B. Roysam⁴; J.F. Guzowski⁵; C.A. Barnes¹

1. NSMA, Univ Arizona, Tucson, AZ, USA
2. Sandia National Labs^†, Albuquerque, NM, USA
3. Neurosci & Neurol, Johns Hopkins Univ, Baltimore, MD, USA
4. ECSE, RPI, Troy, NY, USA
5. Neurosci, Univ New Mexico, Albuquerque, NM, USA

Typical confocal imaging systems are capable of acquiring image stacks through the x, y and z-planes and permit analysis of immediate-early genes (IEGs) within specific neuronal cell populations. Two problems that arise with these systems include the time-consuming nature of acquiring image stacks and the inability to use probes with similar emission spectra. Sandia National Labs has developed a hyperspectral slide scanner (SNL-HSS) capable of acquiring data across the entire visible emission spectrum (560 spectral points, 16 bit dynamic range). Currently, the SNL-HSS can scan a 10 mm x 6 mm brain section in less than 2 minutes at 30 m (low) resolution and 100 m x 200 m section at 0.8 m (high) resolution in ~1 minute. Initial experiments on brain tissue compared hyperspectral and confocal systems using 2 different IEGs, Arc and Homer 1a, with Cy3 and Cy5, respectively, and Propidium Iodide (PI) to label nuclei. These dyes have overlapping emission spectra with peaks at 575 nm for Cy3, 617 nm for PI and 674 nm for Cy5. The additional spectral information provided by the SNL-HSS permits extraction of the overlapping emission spectra and provides accurate dye concentration measurements, even in the presence of high levels of background signal and autofluorescence. The SNL-HSS provides an excellent complementary approach to high-resolution confocal methods and may be appropriate for rapidly determining the colocalization patterns of multiple genes in whole brain sections.

†Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the US DOE under contract DE-AC04-4AL85000.

Support Contributed By: AG09219 & AG18230