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2013-10-09Zeitschriftenartikel DOI: 10.1016/j.jsb.2013.09.024
Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens
dc.contributor.authorSchertel, Andreas
dc.contributor.authorSnaidero, Nicolas
dc.contributor.authorHan, Hong-Mei
dc.contributor.authorRuhwedel, Torben
dc.contributor.authorLaue, Michael
dc.contributor.authorGrabenbauer, Markus
dc.contributor.authorMöbius, Wiebke
dc.date.accessioned2018-05-07T17:57:59Z
dc.date.available2018-05-07T17:57:59Z
dc.date.created2014-10-09
dc.date.issued2013-10-09none
dc.identifier.otherhttp://edoc.rki.de/oa/articles/resqL5vcosxU/PDF/22cULakc5e9A.pdf
dc.identifier.urihttp://edoc.rki.de/176904/1972
dc.description.abstractVolume microscopy at high resolution is increasingly required to better understand cellular functions in the context of three-dimensional assemblies. Focused ion beam (FIB) milling for serial block face imaging in the scanning electron microscope (SEM) is an efficient and fast method to generate such volume data for 3D analysis. Here, we apply this technique at cryo-conditions to image fully hydrated frozen specimen of mouse optic nerves and Bacillus subtilis spores obtained by high-pressure freezing (HPF). We established imaging conditions to directly visualize the ultrastructure in the block face at −150 °C by using an in-lens secondary electron (SE) detector. By serial sectioning with a focused ion beam and block face imaging of the optic nerve we obtained a volume as large as X = 7.72 μm, Y = 5.79 μm and Z = 3.81 μm with a lateral pixel size of 7.5 nm and a slice thickness of 30 nm in Z. The intrinsic contrast of membranes was sufficient to distinguish structures like Golgi cisternae, vesicles, endoplasmic reticulum and cristae within mitochondria and allowed for a three-dimensional reconstruction of different types of mitochondria within an oligodendrocyte and an astrocytic process. Applying this technique to dormant B. subtilis spores we obtained volumes containing numerous spores and discovered a bright signal in the core, which cannot be related to any known structure so far. In summary, we describe the use of cryo FIB-SEM as a tool for direct and fast 3D cryo-imaging of large native frozen samples including tissues.eng
dc.language.isoeng
dc.publisherRobert Koch-Institut, Biologische Sicherheit
dc.subjectAnimalseng
dc.subjectMiceeng
dc.subjectSpores Bacterial/ultrastructureeng
dc.subjectBacillus subtilis/ultrastructureeng
dc.subjectCryoelectron Microscopyeng
dc.subjectFrozen Sectionseng
dc.subjectImaging Three-Dimensionaleng
dc.subjectMicroscopyeng
dc.subjectElectron Scanningeng
dc.subjectOptic Nerve/ultrastructureeng
dc.subject.ddc610 Medizin
dc.titleCryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens
dc.typeperiodicalPart
dc.identifier.urnurn:nbn:de:0257-10037788
dc.identifier.doi10.1016/j.jsb.2013.09.024
dc.identifier.doihttp://dx.doi.org/10.25646/1897
local.edoc.container-titleJournal of Structural Biology
local.edoc.container-textSchertel, A., Snaidero, N., Han, H.-M., Ruhwedel, T., Laue, M., Grabenbauer, M., Möbius, W. Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens (2013) Journal of Structural Biology, 184 (2), pp. 355-360.
local.edoc.fp-subtypeArtikel
local.edoc.type-nameZeitschriftenartikel
local.edoc.container-typeperiodical
local.edoc.container-type-nameZeitschrift
local.edoc.container-urlhttp://www.sciencedirect.com/science/article/pii/S1047847713002657
local.edoc.container-publisher-nameElsevier
local.edoc.container-volume184
local.edoc.container-issue2
local.edoc.container-year2013

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