Laser desorption ionization mass spectrometry (LDI-MS) is used to image brain lipids in the fruit fly, Drosophila, a common invertebrate model organism in biological and neurological studies. Three different sample preparation methods, including sublimation with two common organic matrixes for matrix-assisted laser desorption ionization (MALDI) and surface-assisted laser desorption ionization (SALDI) using gold nanoparticles, are examined for sample profiling and imaging the fly brain. Recrystallization with trifluoroacetic acid following matrix deposition in MALDI is shown to increase the incorporation of biomolecules with one matrix, resulting in more efficient ionization, but not for the other matrix. The key finding here is that the mass fragments observed for the fly brain slices with different surface modifications are significantly different. Thus, these approaches can be combined to provide complementary analysis of chemical composition, particularly for the small metabolites, diacylglycerides, phosphatidylcholines, and triacylglycerides, in the fly brain. Furthermore, imaging appears to be beneficial using modification with gold nanoparticles in place of matrix in this application showing its potential for cellular and subcellular imaging. The imaging protocol developed here with both MALDI and SALDI provides the best and most diverse lipid chemical images of the fly brain to date with LDI.