A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography
Introduction
Cryo-sections of vitreous biological samples are successfully used in several laboratories to study the ultrastructure of cellular organelles and their molecular components in a native hydrated state (Dubochet et al., 1988, Michel et al., 1991, Sitte, 1996, Al-Amoudi et al., 2004, Matias and Beveridge, 2005). Three-dimensional imaging of these structures using electron tomography (ET) can be performed (Hsieh et al., 2002, Hsieh et al., 2004, Hsieh et al., 2006, Frank et al., 2002, Schwartz et al., 2003, Leis et al., 2005), but the yield of good tomograms is low.
Alignment of the images in a tilt series is one of the crucial steps in ET. Two methods are generally used for the alignment: (1) cross-correlation of the collected images and (2) alignment of images using fiducial markers applied to the specimen. When the methods were compared in studies of stained plastic sections, it was shown that cross-correlation gives a higher error than alignment with fiducial markers (Brandt et al., 2001). Furthermore, due to the required low electron dose in studies of vitreous specimens, the signal-to-noise ratio is low, which makes the cross-correlation method even less reliable. Thus, the use of fiducial markers should be the first alternative for high-resolution tomographic reconstructions from vitreous sections.
Colloidal gold particles of 5–20 nm in diameter are conventionally used as fiducial markers to align images of plastic sections for ET. Obviously, gold particles suspended in water cannot be applied directly onto vitreous specimens, which have to be kept at temperatures below −135 °C (Al-Amoudi et al., 2002). To overcome this problem, gold particles have been applied on a support film attached to the grid prior to the shift to the low temperature (e.g. Hsieh et al., 2002). However, in these studies only limited areas of the sections could be aligned and reconstructed since the sections and the support film could be as much as a few hundred nanometers apart (Hsieh et al., 2006).
We have now explored the use of small semiconductor nanocrystals, so-called quantum dots, as fiducial markers for cryo-ET, and provide a simple and effective method to apply them directly onto the cryo-sections. These semiconductor crystals are electron dense and well defined, which have made them suitable for tagging of antibodies in immunoelectron microscopy (Giepmans et al., 2005). Quantum dots are routinely used in life sciences in a water-soluble form, but they are initially manufactured in organic solvents (Murray et al., 2001, Yin and Alivisatos, 2005). In the present study, we have substituted these solvents with other organic solvents so that quantum dot suspensions can be applied onto sections at cryo-temperatures. We have shown that the quantum dots do attach to the section surface under these conditions and can serve as fiducial markers for ET. To evaluate the method, we have performed ET of vitreous cryo-sections from human skin and determined the accuracy of the image alignment. We have studied the 3D reconstructions of a few selected organelles in some detail and estimated the resolution.
Section snippets
Preparation of high-pressure frozen skin samples
Skin biopsies (area, 1 × 1 mm2; thickness, 100–150 μm) were collected from the left forearm of a male Caucasian. To avoid dehydration, the biopsies were immediately placed in 1-hexadecene (Fluka, Buchs, Switzerland). Subsequently, the samples were put in the cavity between two aluminium cylindrical platelets and the cavity space was filled with 1-hexadecene to exclude air. Finally, the samples were vitrified in a high-pressure freezer HPM 010 (Bal-Tec, Balzers, Liechtenstein), at a pressure of 2300
Quantum dots and their deposition onto vitreous cryo-sections
Quantum dots (QDs) differ in size, shape and chemical composition (Murray et al., 2001, Yin and Alivisatos, 2005, Giepmans et al., 2005). We chose PbS core quantum dots of the largest available size, since small particles are known to be difficult to detect in images acquired at high tilt angles. In Fig. 1B, the quantum dots selected for our experiments are shown deposited on a carbon support film at high concentration. The particles are spherical and vary in diameter from 10 to 17 nm (own
Discussion
Electron microscopy of vitreous cryo-sections has revealed the native fine structure of the cell with greater fidelity than can be achieved with plastic sections (Al-Amoudi et al., 2004). Electron tomography of such sections is highly desirable and several successful attempts have been reported (see Section 1). The main obstacle to routine use of electron tomography with vitreous cryo-sections has been related to alignment of the tilt images. When fiducial markers are deposited on the support
Conclusion
We have shown that quantum dots can be applied to vitreous sections and used as fiducial markers for alignment. The average error in the alignment of the images in a tilt series amounted to about 1 nm. Mitochondria, a putative lamellar body and bundles of keratin filaments were reconstructed in three dimensions, and analysis of the tomograms suggested that the resolution was on the order of 5–7 nm. The new fiducial marker method is simple, and makes it possible to carry out cellular tomography on
Acknowledgments
We thank Drs. Lars-Göran Öfverstedt and Ulf Skoglund for discussion and generous support. We are grateful to Dr. K. Tokuyasu for valuable advice. We also thank Kjell Hultenby, Kliniskt forskningscentrum, Karolinska Universitetssjukhuset Huddinge, for technical assistance with the high-pressure freezing. This work was financially supported by the Swedish Research Council (grants to B.D. and O.S.), the European Union “3D-EM” Network of Excellence, the Agouron Institute, the Swedish Foundation for
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