Studying intracellular transport using high-pressure freezing and Correlative Light Electron Microscopy.
Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopy in one experiment. Light microscopy (LM) is especially suited for providing a general overview with data from lots of different cells and by using live cell imaging it can show the history or sequence of events between or inside cells. Electron microscopy (EM) on the other hand can provide a much higher resolution image of a particular event and provide additional spatial information, the so-called reference space. CLEM thus has certain strengths over the application of both LM and EM techniques separately. But combining both modalities however generally also means making compromises in one or both of the techniques. Most often the preservation of ultrastructure for the electron microscopy part is sacrificed. Ideally samples should be visualized in its most native state both in the light microscope as well as the electron microscope. For electron microscopy this currently means that the sample will have to be cryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for using cryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressure freezing in combination with live cell imaging. In addition we examine some of the EM analysis tools that may be useful in combination with CLEM techniques.
Brown
Edward
E
Department of Biochemistry, School of Medical Sciences, University Walk, Bristol, BS8 1TD, United Kingdom.
Mantell
Judith
J
Carter
Debbie
D
Tilly
Gini
G
Verkade
Paul
P
D000818:Animals; D001692:Biological Transport; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D005615:Freezing; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry* / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods*; D011312:Pressure