Nuance 3

Images of paper discs were collected on a Zeiss Axio Zoom V16 Macroscope (magnification 7x) with an AxioCam MRm in a humidified glass chamber or through the bottom of a clear bottom 384-well plate. Collected images were then stitched together using Zeiss Zen software into large composite images for further processing in ImageJ. Experiments were arranged so that images of matching control and treatment paper-based reactions were collected together such that parameters could be adjusted for all samples simultaneously. Once optimized, images of individual paper discs were cropped and arrayed into figures. For GFP expression in the S30 cell-free system, which exhibits a high level of autofluorescence, we used a Nuance camera to collect multispectral images between 500 and 620 nM. Perkin Elmer Nuance 3.0.2 software was then used to unmix the spectral signature of the GFP from that of the cell extract. A similar approach was used to create a absorbance signature (420 to 720 nM) for imaged paper discs with p-nitrophenol, the chitinase cleavage product of 4-Nitrophenyl N,N′-diacetyl-b-D-chitobioside. Images collected with the Nuance camera were scaled using bilinear transformation. For a few composite images, areas around the paper discs were masked to remove extraneous signal.