App. Note: Near-Field Optical Microscopy of TMV

Application Note:
Biological Applications of Scanning Near-field Optical Microscopy

Dr. Patrick J. Moyer Assistant Professor, University of North Carolina, Charlotte(UNCC), Dr. S. P. Marchese-Ragona, and Briggs Christie; TopoMetrix Corp.; Number 2-1193-003 Dec. 1993

SYNOPSIS

Combining the high resolution imaging capabilities of scanning probe microscopy with the contrast mechanisms inherent to optical microscopy provides one with a tool to obtain optical imaging information with resolution not previously afforded with conventional optical characterization techniques. In this Application Note we document the use of the TopoMetrix Aurora (tm) Near-field Scanning Optical Microscope (NSOM) for imaging Tobacco Mosaic Virus (TMV) particles with a resolution of better than 30 nm.

(Fig. 1) Click for closer look (45k)

1痠 x 1痠 scan - Tobacco Mosaic Virus (TMV) is a standard tool for microscopists, with a known and consistent diameter of 18 nm.

As an example of NSOM application to a sample of biological interest, we can image TMV particles as a resolution standard (Figure 1). The TMV particles were covalently attached to aminosilanated mica using glutaraldehyde (0.04-0.08%). After several minutes the glutaraldehyde was removed by washing in distilled water, excess water was removed and the sample was imaged immediately. Figure 1 shows a transmission absorption NSOM image of the above described sample. The image was obtained using an Argon ion laser of wavelength l=488 nm as the light source. The features are clearly imaged with higher localization than can be provided with conventional far-field microscopes. Additionally, as this image is 1.4 x 1.4 痠, individual TMV particles are resolvable with separations of less than 30 nm. Thus, this image clearly shows the resolution capabilities of the NSOM technique.

[NSOM Liquid] High Resolution NSOM in Liquid (Standard sample - Al on spheres)

The NSOM Technique

The effects of far-field diffraction limit the spatial resolution of conventional optical imaging instruments. In practice this limit is no better than half of the wavelength of light being used. Thus, for confocal laser imaging with green light (l=500 nm), resolution is limited to approximately 300 nm. NSOM overcomes this boundary by scanning a sub-wavelength sized light source very close to a sample and building up an optical image of the specimen pixel-by-pixel. The light source is an optical aperture (~25 nm in diameter) fabricated at the tapered apex of an aluminized optical fiber. Using force feedback, the tip of the probe maintains constant separation from the sample (~5 nm). Thus, as the light emanates from the probe tip, it only illuminates a volume of the sample approximately equal to the aperture size. Any collected optical contrast, either transmitted through, or reflected from the sample, originates from this small volume. Hence, resolution is limited to the size of the aperture and not by the wavelength of light.

(Fig. 2) Click for the full view (27k) or click here for a full color diagram from Pat Moyer at (UNCC).

Schematic representation of the Near-field optical effect.

Figure 2 illustrates a schematic diagram of the TopoMetrix Aurora NSOM. As the tip follows the surface of the sample, the data provided includes not only the optical image, but a topographic perspective as well. By controlling the polarization state of the laser light at the aperture and utilizing polarizers and filters of the collected signal from the sample, the same contrast mechanisms used in far-field imaging tools can be used with NSOM. This includes biological fluorescence, absorption and reflection, and polarization contrast imaging. Figure 3 shows the line profile measurements to estimate optical resolution.

Click image for complete line profile measurements.