Hadamard-Multiplexed Multispectral Fluorescence Microscopy

We demonstrate an in-lab built Hadamard-multiplexed light source with a multispectral camera for fluorescence excitation-emission matrix (EEM) microscopy. Various dye emulsions are imaged through a microscope, illuminated by an array of visible-wavelength laser diodes through fiber optic cables coupled to the side of a microscope slide (Fig. 1). The result is a four-dimensional data array consisting of 256x256 “super-pixels” each with eight wavelength channels from 474 – 640 nanometers, and seven excitation wavelengths, creating a 7 × 8 EEM for each of the 65,536 pixels in the image [1]. The fluorescence information for each pixel in the microscope field of view is obtained simultaneously, without raster-scanning the illuminated area.