This work was done In collaboration with Netta Vidavsky lab of Prof. Steve Weiner and Lia Addadi, Department of Structural Biology
Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. Calcium ions pathway along the different stages was constructed using various imaging modalities, from calcium ions in sea water to mineral deposition and integration into the forming spicules. Calcein fluorescence enabled to follow penetration into the membrane, but high resolution correlative imaging using traditional methods was not possible due to signal loss and mineral change during the process of sample preparation. airSEM imaging station allowed direct correlation between fluorescence and energy dispersive spectroscopy (EDX) on a minimally processed sample.
Using the airSEM we confirmed the presence and localization of solid intracellular calcium carbonate in the sea urchin larvae
- Localize the uptake of fluorescent calcein in the sea urchin embryos in its natural state
- Perform elemental analysis and mapping of elements marked by calcein
- Perform minimal sample preparation that will conserve calcein signal and the mineral.
- Correlate calcein flourescent image to backscatter SEM image.
- Perform EDX spectrum and mapping to identify elements identity and distribution in the larva.
A variety of sample preparation methods were tested for identifying the calcium uptake in sea urchin embryos without success:
Fig1: Conventional sample preparation methods failed to enable correlative imaging of Calcein fluorescence signal with high resolution SEM and EDS mapping.
Osmium staining during epon embedding completely masked any fluorescent signal.
High pressure freezing and freeze drying resulted in sample collapse and destruction of morphological structures.
Cryo SEM did not enable EDS mapping for calcium identification.
airSEM™ imaging was the only method to successfully perform direct correlation of fluorescence, SEM and EDS of the same sample without limitation of sample preparation.
The airSEM™ directly imaged the sea urchin embryos in their natural state using only fixation. This together with Fluorescence microscopy and EDX elemental mapping allowed for the first time to prove that the calcium deposits are of solid calcium carbonate
Fig2: Calcium uptake and mineral deposition in sea urchin embryos, cross modality, True Correlative imaging of embryo vibratome slice embedded in gel
airSEM enables working with samples that are not compatible with vacuum such as hydrated gel embedded
Link to PDF
Link to paper: