Herein is described a general sampling protocol that includes culture, differentiation and fixing of cells in their preferred morphology on the one sample substrate (Si3N4) to enable subsequent diverse modern microspectroscopic analyses. The protocol enables unprecedented correlated and complementary information on the intracellular biochemistry of metabolic processes, diseases and their treatment, which offers the opportunity to revolutionize our understanding of cell and tissue biology at a molecular level. The culture of adherent cells onto inexpensive Si3N4 membranes allows microspectroscopic analyses across the electromagnetic spectrum, from hard X-ray fluorescence (both XRF and XANES), through to visible and fluorescence light microscopies, and infrared microspectroscopy without substrate interference. Adherent mammalian cell lines (3T3-L1 adipocytes and H9c2 cardiac myocytes) illustrate the in vitro application of these protocols. The cells adhered strongly to Si3N4 membranes and visually displayed normal proliferative and phenotypic growth; more importantly, rapid alcohol fixation of cells did not affect their structural integrity for subsequent analyses. ; Elizabeth A. Carter, Benjamin S. Rayner, Andrew I. McLeod, Lindsay E. Wu, Craig P. Marshall, Aviva Levina, Jade B. Aitken, Paul K. Witting, Barry Lai, Zhonghou Cai, Stefan Vogt, Yao-Chang Lee, Ching-Iue Chen, Mark J. Tobin, Hugh H. Harris and Peter A. Lay