Semitransparent porous silicon substrates have been developed for pairing nanostructure-initiator mass spectrometry (NIMS) imaging with traditional optical-based microscopy techniques. Substrates were optimized to generate the largest NIMS signal while maintaining sufficient transparency to allow visible light to pass through for optical microscopy. Using these substrates, both phase-contrast and NIMS images of phospholipids from a scratch-wounded cell monolayer were obtained. NIMS images were generated using a spatial resolution of 14 μm. Coupled with further improvements in spatial resolution, this approach may allow for the localization of intact biological molecules within cells without the need for labeling.