TY  - JOUR
TI  - Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in Allium cepa
AU  - Nicolas, William Jules
AU  - Jensen, Grant J
AU  - Meyerowitz, Elliot M
VL  - 12
IS  - 23
PY  - 2022
DA  - 2022/12/05
SP  - e4559
C1  - Bio-protocol 2022;12:e4559
DO  - 10.21769/BioProtoc.4559
UR  - https://doi.org/10.21769/BioProtoc.4559
AB  - Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 &mu;m thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall.
KW  - FIB-milling
KW  - Plant cell wall
KW  - Onion
KW  - Cryo-ET
KW  - Cellulose
KW  - Cell morphology
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False