Cryo-FIB-SEM serial milling and block face imaging: Large volume structural analysis of biological tissues preserved close to their native state.
Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000μm3) at high resolution (5-20nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20μm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues.
Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel.
Netta
N
Vidavsky
Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel.
Anat
A
Akiva
Department of Chemical Research Support, Weizmann Institute of Science, 76100 Rehovot, Israel.
Ifat
I
Kaplan-Ashiri
Department of Chemical Research Support, Weizmann Institute of Science, 76100 Rehovot, Israel.
Katya
K
Rechav
Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel. Electronic address: Lia.addadi@weizmann.ac.il.
Lia
L
Addadi
Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel.
Steve
S
Weiner
Carl Zeiss Microscopy GmbH, Global Applications Support, Oberkochen, Germany.
Andreas
A
Schertel
D058500:Animal Fins / Q000648:ultrastructure*; D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D006056:Golgi Apparatus / Q000648:ultrastructure*; D021621:Imaging, Three-Dimensional; D007814:Larva / Q000648:ultrastructure; D012617:Sea Urchins / Q000196:embryology / Q000648:ultrastructure; D015027:Zebrafish
3D imaging; Biomineralization; Cryo-FIB-SEM; Sea urchin embryo; Zebrafish