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Microscopy 2012: Down to Detail | 2012 Year in Review

12/19/2012
Kristie Nybo, Ph.D.

From an optimized pair of split Venus fragments for bimolecular fluorescence complementation (BiFC) assays to an approach for the high-resolution reconstruction of whole yeast cells in three-dimensions, the BioTechniques editors highlight some of our favorite microscopy advancements published during 2012.


Since 1647 when Anton Van Leeuwenhoek first observed bacteria in a microscope he built, imaging has been central to studies of the molecules and organisms that make up the microscopic world. During the past 365 years, we have definitely come a long way from those first observations with the introduction of technologies including the electron microscope in 1931 and super resolution microscopy in the early 1980s, both of which have vastly increased magnification and resolution and enabled imaging at single nanometer resolutions.

From an optimized pair of split Venus fragments for bimolecular fluorescence complementation (BiFC) assays to an approach for the high-resolution reconstruction of whole yeast cells in three-dimensions, the BioTechniques editors highlight some of our favorite microscopy advancements published during 2012.









These technological developments have moved hand-in-hand with the development of methods to embed and stain samples, the discovery of fluorescent proteins for intracellular labeling, and new techniques for monitoring molecular interactions within living cells, just to name a few. In fact, microscopy technologies change so rapidly nowadays that it is difficult to keep up. With that in mind, the BioTechniques editors would like to highlight some of our favorite microscopy advancements published during 2012. From an optimized pair of split Venus fragments for bimolecular fluorescence complementation (BiFC) assays to an approach for the high-resolution reconstruction of whole yeast cells in three-dimensions, our selections look at different aspects of microscopy, each demonstrating how improving tools can enhance our understanding of what happens on a microscopic level.

Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments
Kazumasa Ohashi, Tai Kiuchi, Kazuyasu Shoji, Kaori Sampei, and Kensaku Mizuno
BioTechniques, Vol. 52, No. 1, January 2012, pp. 45–50

One of the advances in cell biology that occurred as resolution improved was the ability to directly visualize protein-protein interactions in cells. While advances in instrumentation clearly made this feasible, it was the development of fluorescent proteins (FP) along with labeling techniques that made these interaction assays a reality. One such interaction method is BiFC where two non-fluorescent fragments of a FP are attached to a pair of potentially interacting proteins. If the two proteins interact, they will bring together the FP fragments, allowing a functional fluorescent protein to reassemble, thus resulting in fluorescence emissions that can be detected by microscopy. Importantly, the reassembled FP is stable enough that even weak interactions can be seen clearly. In the January 2012 issue of BioTechniques, Kensaku Mizuno and colleagues described a BiFC assay capable of detecting phosphorylation-dependent interactions between cofilin and actin. When initially designing the assay, the authors found that the FP fragments commonly used in BiFC assays were not suitable for their purpose, so they set about designing a new BiFC probe. This was not an easy task, considering that the FP had to: i) be divided at a point where it would re-form into the correct structure when the two parts were brought into proximity; ii) avoid reassembling on its own; and iii) not interfere with the function or localization of the proteins under investigation. Mizuno’s team set to work preparing expression plasmids for 91 different combinations of split Venus pairs, each divided at a different location. The authors eliminated all pairs that were able to fluoresce without attachment to test proteins and then carefully screened the remaining Venus pairs for the ability to distinguish between cells expressing cofilin and cells expressing a cofilin mutant. In the end, one fragment pair demonstrated a clear advantage for assessing cofilin-actin interactions and was successful in detecting additional interactions tested by the authors as well. For the editors, the power of this article was not only in the description of a new split BiFC interaction pair, but also in the extensive data provided by the authors through their testing of all the other sites for splitting Venus proteins, a treasure trove of information to assist researchers in developing new BiFC tools.

High-resolution three-dimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy
Dongguang Wei, Scott Jacobs, Shannon Modla, Shuang Zhang, Carissa L. Young, Robert Cirino, Jeffrey Caplan, and Kirk Czymmek
BioTechniques, Vol. 53, No. 1, July 2012, pp. 41–48

While visualizing molecular interactions has been a major goal for researchers in recent years, 3-dimensional (3D) reconstruction of samples has been a high priority as well. Transmission electron microscopy (TEM) tomography is the current gold standard for high-resolution 3D imaging of biological samples, providing 3nm resolution in 0.5um thick sections. Although many significant contributions have come from using this approach, the thin sections required make reconstructing whole cells challenging. In July 2012, a team led by Kirk Czymmek employed another approach, focused ion beam scanning electron microscopy (FIB-SEM), to create a high-resolution 3D reconstruction of a whole yeast cell and reported their findings in BioTechniques. FIB-SEM functions similar to SEM, but with a scanning beam of ions rather than electrons. As these ions collide with the surface of the sample, secondary ions, neutral atoms, or electrons are thrust from the surface. These signals can be collected for imaging with resolution in the nanometer range. Previously, FIB-SEM had shown resolution limits of 5-100nm in the z-plane, which presents a particular challenge for 3D reconstructions. Czymmek’s method optimized the sample preparation procedures and enhanced electron collection by altering the position of the electron detector to enable the imaging and quantification of sub-cellular structures in single yeast cells at resolutions approaching 3nm in the x-, y-, and even z- directions, thus achieving the highest reported resolution using FIB-SEM to date. This approach was demonstrated by imaging a single yeast cell with 3nm isotropic voxels, a six-fold reduction in z-interval from a previously reported FIB-SEM dataset. From these images, the authors were able to calculate volume, volume percentage, and surface area of subcellular organelles. These modifications will expand researchers' abilities to probe the smallest of cellular processes and understand their behavior in three-dimensions.

We hope you agree that our 2012 article picks nicely highlight some of the diverse and exciting improvements in microscopy and microscopy-based techniques during the past year, and represent advances that are taking us further into the tiny and complex cellular world.