One of few techniques that is able to spatially resolve chemical data, including organic molecules, to morphologicalfeatures in modern and ancient geological samples, is time-of-flight secondary ion mass spectrometry(ToF-SIMS). The ability to connect chemical data to morphology is key for interpreting thebiogenicity of preserved remains in ancient samples. However, due to the lack of reference data for geologicallyrelevant samples and the ease with which samples can be contaminated, ToF-SIMS data may bedifficult to interpret. In this project, we aimed to build a ToF-SIMS spectral database by performing parallelToF-SIMS and gas chromatography–mass spectrometry (GC–MS) analyses of extant photosyntheticmicrobial communities collected from an alkaline silica-depositing hot spring in Yellowstone NationalPark, USA. We built the library by analyzing samples of increasing complexity: pure lipid standards commonlyfound in thermophilic phototrophs, solvent extracts of specific lipid fractions, total lipid extracts,pure cultures of dominant phototrophic community members, and unsilicified phototrophic streamercommunities.The results showed that important lipids and pigments originating from phototrophs were detected byToF-SIMS (e.g., wax esters, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sufloquinovosyldiaglycerol,alkanes, etc.) in the streamer lipid extracts. Many of the lipids were also detected in situin the unsilicified streamer, and could even be spatially resolved to individual cells within the streamercommunity. Together with the ToF-SIMS database, this mapping ability will be used to further exploreother microbial mats and their fossilized counterparts in the geological record. This is likely to expandthe geochemical understanding of these types of samples.