Multispectral imaging has significantly enhanced the analysis of fixed specimens in pathology and cytogenetics. However, application of this technology to in vivo studies has been limited. This is due in part to the increased temporal resolution required to analyze changes in cellular function. Here we present a non-scanning instrument that simultaneously acquires full spectral information (460 nm to 740 nm) from every pixel within its 2-D field of view (200 ìm x 200 ìm) during a single integration time (typically, 2 seconds). The current spatial and spectral sampling intervals of the spectrometer are 0.985 ìm and 5 nm, respectively. These properties allow for the analysis of physiological responses within living biological specimens.