Fig. 4

Correlated SEM and NanoSIMS isotope ratio images for room-temperature and cryogenic workflows. The tissue imaged was from the symbiotic freshwater organism Green Hydra, isotopically labeled through the assimilation of ¹⁵NH₄⁺. A SEM image of a section of conventionally prepared tissue and B a quantified NanoSIMS map (room-temperature) of the corresponding ¹⁵N/¹⁴N ratio. C Cryo-SEM image after the entirely cryogenic sample preparation and D a quantified CryoNanoSIMS map of the corresponding ¹⁵N/¹⁴N ratio. The images were obtained from similar regions in the gastric region of Green Hydra animals that were exposed to identical incubation conditions (cf. Methods), permitting a direct comparison between the two analytical approaches. Note the dramatic difference in appearance between conventionally prepared and cryogenically prepared tissue (A vs. C), i.e., shrinkage and strong deformation (e.g., of vacuoles) vs. no shrinkage/deformation, and the strong ¹⁵N enrichments in the vacuoles in the CryoNanoSIMS image, which are absent in the (emptied) vacuoles after normal sample preparation (B vs. D). The isotopic enrichments shown in B and D are presented by a logarithmic color scale. The isotope ratio images are drift-corrected accumulations of 5 sequential images each consisting of 256 by 256 pixels with a pixel dwelling time of 5 ms; typical analysis time for one image as displayed is about 30 min. Scale bars are 5 μm. C, Chlorella sp. algae; EV, epidermal vacuole; E, epidermis; GV, gastrodermal vacuole; G, gastrodermis; M, mesoglea