Biology Reference
In-Depth Information
13.4.2 Methods .................................................................................... 240
13.4.2.1 Choice of Fixation Protocol ..................................................240
13.4.2.2 Pre-embedding Method ......................................................240
13.4.2.3 Post-embedding Method.....................................................241
13.4.2.4 Ultrathin Cryosectioning Method .........................................241
13.4.3 Results and Considerations ......................................................... 243
13.4.3.1 Pre-embedding Method ......................................................243
13.4.3.2 Post-embedding Method.....................................................244
13.4.3.3 Ultrathin Cryosectioning Method .........................................244
13.5 Freeze-Fracture Electron Microscopy ...............................................................246
13.5.1 Rationale ................................................................................... 246
13.5.2 Method...................................................................................... 246
13.5.3 Results and Considerations ......................................................... 247
13.6 Future Directions ............................................................................................ 248
Acknowledgments ................................................................................................... 248
References ............................................................................................................. 248
Abstract
The lipid droplet (LD) is different from other cellular organelles in that most of its
volume is made of lipid esters and its surface is lined by a phospholipid monolayer.
This uniquely lipid-dominant structure poses a problem for electron microscopy
(EM) because the aldehydes commonly used as a fixative do not react with most
lipids. To circumvent this difficulty and utilize the high resolving power of EM,
many methods have been developed. In this chapter, we discuss methods that have
been used and/or are potentially useful to study LDs. The methods include conven-
tional EM to observe the LD core, cryoelectron microscopy to observe the LD
surface, freeze-substitution, immunoelectron microscopy (pre-embedding, post-
embedding, and cryosectioning methods), and freeze-fracture. Each method has
strong and weak points and therefore some caution is necessary in interpreting the
obtained results. In combination with methods of other disciplines, the electron
microscopic techniques should contribute significantly to solving the remaining
questions on LDs.
INTRODUCTION
The lipid droplet (LD) is a globule of hydrophobic lipid esters covered by a mono-
layer of amphipathic phospholipids (
Suzuki, Shinohara, Ohsaki, & Fujimoto, 2011
).
Because the surface layer is very thin, most of the LD volume is made of lipid esters.
If we suppose an LD of 1
m in diameter and assume the thickness of the surface
m
