Cryopreservation of bacteria requires specific cells pre-adaptation as well as controlled freezing protocol to minimize membrane damage resulting from osmotic cell dehydration. Membrane fluidity has an important role in biophysical events taking place at subzero temperatures, by facilitating or not the exchanges between intracellular and extracellular media. An original approach using Synchrotron fluorescence polarization microscopy was developed to measure membrane fluidity of single bacteria. Cells were labelled with the cytoplasmic membrane probe trimethylammoniumdiphenylhexatriene (TMA-DPH). The degrees of liberty of TMA-DPH inside the bilayer provide a direct marker of membrane fluidity under deep UV excitation. Couples of fluorescence polarization images from zero to forty degrees were recorded by inserting polarizers into the excitation and emission paths. The fluorescence anisotropy of the images was calculated thus allowing the assessment of the cell membrane fluidity. Results indicate intercellular and intracellular heterogeneities with subdomains appearing at low temperature for freeze-sensitive cells.