Capturing in situ fluorescence images of marine organisms presents many technical challenges. The effects of the medium, as well as the particles and organisms within it, are intermixed with the desired signal. Methods for extracting and preparing the imagery for analysis are discussed in reference to a novel underwater imaging system called the low-light-level underwater multispectral imaging system (LUMIS). The instrument supports both uni- and multispectral collections, each of which is discussed in the context of an experimental application. In unispectral mode, LUMIS was used to investigate the spatial distribution of phytoplankton. A thin sheet of laser light (532 nm) induced chlorophyll fluorescence in the phytoplankton, which was recorded by LUMIS. Inhomogeneities in the light sheet led to the development of a beam-pattern-correction algorithm. Separating individual phytoplankton cells from a weak background fluorescence field required a two-step procedure consisting of edge detection followed by a series of binary morphological operations. In multispectral mode, LUMIS was used to investigate the bio-assay potential of fluorescent pigments in corals. Problems with the commercial optical-splitting device produced nonlinear distortions in the imagery. A tessellation algorithm, including an automated tie-point-selection procedure, was developed to correct the distortions. Only pixels corresponding to coral polyps were of interest for further analysis. Extraction of these pixels was performed by a dynamic global-thresholding algorithm.