Probes Enhance Microscopy
Researchers at Max Planck Institut für Biochemie in Martinsried, Germany, have developed a probe design that improves the resolution of near-field scanning optical microscopes. The tip-on-aperture probes combine the benefits of aperture and apertureless probes, and they promise high optical and topographic resolution with low background noise.
A new probe for use in near-field scanning optical microscopy combines the benefits of aperture and apertureless designs. Courtesy of Heinrich G. Frey.
Near-field scanning optical microscopy overcomes the diffraction limit in conventional optics by passing the light through an aperture that is smaller than its wavelength. The illumination source is held at a distance from the sample, which also is smaller than the wavelength, typically through the use of a sharpened and partially metal-coated optical fiber mounted on an atomic force microscope. A benefit of this approach is that it produces both atomic force microscope and optical images. Technical and fundamental issues, however, have kept functional resolutions above 50 nm.
A variant of the technique, called apertureless near-field scanning optical microscopy, illuminates an atomic force microscope probe with far-field-focused laser light polarized parallel to it. Heinrich G. Frey, a graduate student at the institute, explained that the light induces a vertical dipole moment in the tip that interacts with the sample. This changes the scattering at the tip and enables the user to create an image.
The new probe begins with an optical fiber that is etched to a fine point. The researchers clad the point in metal and create an aperture in its end. Using electron-beam deposition, they grow a tiny tip consisting mainly of carbon on the end of the optical fiber. They then deposit more metal on the end at a 45° angle, which closes the aperture everywhere but in the shadow of the tip. This device combines the higher resolutions due to the field concentration at a very fine probe with the controllable, fixed light source of more conventional systems.
Conventional apertureless scanning near-field optical microscopes are sensitive to drifting of the optical far-field focus of the illumination laser relative to the tip, Frey explained, but the position of the light source is fixed for the new probe. More importantly, he said, the area that is illuminated by the light source is smaller than that of apertureless microscopes. "So, we have a smaller background, and fewer dye molecules are photobleached in the environment of the tip," he said.
The probe is too new for the researchers to fully characterize its effects on the resolution of a microscope setup, he added. Resolution, however, is largely limited by the sharpness of the tip. In a report of the technique, they described the imaging of fluorescent beads at an optical resolution of 25 nm, and Frey said that they have achieved 20 nm. "Higher optical resolution with sharper tips may be complicated by quenching effects," he said.
Frey hopes to begin using the technique to image single molecules. He also plans to continue working on ways to sharpen the probes and, consequently, the resolution.
- Acronym for profile resolution obtained by excitation. In its simplest form, probe involves the overlap of two counter-propagating laser pulses of appropriate wavelength, such that one pulse selectively populates a given excited state of the species of interest while the other measures the increase in absorption due to the increase in the degree of excitation.
- 1. In optics, the ability of a lens system to reproduce the points, lines and surfaces in an object as separate entities in the image. 2. The minimum adjustment increment effectively achievable by a positioning mechanism. 3. In image processing, the accuracy with which brightness, spatial parameters and frame rate are divided into discrete levels.
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