TY - JOUR
T1 - Cryo-FIB-SEM serial milling and block face imaging
T2 - large volume structural analysis of biological tissues preserved close to their native state
AU - Vidavsky, N.
AU - Akiva, A.
AU - Kaplan-Ashiri, I.
AU - Rechav, K.
AU - Addadi, L.
AU - Weiner, S.
AU - Schertel, A.
PY - 2016/12/1
Y1 - 2016/12/1
N2 - Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000 μm3) at high resolution (5–20 nm). 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 20 μm 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.
AB - Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000 μm3) at high resolution (5–20 nm). 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 20 μm 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.
KW - 3D imaging
KW - Biomineralization
KW - Cryo-FIB-SEM
KW - Sea urchin embryo
KW - Zebrafish
UR - http://www.scopus.com/inward/record.url?scp=85000613273&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2016.09.016
DO - 10.1016/j.jsb.2016.09.016
M3 - Article
C2 - 27693309
AN - SCOPUS:85000613273
SN - 1047-8477
VL - 196
SP - 487
EP - 495
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -