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Super-resolution microscopy: the smaller, the better

Janice Y. Ahn

Super-resolution microscopy techniques are allowing researchers to image small biological structures beyond the diffraction limit of light. Now, the main limitations are the fluorescent probes used in these techniques. Janice Ahn investigates efforts to create smaller, brighter, and faster switching fluorophores. 

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Light is a wave, subject to diffraction, and thereby imperfectly focused by a microscope lens. The diffraction limit of light prevents resolving anything smaller than about 250 nm, but the majority of subcellular structures and proteins are at least two orders of magnitude smaller, on the scale of 10-100 nm (1). Researchers are now attempting to push the boundaries of light’s diffraction limit with super-resolution microscopy.

“We achieve super-resolution mainly through two principles: high-accuracy localization of the fluorophore and photoswitching,” Miriam Bujny told BioTechniques. Bujny is a post-doctoral fellow in the laboratory of Xiaowei Zhuang at Harvard University, where the super-resolution imaging technique called STORM (stochastic optical reconstruction microscopy) was developed.

A thin section through a lysosome. The marker is PA-GFP tagged to a lysosomal membrane protein. Source: Jennifer Lippincott-Schwartz

Researchers have modified classic ‘far-field’ microscopy to reduce blurriness, and also included the use of fluorescence probes (fluorophores) to directly visualize nanoscale cellular structures such as mitochondria and clathrin-coated pits. But the one shared tactic of all super-resolution imaging techniques, in combating diffraction limits, is to spatially or temporally separate a fluorophore’s ‘on’ state from its ‘off’ state.

Although a single fluorophore can be localized with high accuracy, a normal biological sample contains many fluorophores crowded together, so they appear within a diffraction-limited spot. With STORM (2), along with a similar approach called PALM (photoactivation localization microscopy) (3,4), only a few molecules are turned on during a single imaging event in order to overcome the problem of high molecular density. To avoid interference from neighboring fluorescent molecules and ensure separation of bright molecules from dark ones, researchers image only a small subset of molecules at a time (less than 0.001% of the population). This is accomplished by using fluorophores that cycle on and off.

“Both PALM and STORM are essentially identical in that images are built up from single, stochastically switched-on molecules. It’s a kind of pointillism…you are building an image based on lots of single points,” says Jennifer Lippincott-Schwartz at the National Institutes of Health (NIH), who has been using super-resolution microscopy to study the endomembrane system of cells.

“If you switch one (fluorophore) on and localize it with high accuracy, switch it off, then switch the next (fluorophore) on,” explains Bujny, “you can accurately reconstruct the image.”

A third approach, called STED (stimulated emission depletion) microscopy (5), achieves super-resolution by quenching or dampening the fluorescence light emitted from the periphery of a spot. This technique uses two laser pulses, one to excite a fluorophore and a second doughnut-shaped erase beam (called a STED beam) to de-excite the fluorophore back to the ground state.

“You can think of it as taking a very smooth hill and chopping away its sides to leave a mesa of (your) population” says NIH’s Jay Knutson, who studies optical imaging methods. STED microscopy has achieved resolution as fine as 20 nm in a single plane. It’s enabled the visualization of mitochondrial proteins (6), and has even been used in live-cell imaging of synaptic vesicles in neurons (7) and endoplasmic reticulum trafficking in mammalian cells (8).

One of the difficulties with STED microscopy is that trimming away the edges of the blurry spot takes a lot of power. The Knutson lab is currently collaborating with an organic probe design group at NIH to engineer STED-like probes that are all different in color, yet able to be simultaneously deactivated. This would allow STED to not only become a multi-colored technique, but also a lower-powered process.

Probe problems

Because photoactivatable proteins are the source of fluorescence, researchers can be certain that the center of the blurry spot is their molecule of interest. In PALM, researchers can quantify the number and density of molecules within a structure because the fluorescent proteins are irreversibly photobleached and cannot be turned back on. Lippincott-Schwartz’s lab has been using PALM for just this purpose: to correlate the number of molecules mapped with the actual number of molecules that are being expressed, which provides information on the molecular ratios at specific cellular sites.

However, even the brightest fluorescent proteins are still dimmer than small-molecule fluorophores. The development of brighter fluorescent proteins has been challenging because a strict conformation (of the chromophore within a β-barrel) of the protein is required. Also, new fluorescent proteins must first be identified from other species to allow for multicolor imaging.

On the other hand, small-molecule fluorophores—such as the Cy3/Cy5 dyes attached to antibodies used in STORM—are photostable and can be paired together to facilitate photoswitching, increasing the color palette available in multicolor super-resolution imaging. In fact, simultaneous visualization of microtubules and clathrin-coated pits has already been accomplished through STORM using this dye pair (9).

But the large size of the antibody-conjugated fluorophores adds an extra 10–20 nm of uncertainty in measurements and makes the probes unsuitable for live-cell imaging because antibodies cannot permeate cell membranes. One approach to make these super-resolution probes smaller with higher labeling efficiencies is to tag a protein of interest with a fluorescently tagged short peptide instead of an antibody. Examples of these include tetraCys, hexaHis and polyAsp (1).

Promising probes in development
Although the current probes used in super-resolution microscopy have their flaws, there are several groups working to make better ones.

The Zhuang group is collaborating with Alice Ting at the Massachusetts Institute of Technology (MIT) to develop one set of promising probes called quantum dots. Quantum dots are inorganic nanocrystals that are very bright and photostable—they last for hours, making them ideal for single-molecule imaging (10). However, they are still fairly large in size, unable to permeate the cell membrane, and difficult to target to specific cellular proteins. The Ting lab has been working to remove each of these barriers, so that quantum dots can be used routinely for single-molecule imaging in live cells.

At the moment, at least a half-dozen labs worldwide are actively working to engineer more photo-switchable probes for both STED and PALM/STORM, including Enrico Gratton’s group at the University of California, Irvine, where he has established the first national facility dedicated to fluorescence spectroscopy: the Laboratory for Fluorescence Dynamics (LFD). The problem with improving photoswitching probes is that no one knows how the switching mechanism of these dyes actually works.

“(The field is) not used to thinking at this scale,” comments Lippincott-Schwartz. “It’s just been such an exciting time to be at the interface between physics, chemistry, and biology.”


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