Scientific Method / Science & Exploration

Diffraction limit? What diffraction limit?

Microscopic technique allows biologists to beat the diffraction limit.

Biologists love to have detailed images of cellular processes.  Unfortunately, the rules of physics limit how closely two objects can be before they become indistinguishable, this limit corresponds to about the wavelength of light.  Thus, a really good microscope, using laser induced fluorescence as its light source can manage to distinguish objects about 200 nm apart.  One way to increase the resolution is to use a shorter wavelength or electrons.  The problem with this approach is that the higher energy associated with short wavelength photons and electrons tends requires a vacuum and tends to kill cells, eliminating any chance of observing dynamic processes.

Enter stimulated emission depletion microscopy (STED), a modification of laser induced fluorescence microscopy.  Laser induced fluorescence microscopy requires that the biologist stains the sample with a dye that's linked to an antibody which directs it to a specific protein.  When illuminated with a laser the dye glows, giving the biologist a high contrast image of the dye concentration.  Given some knowledge about what the dye will bond to, information about cellular structures can be obtained.  STED modifies this technique by using two lasers, one suitable for exciting the dye so that it will glow.  The second laser has the same color as the glow produced by the dye and has a donut beam (i.e., if you shine it on the wall you see a donut shape).  To increase the resolution the dye is excited by a pulse of light from the first laser, the second laser then illuminates the sample briefly.  The second laser stimulates the dye, causing it to emit the stored energy.  That leaves only the hole in the center glowing when the camera is turned on.  It is worth noting that this technique does not beat the diffraction limit, the spot is still about 200 nm wide, however because the size of the spot causing the glow is known, the image can be refined to resolve details well below that of the diffraction limit - 50 nm in this case.

This case being the technique's first use, where it was applied to probe the details of how nerve cells transmit signals that inform muscles that it's time to contract.  At the nerve-muscle junction, called a synapse, electrical impulses from the nerve enter in the form of Calcium ion fluxes, which cause the nerve to send out signals in the form of small molecules.  The new visualization technique allowed the researchers to visualize both the protein channels that allow Calcium ions to enter the cell, and proteins that sit on the surface of the structures that store the small molecule signals, called vesicles.  They found that the calcium ions were clustered at the periphery of a donut-shaped structure, while the vesicles were poised near the edge of the nerve cell in the center of the donut.  They then tested a mutation which fails to efficiently transfer nerve impulses to the muscle, and found that it lacked the clustering of Calcium channels normally seen, demonstrating that this organization is necessary for the proper function of the synapse.  As all of these structures are smaller than the wavelength of the light used, it would have been impossible to examine this prior to the development of this technique.

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