Fluorescence Excitation-Emission-Matrix Imaging .

We present a 4-dimensional (4D) fluorescence imaging system in which each of the 65,536 pixels in the image array contains an excitation-emission-matrix spectrum with 31 excitation wavelengths and 8 emission wavelengths ( x , y , λ exc , λ em ). Hadamard-transform multiplexing of the excitation light from a 31-channel programmable light source allows for an increase in the data acquisition rate so that each 65,536-pixel image can be obtained within 8 s. The system is demonstrated and characterized using, first, a 4D image of 10 capillaries filled with four dye solutions and their binary and ternary mixtures, and, second, using a sequence of about 100 images of layered fluorescent dye solutions and their changing fluorescence as a function of temperature. Multivariate analysis using parallel factor analysis produces images of the spatial distribution of the fluorophores together with their relative intensity as a function of time.