Nonlinear Optical Microscopy (NLOM) has been shown to be a valuable tool for noninvasive imaging of complex biological systems. An effective approach for multicolor molecular microscopy is simultaneous excitation of multiple fluorophores by broadband sub-10-fs pulses. This dissertation will discuss the development of two spectral imaging systems using the principles of nonlinear optical microscopy for pixel-by-pixel spectral segmentation of multiple fluorescent spectra. The first spectral system is reliant on a fiber-optic cable to transmit fluorescent signal to a spectrometer, while the second is based on a spectrometer with an aberration-corrected concave grating that is directly coupled to the microscope. A photon-counting, 16-channel multianode photomultiplier tube (PMT) is used for both systems. Custom software developed in LabVIEW controls multiple counter cards as well as a field-programmable gate array (FPGA) for 1 Hz acquisition of 256x256x16 spectral images. Biological specimen consisting of multicolor endothelial cells and zebrafish will be used for experimental verification. Results indicate successful spectral segmentation of multiple fluorophores with a decrease in signal-to-noise ratio in the FPGA-based imaging system.