Sid Ragona and Phil Haydon, Laboratory of Cellular Signaling, Dept. of Zoology and Genetics, Iowa State University, Ames, IA 50011

Click each image for a closer look (JPG)

Figure 1 (left), Phase contrast optical image of interband region of polytene chromosome; Figure 3 (middle), Far-field image of a 5µm² area; Figure 4 (right) Near-field image of a 5µm² area.

Unlike conventional optical microscopy, in which the resolution is limited by the wavelength of light used, the resolution of a Near-field Scanning Optical Microscope (NSOM) is primarily dependent upon the size of a sub-wavelength aperture that is brought within a few nanometers of the surface.

The data presented here demonstrates this remarkable improvement in resolution. The test sample used for this experiment is the interband region of a polytene chromosome.

Figure 1 shows the sample as seen with an optical resolution of 700nm, close to the limit using conventional optical microscopy. Using a TopoMetrix Aurora™ NSOM, a small area 5µm² was then imaged with a probe which had an aperture of approximately 25 nm. The experimental setups are shown in Figure 2. When the tip-sample separation is 1.8 microns, providing far-field illumination, the resolution is about 700 nm (Figure 3).

Figure 2 (left), Phase contrast optical image of interband region of polytene chromosome; Figure 5 (right) Line profiles across far-field (red) and near-field image (blue).

A dramatic improvement is shown when the same area is imaged with a separation of only 2-5 nm, which is obtained when shear force feedback is used to position the aperture (Figure 4). In this case, the sample is within the near-field, and resolution is limited by the size of the aperture.

Line profiles (Figure 5) across the images clearly demonstrate the increase in resolution.

NSOM of Polytene Chromosomes

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