Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2 x 2 fan-out element.

We have developed a multifocal diffractive-optical fluorescence correlation spectroscopy system for parallel excitation and detection of single tetramethylrhodamine biomolecules in microarrays. Multifocal excitation was made possible through the use of a 2 x 2 fan-out diffractive-optical element with uniform intensity in all foci. Characterization of the 2 x 2 fan-out diffractive-optical element shows formation of almost perfect Gaussian foci of submicrometer lateral diameter, as analyzed by thermal motion of tetramethylrhodamine dye molecules in solution. Results of parallel excitation and detection in a high-density microarray of circular wells show single-biomolecule sensitivity in all four foci simultaneously.