Cryo-Electron Microscopy (Cryo-EM) of vitrified biological samples has proven to be an effective technique to investigate the structure of native cells with macromolecular resolution. Cryo-preservation of biological specimens ensures preservation of the whole cellular integrity. On the other hand, the high magnification available with electron microscopy (EM) comes with a limited field of view. However, investigations of frozen hydrated specimens with the electron microscope comes at a cost; the very low contrast of unstained and vitrified biological specimens and the need to minimize the exposure to electron radiation make the identification of specific structures a difficult and time consuming task. Therefore one needs a gentle and time efficient way to locate structures of interest.
Correlative Microscopy combines the advantages of light/fluorescence microscopy (LM) - namely the ability to rapidly scan large and heterogeneous sample areas - with the high resolving power of electron microscopy, to navigate to an area of interest and obtain ultrastructural cellular detail. It allows for examination of vitrified biological samples by both light and electron microscopy and offers a means for studying large and complex cellular structures in three dimensions and with nanometer-scale resolution.
Light microscopy provides a survey over large cellular landscapes along with a positive identification of the biological features of interest when combined with fluorescent probes. Using techniques of correlative microscopy in combination with a cryogenic stage, ice embedded samples can first be examined at the LM level. The sample and the regions of interest identified at the LM level are then transferred to the EM and the identified areas of interest are relocated using suitable software routines to quickly navigate to these pre-selected regions of interest. Electron microscopy and electron tomography then allow investigations of the identified structures at a much higher resolution and in three dimensions.
The Correlative Stage
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| Correlative stage device |
The correlative cryo-stage has been designed for use with an inverted light microscope equipped with a motorized stage. It maintains the vitreous state of frozen hydrated samples by keeping them at liquid nitrogen temperature and furthermore it provides a suitable shielding from potential contamination. The stage is equipped with an automated liquid nitrogen filling system for extended screening. Up to four EM-grids can be placed in the correlative stage and the design uses only one glass coverslip in the optical path. The correlative cryo-stage works with long working distance (LWD) objective lenses. To avoid condensation on the objective lens, the area around the objective is flushed with dry nitrogen gas. The correlative stage has been designed and built at the Max-Planck-Institute (MPI) of Biochemistry, Martinsried, Germany and has been in use in the laboratory of Prof. Wolfgang Baumeister since 20061.
Coordinate transfer between Light Microscopy and Electron Microscopy
Structures of interest are identified on the LM overview image (generated by the software from the light microscope). After transfer of the sample to the electron microscope a low magnification overview image is generated with the grid scan software (integrated into the Matlab based TOM toolbox2). The coordinates of the identified structures in the LM overview image can then be transferred to EM stage positions using the included software routines.
An Academic-Industry Partnership
FEI, a leading provider of high-resolution imaging and analysis systems, and Max-Planck-Institute of Biochemistry, a leading biochemical research institution, will collaborate on a correlative microscopy solution enabling scientists to quickly and easily acquire high-resolution transmission electron microscope (TEM) images of molecular entities found using optical microscopy techniques. Workshops will be conducted to allow users of the correlative stage to present their correlative microscopy experimental results and discuss requirements for future solutions in correlative microscopy.
References
"Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography"
Sartori, A.; Gatz, R.; Beck, F.; Rigort, A.; Baumeister, W.; Plitzko, J.M.
Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany
J. Struct. Biol. 2007, 160(2), 135-45
[PubMed Abstact]
"Correlative light and electron microscopy using immunolabeled resin sections"
Schwarz, H.; Humbel, B.M.
Max-Planck-Institut fur Entwicklungsbiologie, Tuebingen, Germany
Methods Mol. Biol. 2007, 369, 229-56
[PubMed Abstract]
Additional References