PMID- 37502891
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20230904
DP  - 2023 Jul 12
TI  - Integrated Fluorescence Microscopy (iFLM) for Cryo-FIB-milling and In-situ 
      Cryo-ET.
LID - 2023.07.11.548578 [pii]
LID - 10.1101/2023.07.11.548578 [doi]
AB  - Correlative cryo-FLM-FIB milling is a powerful sample preparation technique for 
      in situ cryo-ET. However, correlative workflows that incorporate precise 
      targeting remain challenging. Here, we demonstrate the development and use of an 
      integrated Fluorescence Light Microscope (iFLM) module within a cryo-FIB-SEM to 
      enable a coordinate-based two-point 3D correlative workflow. The iFLM guided 
      targeting of regions of interest coupled with an automated milling process of the 
      cryo-FIB-SEM instrument allows for the efficient preparation of 9-12  approximately 200 nm 
      thick lamellae within 24 hours. Using regular and montage-cryo-ET data collection 
      schemes, we acquired data from FIB-milled lamellae of HeLa cells to examine 
      cellular ultrastructure. Overall, this workflow facilitates on-the-fly targeting 
      and automated FIB-milling of cryo-preserved cells, bacteria, and possibly high 
      pressure frozen tissue, to produce lamellae for downstream cryo-ET data 
      collection.
FAU - Yang, Jae
AU  - Yang J
AD  - Department of Biochemistry, University of Wisconsin, Madison, WI USA.
AD  - Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, 
      University of Wisconsin, Madison, WI USA.
FAU - Vrbovska, Veronika
AU  - Vrbovska V
AD  - Thermo Fisher Scientific Brno, Brno, Czech Republic.
FAU - Franke, Tilman
AU  - Franke T
AD  - Thermo Fisher Scientific Brno, Brno, Czech Republic.
FAU - Sibert, Bryan
AU  - Sibert B
AD  - Department of Biochemistry, University of Wisconsin, Madison, WI USA.
AD  - Cryo-Electron Microscopy Research Center, Department of Biochemistry, University 
      of Wisconsin, Madison, WI USA.
AD  - Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, 
      University of Wisconsin, Madison, WI USA.
FAU - Larson, Matthew
AU  - Larson M
AD  - Department of Biochemistry, University of Wisconsin, Madison, WI USA.
AD  - Cryo-Electron Microscopy Research Center, Department of Biochemistry, University 
      of Wisconsin, Madison, WI USA.
AD  - Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, 
      University of Wisconsin, Madison, WI USA.
FAU - Hall, Alex
AU  - Hall A
AD  - Thermo Fisher Scientific Brno, Brno, Czech Republic.
FAU - Rigort, Alex
AU  - Rigort A
AD  - Thermo Fisher Scientific Brno, Brno, Czech Republic.
FAU - Mitchels, John
AU  - Mitchels J
AD  - Thermo Fisher Scientific Brno, Brno, Czech Republic.
FAU - Wright, Elizabeth R
AU  - Wright ER
AD  - Department of Biochemistry, University of Wisconsin, Madison, WI USA.
AD  - Cryo-Electron Microscopy Research Center, Department of Biochemistry, University 
      of Wisconsin, Madison, WI USA.
AD  - Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, 
      University of Wisconsin, Madison, WI USA.
AD  - DOE Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI 
      USA.
AD  - Morgridge Institute for Research, Madison, WI, USA.
LA  - eng
GR  - R01 GM114561/GM/NIGMS NIH HHS/United States
GR  - U24 GM139168/GM/NIGMS NIH HHS/United States
PT  - Preprint
DEP - 20230712
PL  - United States
TA  - bioRxiv
JT  - bioRxiv : the preprint server for biology
JID - 101680187
UIN - Microsc Microanal. 2023 Jul 22;29(Supplement_1):1055-1057. PMID: 37613109
PMC - PMC10369943
EDAT- 2023/07/28 06:43
MHDA- 2023/07/28 06:44
PMCR- 2023/07/26
CRDT- 2023/07/28 04:24
PHST- 2023/07/28 06:44 [medline]
PHST- 2023/07/28 06:43 [pubmed]
PHST- 2023/07/28 04:24 [entrez]
PHST- 2023/07/26 00:00 [pmc-release]
AID - 2023.07.11.548578 [pii]
AID - 10.1101/2023.07.11.548578 [doi]
PST - epublish
SO  - bioRxiv [Preprint]. 2023 Jul 12:2023.07.11.548578. doi: 
      10.1101/2023.07.11.548578.