Electron and scanning probe microscopy have high spatial resolutions and thus can resolve molecules, but these techniques mostly are used to examine nonliving matter. On the other hand, fluorescence microscopy is less perturbative and thus is more suited for live-cell imaging, but its resolution is limited by the diffraction of light waves to ~300 nm, one to two orders of magnitude above the length scales of molecules inside cells. Recently developed superresolution optical imaging techniques have achieved resolutions down to 20 to 50 nm, depending on the experimental conditions.This two-color STORM image of a cell is presented in very high resolution.To achieve higher clarity, a super-resolution optical imaging technique called stochastic optical reconstruction microscopy (STORM) distinguishes individual fluorophores with spatially overlapping fluorescence signals. Fluorescence emission overlaps when neighboringfluorophores are illuminated simultaneously, and STORM solves this problem using photoswitchable fluorophores. By activating only subsets of photoswitchable fluorophores at a time, one can determine the locations of individual fluorophores. The locations can be used to reconstruct super-resolution images. STORM was invented by Xiaowei Zhuang and colleagues at Harvard University in Cambridge, Mass. Last year, they demonstrated the microscopy technique in a single color. Now they have achieved 20- to 30-nm resolution optical imaging of DNA samples and of fixed mammalian cells while using fluorophores of more than one color. To perform multicolor imaging, the researchers needed to use several photoswitchable fluorophores, so they investigated — in both previous and current experiments — whether cyanine fluorophores are photoswitchable. “We actually discovered a whole family of photoswitchable fluorophores,” Zhuang said. Researchers improved their super-resolution optical imaging technique, STORM, and they used it to image DNA molecules in three colors. Each colored spot in this image represents a cluster of localizations from a single DNA molecule. The images on the right are higher-magnification views of the boxed regions in the left images, and they are examples of closely spaced DNA molecules. Reprinted with permission of Sciencexpress.They found that photoswitchable cyanine fluorophores can serve as “reporters,” while other fluorophores, not necessarily cyanine, can serve as “activators.” After an activator is excited with a laser, the activator strongly promotes a neighboring reporter to switch on. Once that is done, another laser is used to cause the reporter to fluoresce. In principle, one can have nine colors by pairing three activators and three reporters in all possible combinations, according to Zhuang. The researchers asserted that the reporters typically can be switched on and off for hundreds of cycles before permanently photobleaching.Multicolor imagingTo achieve multicolor imaging with the photoswitchable fluorophores bound to DNA samples and with those in mammalian cells, the researchers performed total internal reflection fluorescence (TIRF) imaging using an Olympus inverted microscope. Zhuang said that, although the researchers used TIRF geometry, the strategy is not a requirement for creating STORM images. They even could have used basic epifluorescence, she said. They used an Olympus 60×, 1.2-NA water-immersion objective for the DNA samples and an Olympus 100×, 1.4-NA objective for the mammalian cells. The fluorescence signal was collected with an Andor Technology electron-multiplying CCD camera at ~20 Hz.To obtain a three-color image of a model DNA sample, they turned on three photoswitchable activator-reporter pairs, AlexaFluor 405-Cy5, Cy2-Cy5 and Cy3-Cy5, bound to the DNA by a Coherent 405-nm (violet) diode laser, by the 457-nm (blue) line of a Melles Griot argon-ion laser and by a Crystalaser 532-nm (green) diode-pumped solid-state laser, respectively. The laser emission wavelengths corresponded to the absorption wavelengths of the activators, AlexaFluor 405, Cy2 and Cy3. For imaging, the activated reporter, Cy5, was excited using a Melles Griot 633-nm HeNe laser.To obtain a two-color image of microtubules and clathrin-coated pits in cells, the researchers used the 457- and 532-nm lasers to activate the Cy2- and Cy3- AlexaFluor 647 pairs and imaged the activated reporter dye, AlexaFluor 647, using a Crystalaser 657 (red) diode-pumped solid-state laser. The super-resolution images of microtubules (green, lower panels) in a cell resolve individual microtubule filaments significantly better than the conventional-resolution images (blue, upper panels). Reprinted with permission of Sciencexpress.In cells, they used antibody labeling because it is a familiar method for cell biologists, Zhuang said. Likewise, they chose microtubules and clathrin-coated pits because those structures are important for cellular biology. For both DNA and cell imaging, the investigators operated the activation lasers during one frame and the imaging laser during nine frames, and the images were recorded at approximately 20 Hz. They typically reconstructed 2000 to 100,000 frames to produce the final super-resolution images. For three-color imaging of model DNA samples, the researchers achieved ~25-nm resolution. Single- and dual-color super-resolution imaging in cells resolved individual microtubule filaments and clathrin-coated pits and revealed ultrastructural information that conventional fluorescence imaging cannot show. Measurements of microtubule width from single-color super-resolution images were comparable to measurements made with electron microscopy. Measurements of microtubules and clathrin-coated pits from dual-color super-resolution images also were similar to measurements made with electron microscopy. The technique resolved clathrin-coated pits (red) and microtubules (green) in cells, achieving a resolution beyond that of traditional fluorescence imaging. The image on the bottom left is a higher-magnification view of the boxed region in the top image, and the image on the bottom right is an even higher magnification image of the boxed region in the bottom left image. Reprinted with permission of Sciencexpress.The researchers took the STORM images of cells in 2 to 30 min and pointed out that super-resolution STORM images of cellular structures typically began to emerge in only 10 to 20 s. In summary, they discovered a family of photoswitchable fluorophores, they improved the resolution and speed of their imaging technique, and they performed multicolor super-resolution imaging. This work is detailed in the Aug. 16 online edition of Sciencexpress.“[Ten seconds] is one of the fastest super-resolution imaging speeds already,” Zhuang said, “but, in many cases, it is still not fast enough for live-cell imaging of cellular processes.” She said that a faster camera, an improved data acquisition scheme and an improved image-analysis algorithm all could improve the imaging speed. She believes that scientists other than super-resolution imaging experts can do STORM imaging on their own, as long as they know how to perform immunofluorescence imaging and single-molecule imaging and analysis, which are commonly used procedures.