A solitary piling sticking up out of the sea a few dozen meters from the beach is the epitome of loneliness. Incoming waves sweep around it with just a momentary ruffling of their crests, and the diffraction limit makes it invisible from the sand.
The same phenomenon prevents conventional light microscopes from resolving any object smaller than about half the wavelength of whatever light they use. Like the ocean waves, light waves bend around small objects, neither reflecting nor blocking enough energy to reveal their outlines.
The diffraction limit began to drop in the 1990s, when researchers at the Max Planck institute invented "super-resolution" microscopy with stimulated-emission-depletion fluorescence microscopy. This brought the resolution limit down below the half-wavelength mark, but required that fluorescent labels be bound to the target particles or molecules.
Now Pu Wang, Ji-Xin Cheng, and their Purdue University collaborators, have developed the saturated transient absorption microscope (STAM), a tool for seeing objects tinier than a half wavelength without the need for secondary labels.
The method uses a succession of three laser beams to create a sharply defined spot of illumination just 225 nanometers wide. The spot sweeps across a sample on a slide, creating a transmission image that reveals objects in the 100-nm range more clearly and quickly that ever possible in a far-field image. (Far-field techniques, such as conventional microscopes, let researchers record images at a distance from the sample. Techniques like near-field scanning optical microscopy, NFSOM, and scanning tunneling microscopy, STM, have resolutions of about 20 nm and 0.1 nm, respectively; these, however, rely on very short range quantum mechanical phenomena and require that the detector be positioned within about one wavelength of the sample.)
STAM is possible because some materials change transparency as their energy states alter. The Purdue technique exploits this with a three-step laser system: a pump pulse, a saturation pulse, and a probe pulse.
If the probe pulse is used on its own, it will expend much of its energy lifting atoms and molecules to higher energy states, so the material absorbs photons and appears partially opaque. If the system is first mildly stimulated with a pump-laser beam, some of it will already be in a high energy state when the probe beam arrives; therefore, less of the probe light's energy is absorbed and the material appears more transparent. Finally, if a longer and stronger saturating light is applied, it pushes the whole system into a high energy state. When this happens, none of the probe pulse's energy is absorbed and the material appears almost completely transparent.
The key to STAM is the saturation beam. A programmable spatial light modulator (SLM) turns the saturation pulse into what the developers call a "doughnut"—a 225-nm-diameter ring of very bright light surrounding a dark hole. (By contrast, the illumination window of a diffraction-limited pump-probe scan is almost 400 nm wide.) When the pulses are properly timed (close enough together so that the pumping energy doesn't bleed away, but far enough apart so that the wave trains don't trip over one another), the method produces a tight, sharply defined spotlight. Rastering mirrors sweep the spotlight across the sample at very high speed (fast enough that the beam does not start to eat away at the sample), and a photodiode detector assembles the image.
The technique, say the researchers, offers contact-free imaging that is much faster than STM or NFSOM, and is inherently adapted to three-dimensional super-resolution imaging. The researchers add that the method has a wide array of potential applications in "studying nanostructues in biological environments or inside functional materials," and can be applied to any nanomaterial with "saturable [light] absorption, such as single-walled carbon nanotubes or iron oxide and zinc oxide nanoparticles...."
Image: Weldon School of Biomedical Engineering, Purdue University