Technical Abstract: Infrared microspectroscopy (IMS) and chemical imaging is ideal for measuring nutrient distribution in single algal cells on a cellular and subcellular level. The study of small algal cells, or cells within a colony requires enhanced spatial resolution IMS. Synchrotron infrared microspectroscopy with confocal image plane masking provides spatial resolution at the diffraction limit, which allows the study of micro-ecological interactions with a minimum target width of 5 µm. Many species that contribute substantially to ecosystem productivity and nutrient cycling require this optical advantage. Also, this spatial resolution allows the measurement of individuals within a colony, providing insight into physiological advantages of colony formation. We used synchrotron confocal IMS to measure the response of two dominant algal species from a stream biofilm to a reduction in nutrient availability: Achnanthes affinis, a single cell (5 ' 10 µm) diatom, and Fragilaria virescens, a colonial diatom (5 ' 15 µm cells connected lengthwise). Both species initially had similar variation in macromolecular content but had differing responses to nutrient limitation in carbohydrate, phosphorous compound, and lipid distribution over time. The response of F. virescens colonies was at the cellular level, but the cellular response of individuals varied with colony length, as long colonies had a stronger response than short colonies. Better IMS spatial resolution allows the biochemical analysis of smaller, more closely associated algae and increases our ability to predict how algal communities will be affected by changing environmental conditions.