Combo fluorescence microscopy approach enhances cell interior insight

Combining two very advanced fluorescence microscopy techniques enables greater insights into a cell's interior.

Jun 1st, 2017
Content Dam Bow Online Articles 2017 06 Pm 103  Stelzer Guffm Web

Researchers at Goethe University Frankfurt (Germany) have combined two very advanced fluorescence microscopy techniques, enabling greater insights into a cell's interior.

Related: Advanced light microscopy enables rapid mapping of brain structure and function in high resolution

Using the light-sheet microscopy technology invented and developed by Goethe University Frankfurt professor Ernst Stelzer, it was already possible to observe organisms in a very precise and vivid way during cell differentiation. His research group has now combined light sheets with a technique that so far only allowed very high spatial resolutions (<100 nm) on a cell's surface. Combining both methods makes it possible to obtain a three-dimensional (3D) insight into a cell with a high resolution.

Light-sheet-based fluorescence microscopy (LSFM) is the most recent 3D fluorescence microscopy technique. In fluorescence microscopy, a fraction of a cell's molecules is labeled with fluorescent markers, which are illuminated with a beam of light. A camera records the 3D distribution of the fluorescing molecules (fluorophores). The advantage of LSFM is that even sensitive samples such as fish embryos survive observation. This is a major advancement since conventional methods, which illuminate the whole sample, expose the specimens to much more energy, and destroy the cells in a very short period of time.

Stelzer, professor at the Institute of Cell Biology and Neuroscience and a principal investigator in the Macromolecular Complexes Cluster of Excellence at Goethe University Frankfurt, explains that LSFM does not illuminate the entire sample, but only micrometer-thin light sheets. "Since we examine the biological specimens under conditions that are as natural as possible, we achieve very precise results," he says. Static images of cells, as well as dynamic changes in their environment or genetic mutations, can be measured in direct comparisons.

Bo-Jui Chang, Victor Perez Meza, and Stelzer have now improved the technique further: combining LSFM with coherent structured illumination microscopy (SIM), a super-resolution technique that produces several images, which are combined digitally. As a result, resolution is improved in the physical sense. The technical approach is to excite a fluorescing sample with a very specific illumination pattern. Sub-100 nm resolutions with this method are limited to surfaces, but the technique has major advantages. It is fairly moderate in the excitation of the fluorescence, allows very fast imaging, and can be used with all fluorescing molecules for high-resolution purposes.

Live yeast cell embedded in agarose imaged using conventional fluorescence (left), conventional treatment (center), and csiLSFM (right). (Scale bar: 1 µm)

"In the new microscope, which we call csiLSFM, we have developed the principle of SIM further in such a way that sub-100 nm resolutions are no longer limited to surfaces, but can also be used in extensive three-dimensional objects. Here, two counterpropagating light sheets interfere at an angle of 180° so that they form the smallest possible linear interference pattern. As a result, we achieve an optimal resolution of less than 100 nm," Stelzer explains. The new instrument has three objective lenses. It works via the flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet.

Full details of the work appear in the Proceedings of the National Academy of Sciences; for more information, please visit http://dx.doi.org/10.1073/pnas.1609278114.

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