The Maestro 2.4 software for in vivo fluorescence imaging is available from Cambridge Research & Instrumentation Inc. It enables users to build spectral libraries and to remove autofluorescence from specimens. The Compute Spectra tool automatically unmixes combined/overlapping spectral signatures, and the Real Component Analysis algorithm automatically identifies them. Quantitative analysis capabilities include detection and quantification of colocalized signals, component segmentation, region-of-interest analysis and image export to third-party image analysis software. The software acquires spectral images accurately, detects fluorophores with different excitation wavelengths and generates pseudocolor unmixed composite images in selectable colors.