Dual-wavelength microarray fluorescence detection system using volume holographic filter

Abstract

A dual-wavelength microarray scanner based on laser confocal principle is constructed to acquire the laser-induced two-color fluorescence from dyes Cy3 and Cy5. Unlike most commercially available micoarray scanners whose lasers are continuous wave, filters are bandpass and the channels are multiple photomultiplier tubes (PMTs), the system mainly consists of two diode lasers, four volume holographic band-stop filters, and one PMT. The central wavelengths of two kinds of volume holographic band-stop filters are equal to those of the two laser, respectively. The fluorescence beams can pass through the filters, while laser beams are blocked by them because the filters are cut off. The two fluorescences can pass through a common optical path. Thus, only one pinhole and one PMT are required. The two fluorescences are acquired by the PMT with time sharing. The two respective fluorescences can be gathered when two diode lasers work asynchronously. If lasers work synchronously, the two wavelength fluorescences could be acquired simultaneously by the same PMT. A synthesis of a two-color fluorescence image can be realized straightforwardly in this manner. An experiment is conducted and Cy3 and Cy5 fluorescences are captured. Gridding of the fluorescent images based on basic morphological erosion appropriately locates the boundary between adjacent spots.