Highly multiplexed optically sectioned spectroscopic imaging in a programmable array microscope

We report the construction of a spectroscopic programmable array microscope capable of optical sectioning using Hadamard transform methods. Previously described Hadamard transform imaging consisted of first-order systems in which the mask was used for either illumination or detection. In this paper, a second-order Hadamard transform system is described in which a single one-dimensional mask serves for both illumination and detection. The system verifies earlier predictions that the optical spreading function along the transform axis can be inferred from the echoes arising in second-order systems using sequence lengths of 2(n)-1. With a one-dimensional Hadamard mask, the system has an axial resolution of 1.3 mum and may be used with or without the subtraction of an appropriately scaled wide field image depending on the degree of sectioning required. The system was tested using thin films, fluorescent beads, and a fixed biological specimen.