Two-Laser, Large-Field Hyperspectral Microarray Scanner for the Analysis of Multicolor Microarrays

Abstract

We describe the development and operation of a two-laser, large-field hyperspectral scanner for analysis of multicolor genotyping microarrays. In contrast to confocal microarray scanners, in which wavelength selectivity is obtained by positioning band-pass filters in front of a photomultiplier detector, hyperspectral microarray scanners collect the complete visible emission spectrum from the labeled microarrays. Hyperspectral scanning permits discrimination of multiple spectrally overlapping fluorescent labels with minimal use of optical filters, thus offering important advantages over standard filter-based multicolor microarray scanners. The scanner uses two-sided oblique line illumination of microarrays. Two lasers are used for the excitation of dyes in the visible and near-infrared spectral regions. The hyperspectral scanner was evaluated with commercially available two-color calibration slides and with in-house-printed four-color microarrays containing dyes with spectral properties similar to their commercial genotyping array counterparts.

Designing microarray phantoms for hyperspectral imaging validation

“…Additionally, the narrow bandwidths and numerous channels of hyperspectral imagers allow resolution of fluorophores with similar emission maxima when compared to optical filters [3]. For example, Erfurth et al [4] calculated a cross-talk of typically <1% between a series of fluorophores after hyperspectral processing. Our ultimate goal is to use hyperspectral imaging techniques for cell and tissue imaging [5].…”

Oligonucleotide Array-in-Well Platform for Detection and Genotyping Human Adenoviruses by Utilizing Upconverting Phosphor Label Technology

“…A number of instruments, based on fluorescence readout, have been reported for detection of hybridized targets . The most widely used imaging method in microarray analysis is a laser-based confocal microscope scanner, that detects the fluorophore emission corresponding to the band-pass filter utilized for each fluorophore. ,, Recently, a two-laser hyperspectral microarray scanner has been developed for the multicolor microarray analysis . The hyperspectral imaging is able to identify multiple fluorescent dyes within a single scan.…”

“…10,20,21 Recently, a two-laser hyperspectral microarray scanner has been developed for the multicolor microarray analysis. 22 The hyperspectral imaging is able to identify multiple fluorescent dyes within a single scan. Because of the high cost of a confocal microscope scanner, development of new microarray detection methods is gaining more importance in the field of diagnostics.…”

Quasi-Confocal, Multichannel Parallel Scan Hyperspectral Fluorescence Imaging Method Optimized for Analysis of Multicolor Microarrays

“…Moreover, hyperspectral scanning permits minimal use of optical filters for analysis of multicolor microarrays due to recording of spectra, thus offering advantages over filter-based scanners. The wealth of information that is available from spectrally resolving an image makes hyperspectral imaging an extremely attractive technique, and various hyperspectral instruments have been reported over the past years. − In these papers, hyperspectral fluorescence imaging systems are employed mainly for imaging biological samples, and some efforts have been devoted to microarray research, while we attempt to develop a system further improved and optimized for microarray readout.…”