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Microscopy: Looking Deeper | 2012 Year in Review

Kristie Nybo, Ph.D.

With the challenge of trying to pin down specific areas where microscopy developers could make significant strides in 2013, the editors at BioTechniques needed to huddle together and discussing (as well as debate) our various predictions.

Microscopy is an ever changing field, with new and significant advances introduced on a regular basis. When it comes to areas such as fluorescent proteins, resolution, data processing, automation, and unique applications for these techniques, there is no doubt that 2013 will continue to bring advances in each of these areas.

With the challenge of trying to pin down specific areas where microscopy developers could make significant strides in 2013, the editors at BioTechniques needed to huddle together and discussing (as well as debate) our various predictions. Source: RIKEN

The BioTechniques editors look forward to many changes in this field, among them new technologies and refinement of existing approaches to close the gap between light and electron microscopy capabilities. Methods for sample preparation and tools for imaging specific targets seem poised for significant advances in the coming months as well.

Closing the gap

In 1873, Ernest Abbe recognized that the resolving power of light microscopy was limited such that two points could only be distinguished if they were separated by more than half of the wavelength of light. This barrier was overcome when optical laws were applied to electrons and the electron microscope (EM) was created, arguably the most important advance in microscopy since its introduction. With wavelengths 100,000 times smaller than light, electron microscopes allowed for picometer resolution. Rather than replacing light microscopy, EM has become its valuable companion. Light microscopy maintains several advantages, such as the ability to image live cells and amenability to 3D reconstruction of images that keeps this technology at the front of engineers' and biologists' minds. In fact, significant efforts are now focused on closing the resolution gap between these types of microscopy.

Since Stefan Hell first overcame Abbe’s barrier by introducing the stimulated emission depletion (STED) microscope in 2000, with which he obtained fluorescent images with nanometer resolution, many other super-resolution microscopes based on differing strategies have been developed and refined. Initially, these microscopes were confined to specialized laboratories requiring trained microscopists, but recent years have brought us a little closer to readily accessible instruments that can be used by non-specialists. In 2012 for example, Liu et al. modified Continuous Wave STED, a variation of STED that does not require precise synchronization between the pulsed excitation and STED lasers. They used a Ti:Sapphire oscillator, which is more accessible than the lasers commonly required for STED, and optimized the depletion wavelength with different dyes, and were able to achieve super-resolution of 71nm, sufficient to image cytoskeletal filaments and to visualize individual RNA granules of DYlight 650-MTRIP-labeled viral genomic RNA of human respiratory syncytial virus in Hep2 cells for the first time (1).

Alternative approaches for super-resolution microscopy based on molecule localization schemes also saw notable achievements in 2012. Some of these include a solution for handling data from photoactivated localization microscopy (PALM) using statistical algorithms to quantitatively describe the spatial organization of molecules (2) and compressed sensing for images with overlapping fluorescent spots, which allows for a density of activated fluorophores an order of magnitude higher than previously accomplished (3). As more super-resolution imaging tools are refined and their ease of use enhanced, we expect new users to dip into the super-resolution imaging waters in the coming months, resulting in new molecular insights and new challenges for developers that will serve to further push super-resolution imaging to the forefront of cell biology during 2013.

Clearing the way

While there are numerous technologies available now for imaging cellular processes (confocal microscopes, super-resolution systems, total internal reflectance, etc.), these did not develop in a vacuum, but side-by-side with methods for preparing the samples to be examined. It's hard to say which side has provided the bigger driving force since specimen preparation improves to exploit the full potential of the microscope while microscopes evolve to broaden and deepen what samples can be examined. Because these two sides of microscopy are so intertwined, we can hardly predict advances in technology without also expecting new approaches to prepare those cells, organs, and organisms to be photographed.

Imaging a cell in its native context and environment is ideal for understanding cellular function. Atsushi Miyawaki and his colleagues recently brought us one step closer to this goal by introducing a urea-based reagent, Scale, that can render a biological sample transparent, thus avoiding light scattering that prevents imaging deep within tissues, without affecting fluorescent signals. In mouse brains prepared with Scale, fluorescently labeled neurons were imaged at subcellular resolution in situ at a depth of several millimeters, permitting 3D reconstructions of neural projections (4).

Of course, this approach depends on fluorescent protein labeling of the cells under investigation as well. The array of proteins available for fluorescent labeling has increased significantly since the 1990s when GFP was introduced and continues to expand today. 2012 saw the development of cysteine-free fluorescent proteins that maintain their proper conformations when expressed in the endoplasmic reticulum (5), a new cyan fluorescent protein (CFP) with the highest quantum yield yet reported for a monomeric fluorescent protein and which was optimized following structural analysis of currently available CFP, and other reports on the development of far red fluorescent proteins, photoswitchable proteins, and a variety of new applications.

There is no question that 2013 will bring many more advances in microscopy and we are eager to see each and every one. But we are even more excited to see those unknown, unpredictable advances that are sure to come. Will we see a new super-resolution approach or an reagent akin to Scale come out in 2013? Only time will tell.


1. Liu, Y., Y. Ding, E. Alonas, W. Zhao, P. J. Santangelo, D. Jin, J. A. Piper, J. Teng, Q. Ren, and P. Xi. 2012. Achieving λ/10 resolution CW STED nanoscopy with a Ti:Sapphire oscillator. PloS one 7(6).

2. Sengupta, P., and J. Lippincott-Schwartz. 2012. Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair-correlation analysis. BioEssays : news and reviews in molecular, cellular and developmental biology34(5):396-405.

3. Zhu, L., W. Zhang, D. Elnatan, and B. Huang. 2012. Faster STORM using compressed sensing. Nature methods9(7):721-723.

4. Hama, H., H. Kurokawa, H. Kawano, R. Ando, T. Shimogori, H. Noda, K. Fukami, A. Sakaue-Sawano, and A. Miyawaki. 2011. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nature neuroscience 14(11):1481-1488.

5. Suzuki, T., S. Arai, M. Takeuchi, C. Sakurai, H. Ebana, T. Higashi, H. Hashimoto, K. Hatsuzawa, and I. Wada. 2012. Development of Cysteine-Free fluorescent proteins for the oxidative environment. PLoS ONE 7(5):e37551+.

6. Goedhart, J., D. von Stetten, M. Noirclerc-Savoye, M. Lelimousin, L. Joosen, M. A. Hink, L. van Weeren, T. W. Gadella, and A. Royant. 2012. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nature communications 3.