Near-Field Scanning Optical Microscopy (NSOM)

NSOM uses specially fabricated, tapered fiber optic probes to deliver light to the nanometric dimension. By scanning NSOM tips near a sample surface, high-resolution optical images can be measured.

Near-Field Scanning Optical Microscopy (NSOM)

The diffraction of light limits the size of a spot that can be formed when focused with a lens. This diffraction limit dictates the maximum resolution possible in conventional microscopy and is often approximated by l/2 where l is the wavelength of the excitation light. When working in the visible region of the spectrum, therefore, spatial resolution is limited to several hundred nanometers. For many applications in the biological sciences, this proves restrictive and led to the development of higher resolution techniques such as electron microscopy and scanning probe microscopy. The enhanced resolution, however, often comes at a cost in flexibility of sample imaging conditions, sample preparation, and information content. This has led to the development of near-field scanning optical microscopy (NSOM or SNOM) which combines the favorable attributes inherent to optical microscopy with the high spatial resolution of the other techniques.

There are several ways that NSOM can be implemented. In aperture NSOM, light is delivered to the nanometer dimension using specially fabricated tapered single-mode optical fibers coated with a reflective coating around the tapered region. Figure 1 shows magnified views of a typical NSOM probe. In the electron microscopy image shown in Fig. 1a, the aperture at the distal end can be seen along with the metal coating around the sides of the taper. Light emerging from the end of the NSOM probe can be seen in the magnified optical image shown in Fig. 1b. The aperture size and transmission characteristics are strongly tied to the exact geometry of the NSOM probe, but typically probe apertures have sub-100 nm diameters and can deliver hundreds of nanowatts of light.

To achieve high spatial resolution, the NSOM probe must be positioned and held within nanometers of the sample surface as the probe is scanned across the surface. Numerous feedback systems have been introduced to achieve this, with most based on force interactions between the probe and the surface. Because of this force feedback, a topography image of the surface is also generated along with the NSOM fluorescence information, which can be particularly informative for biological applications.

NSOM Tip

Tapered fiber optic probes are coated with a thin layer of aluminum to confine light such that it only exits the aperture at the distal end. By positioning the probes near a sample surface, the emerging light interacts with the surface before diffracting - leading to high spatial resolution. SEM of a coated NSOM tip. Grains in the aluminum and the aperture at the end are visible.

NSOM Microsope

A modified Digital Instruments Bioscope is used to implement NSOM measurements. The NSOM probe is held in a modified Dimension head and mounted on an inverted fluorescence microscope. The sample is scanned below the tip using a separate xy piezo scanning while the z-tube adjust the tip over sample topography. Fluorescence excited by the NSOM tip is collected from below and detected on an avalanche photodiode.

Single Molecule Fluorescence

Single molecule fluorescence measured with NSOM. Each feature has a FWHM of 28 nm illustrating the sub-diffraction limited resolution possible with NSOM.

NSOM Measurements of Lipid Monolayers

NSOM topography (left) and fluorescence (right) of a DPPC monolayer transferred onto a substrate using the LB method. The lipid monolayer was transferred in the LC/LE phase correspondence region. The 5 Angstrom height difference in the NSOM topography image reflects the difference between the more packed LC regions and lower topography LE regions. The NSOM fluorescence reveals bright regions where a dye has partitioned into the LE regions.

Selected papers

Xie, X. S.; Dunn, R. C., Probing Single-Molecule Dynamics. Science 1994, 265 (5170), 361-364.

Talley, C. E.; Cooksey, G. A.; Dunn, R. C., High resolution fluorescence imaging with cantilevered near-field fiber optic probes. Appl Phys Lett 1996,69 (25), 3809-3811.

Hollars, C. W.; Dunn, R. C., Submicron fluorescence, topology, and compliance measurements of phase-separated lipid monolayers using tapping-mode near-field scanning optical microscopy. J Phys Chem B 1997,101 (33), 6313-6317.

Hollars, C. W.; Dunn, R. C., Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy. Biophys J 1998,75 (1), 342-353.

Hollars, C. W.; Dunn, R. C., Evaluation of thermal evaporation conditions used in coating aluminum on near-field fiber-optic probes. Rev Sci Instrum 1998,69 (4), 1747-1752.

Shiku, H.; Dunn, R. C., Direct observation of DPPC phase domain motion on mica surfaces under conditions of high relative humidity. J Phys Chem B 1998,102 (19), 3791-3797.

Talley, C. E.; Lee, M. A.; Dunn, R. C., Single molecule detection and underwater fluorescence imaging with cantilevered near-field fiber optic probes. Appl Phys Lett 1998,72 (23), 2954-2956.

Dunn, R. C., Near-field scanning optical microscopy. Chem Rev 1999,99 (10), 2891-+.

Shiku, H.; Dunn, R. C., Near-field scanning optical microscopy studies of L-alpha-dipalmitoylphosphatidylcholine monolayers at the air-liquid interface. J Microsc-Oxford 1999,194, 461-466.

Shiku, H.; Dunn, R. C., Domain formation in thin lipid films probed with near-field scanning optical microscopy. J Microsc-Oxford 1999,194, 455-460.

Shiku, H.; Dunn, R. C., Near-field scanning - Optical microscopy. Anal Chem 1999,71 (1), 23a-29a.

Shiku, H.; Krogmeier, J. R.; Dunn, R. C., Noncontact near-field scanning optical microscopy imaging using an interferometric optical feedback mechanism. Langmuir 1999,15 (6), 2162-2168.

Vickery, S. A.; Dunn, R. C., Scanning near-field fluorescence resonance energy transfer microscopy. Biophys J 1999,76 (4), 1812-1818.

Clancy, C. M. R.; Krogmeier, J. R.; Pawlak, A.; Rozanowska, M.; Sarna, T.; Dunn, R. C.; Simon, J. D., Atomic force microscopy and near-field scanning optical microscopy measurements of single human retinal lipofuscin granules. J Phys Chem B 2000,104 (51), 12098-12101.

Hollars, C. W.; Dunn, R. C., Probing single molecule orientations in model lipid membranes with near-field scanning optical microscopy. J Chem Phys 2000,112 (18), 7822-7830.

Vickery, S. A.; Dunn, R. C., Direct observation of structural evolution in palmitic acid monolayers following Langmuir-Blodgett deposition. Langmuir 2001, 17 (26), 8204-8209.

Sibug-Aga, R.; Dunn, R. C., High-resolution studies of lung surfactant collapse. Photochem Photobiol 2004,80 (3), 471-476.

Erickson, E. S.; Dunn, R. C., Sample heating in near-field scanning optical microscopy. Appl Phys Lett 2005,87 (20).

Dickenson, N. E.; Erickson, E. S.; Mooren, O. L.; Dunn, R. C., Characterization of power induced heating and damage in fiber optic probes for near-field scanning optical microscopy. Rev Sci Instrum 2007, 78 (5).

Dickenson, N. E.; Armendariz, K. P.; Huckabay, H. A.; Livanec, P. W.; Dunn, R. C., Near-field scanning optical microscopy: a tool for nanometric exploration of biological membranes. Anal Bioanal Chem 2010, 396 (1), 31-43.