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Twice the resolution, using multiple beams

Aug 2008
High resolution achieved with high speed.

Hank Hogan

For those who want an up-close yet targeted picture of biology, three-dimensional structured illumination microscopy may be the answer, according to a group of researchers. In a study of cells, they successfully used the technique to simultaneously image chromatin, the nuclear lamina and the nuclear pore complex, revealing details hidden from standard light microscopy. The investigators were able to do so because the new technique provides a resolution better than the 200-nm limit of classical microscopy.

Microscopy is revealing finer details, thanks to structured illumination. Here a mouse myoblast cell is triple-stained for DNA (red, DAPI stain), nuclear lamina (blue, immunofluorescence) and nuclear pores (green, immunofluorescence). It is viewed with 3-D structured illumination microscopy, which is capable of about 100-nm resolution, or twice that of classical microscopy. The 3-D images have been partially projected to reveal the interior of the nucleus (where the blue and green staining is “cut away” from the rest). The large red blobs are heterochromatin — condensed DNA that stains more intensely with DAPI. Courtesy of Lothar Schermelleh and Peter M. Carlton, University of California, San Francisco.

“It’s about 100-nm resolution in X and Y and 200 to 300 in Z,” said team co-leader John W. Sedat, a professor of biochemistry and biophysics at the University of California, San Francisco.

Others in the group were from the Max Delbrück Center for Molecular Medicine in Berlin, from the University of Florida in Gainesville, and from Ludwig Maximilians University in Munich. The team from Munich operated under the direction of the other team co-leader, professor Heinrich Leonhardt. Those in California developed the microscopy system, while the team members in Germany concentrated on the biology.

The resolution of a standard light microscope is limited by diffraction to about half the wavelength of the light. For a visible source, that translates into a best-case resolution of between 200 and 300 nm — a figure too big for many subcellular studies.

Electron microscopy has a resolution that is hundreds of times finer. However, it lacks specificity, which is achieved in light microscopy through the use of fluorescence and other methods that highlight specific structures. Electron microscopy also cannot image live cells because it’s done in a vacuum and often entails coating samples with a conductor such as thin-film gold.

Researchers, therefore, have been looking for methods that have greater resolution than standard light microscopy but that retain its capabilities. One such approach is structured illumination microscopy, which was first demonstrated about a decade ago by Mats G.L. Gustafsson of the University of California, San Francisco.

As the name implies, the technique makes use of patterned illumination created by multiple interfering beams of light. The structured light thus produced allows for finer features to be extracted from a series of raw images. It’s a process that can be computationally intensive, noted University of California, San Francisco researcher Peter M. Carlton.

To generate the pattern, the team used a grating to diffract incident light into multiple orders. They focused and recollimated the innermost three orders through the use of optics. These beams, in turn, intersected and interfered with each other at the sample. Getting the complete picture required five phases of the pattern at three orientations or 15 images, with exposure time of 100 to 500 ms at each focal plane.

The lateral resolution achievable with the technique is about twice that of classical microscopy, or between 100 and 150 nm. The exact value depends on the lenses and the wavelength of the original source light. Adding more beams or using a finer diffraction grating will not improve the resolution because it is constrained by the same effects that limit standard microscopy.

In their setup, the researchers used light from one of three lasers as a source, with the available wavelengths 405, 488 or 532 nm. They captured the resulting fluorescent emission light from the sample at 450, 515, 590 and 685 nm using four dichroic mirrors and the same number of Andor Technology electron-multiplying CCD cameras. One computer operated each camera. Another controlled the stage components from Applied Precision Inc. of Issaquah, Wash. A sixth computer handled the submillisecond timing of image acquisition, shutters and stage motion.

In a demonstration of the system’s capabilities, the researchers studied the mammalian nucleus, selecting mouse cells highlighted by Alexa488 for green fluorescence and Alexa555 or Alexa568 for red fluorescence. They compared the results obtained with their three-dimensional structured illumination microscope with those from conventional wide-field epifluorescence and confocal microscopes.

The investigators found that the new system revealed features that the conventional ones did not. For example, structured illumination microscopy showed that DNA was excluded from some of the nuclear pore complexes, a phenomenon known from electron microscopy studies but not previously seen using light microscopy. With the new technique, the researchers visualized the folding of the nuclear lamina around what they presumed to be microtubules, again finding something known to be the case from electron microscopy but heretofore not seen using visible light.

Finally, they calculated the density of nuclear pore complexes in both the structured illumination and confocal images, using an algorithm and identical criteria to sift through the data. On average they found 5.6 complexes per μ2 using the new technique but only 2.8 using the conventional one. Thus, the density ratio between the two imaging techniques tracked the resolution ratio between them. The work was published in the June 6, 2008 issue of Science.

Results from 3-D structured illumination microscopy (on right) and from classical microscopy (on left) of the actin cytoskeleton of a mammalian cell stained with Alexa488-phalloidin are shown. The twofold improvement in resolution of structured illumination as compared with classical microscopy can easily be seen. Courtesy of Peter M. Carlton, University of California, San Francisco.

Leonhardt noted that studies done with electron microscopy on other cell types found a density of 12. Because of the difference in cell types, he said, a direct comparison with the electron microscopy studies isn’t possible. In considering the electron microscopy results, however, he noted, “It indicates that the higher 3-D structured illumination microscopy numbers are in good company and probably closer to the truth.”

As for the future, work in this area continues. While the team’s instrument was a custom-built affair, a commercial version is being developed by Applied Precision. In the meantime, high-resolution studies of the nucleus are being done with the custom setup. Other basic cell biology questions also are being investigated.

With regard to such studies, the new technique offers a particular advantage. The acquisition rate can be quite high, given the right hardware. Sedat said that imaging speed is particularly important for a variety of reasons. “The faster you can do it, the less drift there is in the sample and everything else. For the live information, it’s critical.”

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