Prof. Jürgen M. Plitzko
Phone:+49 89 8578 2645

Members

Dr. Felix Bäuerlein
Tim Laugks
Rudolf Gatz
Email:gatz@...

Alumni

Dr. Elizabeth Villa
Alexander Schwarz
Dr. Alexander Rigort
Dr. Julia Mahamid

Correlative Microscopy & FIB

Correlative Microscopy at cryo-temperatures

Integrated or correlative microscopy attempts to combine multidimensional information from complementary techniques to bridge the various resolution gaps and thus to be able to integrate structural information gathered from multiple levels of the biological hierarchy into one common framework. Cryo-fluorescence microscopy for example can be exploited to navigate the cellular landscapes for features of interest before zooming in on these areas by cryo-electron tomography. It offers an independent and unambiguous confirmation of the identity of the investigated features.

Correlative microscopy of mammalian cells. Illustrative example for correlating a cellular region from the scale of the light microscope to the high magnification and relatively small field of view of the electron microscope. The red arrows indicate areas enlarged in subsequent panels (Figure adapted from Lucic et al., J Struct Biol 160, 2007).

Cryo-holder for correlative microscopy

The main prerequisite for a successful investigation at cryo-temperatures with the light/fluorescence microscope is a dedicated cryo-holder capable of maintaining the frozen-hydrated EM grid in its vitrifed state. We have developed a cryo-holder for an inverse optical systems (CAD drawing of the holder on the right). This systems allows investigations of multiple frozen hydrated samples (up to 4) without any temperature or time constraints enabled by an automated liquid nitrogen supply system. The objective lens is not immersed in liquid nitrogen, such that this type of cryo-holder can be used almost universally at any kind of conventional inverse light microscope. The stage is meanwhile available from FEI (licensed production, see: FEI Company). Software to assist in the correlation of EM maps with LM or FM maps has been developed within the TOM toolbox and a self executable version is meanwhile available for download (see Software development).

Focused Ion Beam (FIB) micromachining

Frozen hydrated samples for cellular cryo-electron tomography have to be less than 500 nm thick if imaged with 300 keV electrons. Most eukaryotic cells exceed this thickness requirement and are only accessible at suitable thin peripheral regions. Mechanical sectioning with a cryo-ultramicrotome is todays method of choice to generate samples with adequate thicknesses for electron tomography (see vitreous cryosectioning). With a focused ion beam (Gallium ions) we are exploring the possibility of thinning frozen hydrated samples in a controlled and artefact free manner. For that purpose we are utilizing a FEI Quanta 3D FEG instrument (see image on the right) equipped with a Quorum cryo-SEM preparation system (Quorum Technologies, Ringmer, UK). This development is integrated in PROSPECTS, a large collaborative project funded by the European Comission in its Research Framework Programme 7 (see: PROSPECTS). The use of focused ion beam (FIB) technology promises a significant improvement over conventional preparatory methods for vitrified ice samples needed for cryo-electron tomography.

Publications

Rigort A., Bäuerlein F., Leis A., Gruska M., Hoffmann C., Laugks T., Böhm U., Eibauer M., Gnaegi H., Baumeister W. and Plitzko J.M.: Micromachining tools and correlative approaches for cellular cryo-electron tomography. Journal of Structural Biology 172(2):169-79, 2010

Plitzko J.M., Rigort A., Leis A.: Correlative cryo-light microscopy and cryo-electron tomography. Current Opinion in Biotechnology 20:83-89, 2009

Lucic V., Kossel A.H., Yang T., Bonhoeffer T., Baumeister W. and Sartori A.: Multiscale imaging of neurons grown in culture: From light microscopy to cryo-electron tomography. Journal of Structural Biology 160:146-156, 2007

Sartori A., Gatz R., Beck F., Rigort A., Baumeister W. and Plitzko J.M.: Correlative microscopy: Bridging the gap between fluorescence light microscopy and cryo-electron tomography. Journal of Structural Biology 160:135-145, 2007

 
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