FIB-SEM serial block face milling (SBFM) and imaging is a volumetric technique for 3D reconstruction of cells and tissue at the ultrastructural resolution. The sample preparation for standard SBFM requires embedding of the specimen into resin block which is a lengthy multi-step process. In addition, the procedure involves sample dehydration and staining with heavy metal ions which may adversely affect the structure of the biological samples. Direct imaging of the cryo-fixed samples (prepared by e.g. high-pressure freezing)[1, 2] provides an alternative for sample image at near-native state which offers simple sample preparation workflow and does not involve sample processing steps which would influence the sample structure. Thanks to the recent developments in the field of high-resolution SEM imaging, the cryoFIB-SEM imaging of high-pressure frozen biological specimens can be used for 3D volume reconstruction at ultrastructural resolution. Here, we demonstrate the potential of cryo-FIB/SEM block face milling for reconstruction of the whole cells.
The cells were cultured, and resuspended in cultivation medium. The suspension was vitrified by high pressure freezing on 3mm carrier. Subsequently it was transferred to FIB-SEM microscope (Helios Hydra 5 CX, ThermoFisher Scinetific) equipped with cryo-stage. For the data acquisition the sample was milled out in the shallow angle 38 degrees with respect to the sample plane. This geometry increases detection rate of secondary electrons and improves image contrast. Milling was done by Oxygen plasma ions (30kV, 1,7nA). Oxygen ions create milling face with low surface distortion (curtaining). For imaging we used immersion mode with deceleration voltage that results of electrons landing energy 2kV. The selected voltage balances the charging artefacts visible on higher voltages and insufficient contrast induced by lower voltage. The consecutive milling and imaging was orchestrated by Auto Slice & View 5 (ThermoFisher Scientific). Images were processed by line-by-line high pass filter, histogram normalization and the dataset where drift corrected by AMST algorithm [3].
We have acquired data for the reconstruction of the whole INS-1E cell (volume 6,786um3 , voxel size 10x10x10nm) in telophase and carried out volume segmentation to analyse the level of detail which can be obtained from the data.