Three-dimensional spectral imaging by Hadamard transform spectroscopy in aprogrammable array microscope

We report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolu tion. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chr omosome spread from Drosophila melanogaster doubly labelled for the Polyhom eotic protein by indirect immunofluorescence labelling with Alexa594 and fo r DNA with YOYO-1. The resulting four-dimensional data set consisted of the xyz spatial dimensions (898 x 255 x 16) with a 26-point spectrum at each s patial location. The total exposure time to the sample was 34 min. The syst em requires the acquisition of multiple images, and thus works best with fl uorophores that are resistant to photobleaching. Image deconvolution reduce d the amount of out-of-focus blur by up to a factor of 8, resulting in a dr amatic improvement in the visualization of the chromosome backbone and loca lization of the specific Polyhomeotic domains.