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Second-harmonic imaging with minimal damage

Apr 2006
Hank Hogan, Contributing Editor,

When researchers at Cornell University in Ithaca, N.Y., and the University of Florence in Italy set out to optimize second-harmonic-generation microscopy, they did so to improve measurements of neuronal membrane potentials. For this to work, though, they had to overcome the photodamage that is a byproduct of second-harmonic generation. They succeeded and uncovered some unexpected effects.

“We were surprised by two aspects of the photodamage: the significant decrease on slowing the exposure rate and the cubic intensity dependence,” said Watt W. Webb, research team leader and a professor of applied physics at Cornell.

Unlike electrode-based methods, second-harmonic-generation microscopy can measure membrane potential from many neurons at the same time, and it does so with micron and millisecond resolution. What’s more, the signal originates only from dye molecules that are properly ordered in the plasma membrane, and the technique allows deep-tissue, high-resolution imaging.

By tweaking their setup, researchers imaged neurons of the sea slug Aplysia with second-harmonic generation but without photodamage. They used a membrane potential sensitive second-harmonic-generating dye at the right concentration and illuminated it with the proper laser power. The electrical activity of the neurons changed the intensity of the emitted second-harmonic signal and allowed them to optically record neural activity in real time. This image measures about 150 x 450 μm. Courtesy of L. Sacconi and D.A. Dombeck.

Because the second-harmonic-generation signals that track voltage responses are small, achieving an acceptable signal-to-noise ratio typically requires intense illumination and/or high dye concentrations. One solution is to average multiple line scans, but that doesn’t work in some situations.

According to the researchers, photodamage probably results from multiphoton excitation that accompanies second-harmonic generation.

The researchers set out to find a recipe for conditions that maximize second-harmonic generation while minimizing photodamage. They did this by comparing the resting potential of Aplysia neurons as measured by an electrode before, during and after illumination under various conditions.

They measured photodamage by its effect on resting membrane potential during line- and image-scanning modes, varying laser illumination from 0 to 100 mW and dye dose concentration from 0 to 25 μM. They also used OxyFluor, an oxygen scavenging enzyme, from Oxyrase Inc. of Mansfield, Ohio, to examine the dependence of photodamage on oxygen. Webb noted that varying all these parameters involved quite a bit of work.

For staining, the scientists used FM4-64 dye. The imaging system consisted of a fiber laser from Fianium Inc. of Eugene, Ore., coupled to a Bio-Rad scan box and an Olympus microscope, with Hamamatsu gallium-arsenide-phosphide photomultipliers sitting behind an optical filter for a detector.

These measurements showed that the photodamage increased linearly with the dye concentration but grew with the cube of laser intensity. The latter might be due to a third-order photochemical process, such as three-photon dye excitation, but this has yet to be pinned down. Adding the appropriate antioxidants before the laser scan lessened photodamage.

Applying these results, the researchers used second-harmonic-generation microscopy to record membrane action potentials in a single trial, a technique that could prove useful for real-time investigations. They also recorded action potentials along neuron neurites, although they had to cut laser power approximately in half to do this.

These findings help point the way to the researchers’ ultimate goal of optimizing optical imaging of fast changes in membrane potential anywhere in a neural system. Webb said that achieving this might become easier with advances in technology. “There is always the hope of developing better dyes,” he said.

PNAS, Feb. 28, 2006, pp. 3124-3129.

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