There is immense cellular and molecular heterogeneity in biological systems. Here, we demonstrate the utility of integrating an inverted light microscope with an ambient ionization source, nanospray electrospray desorption ionization, attached to a high-resolution mass spectrometer to characterize the molecular composition of mouse spinal cords. We detected a broad range of molecules, including peptides and proteins, as well as metabolites such as lipids, sugars, and other small molecules, including S-adenosyl methionine and glutathione, through top-down MS. Top-down analysis revealed variation in the expression of Hb, including the transition from fetal to adult Hb and heterogeneity in Hb subunits consistent with the genetic diversity of the mouse models. Similarly, temporal changes to actin-sequestering proteins β-thymosins during development were observed. These results demonstrate that interfacing microscopy with ambient ionization provides the means to perform targeted in situ ambient top-down mass spectral analysis to study the pattern of proteins, lipids, and sugars in biologically heterogeneous samples.