Biomineralization pathways in a foraminifer revealed using a novel correlative cryo-fluorescence-SEM-EDS technique. 
Foraminifera are marine protozoans that are widespread in oceans throughout the world.  Understanding biomineralization pathways in foraminifera is particularly important because their calcitic shells are major components of global calcium carbonate production.  We introduce here a novel correlative approach combining cryo-SEM, cryo-fluorescence imaging and cryo-EDS.  This approach is applied to the study of ion transport processes in the benthic foraminifer genus Amphistegina.  We confirm the presence of large sea water vacuoles previously identified in intact and partially decalcified Amphistegina lobifera specimens.  We observed relatively small vesicles that were labelled strongly with calcein, and also identified magnesium (Mg)-rich mineral particles in the cytoplasm, as well as in the large sea water vacuoles.  The combination of cryo-microscopy with elemental microanalysis and fluorescence imaging reveals new aspects of the biomineralization pathway in foraminifera which are, to date, unique in the world of biomineralization.  This approach is equally applicable to the study of biomineralization pathways in other organisms.