Biology Reference
In-Depth Information
CHAPTER
13
Toyoshi Fujimoto, Yuki Ohsaki, Michitaka Suzuki, and Jinglei Cheng
Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of
Medicine, Nagoya, Japan
Imaging Lipid Droplets by
Electron Microscopy
CHAPTER OUTLINE
Introduction ............................................................................................................ 228
How Images are Generated in TEM .......................................................... 229
Sample Preparation for Conventional EM.................................................. 229
How Do LDs Look in Conventional TEM .................................................... 231
13.1 Probing the LD Core ........................................................................................ 231
13.1.1 Rationale ................................................................................... 231
13.1.2 Methods .................................................................................... 231
13.1.2.1 Observation of Small LDs ................................................... 231
13.1.2.2 Incorporation of Newly Synthesized TG into Existing LDs..... 233
13.1.2.3 Sample Preparation and Objective Measurement of the
Relative Electron Density .................................................................. 233
13.1.3 Results and Considerations ......................................................... 234
13.1.3.1 Observation of Small LDs ................................................... 234
13.1.3.2 Incorporation of Newly Synthesized TG into Existing LDs..... 234
13.2 Observation of the LD Surface..........................................................................235
13.2.1 Rationale ................................................................................... 235
13.2.2 Methods .................................................................................... 235
13.2.2.1 Isolation of LDs.................................................................. 235
13.2.2.2 Cryoelectron Microscopy .................................................... 236
13.2.3 Results and Considerations ......................................................... 236
13.3 Freeze-Substitution ......................................................................................... 237
13.3.1 Rationale ................................................................................... 237
13.3.2 Methods .................................................................................... 237
13.3.3 Results and Considerations ......................................................... 238
13.4 Immunoelectron Microscopy of LD-Associated Proteins .....................................238
13.4.1 Rationale ................................................................................... 238
13.4.1.1 Pre-embedding Method ..................................................... 239
13.4.1.2 Post-embedding Method.................................................... 239
13.4.1.3 Ultrathin Cryosectioning Method ........................................ 239
 
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