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Cell biology beyond the diffraction limit: near-field scanning optical microscopy

¹ Department of Tumor Immunology, University Medical Center Nijmegen, NCMLS/187 TIL, PO Box 9101, 6500HB Nijmegen, The Netherlands
² Applied Optics Group, Faculty of Applied Physics and MESA+ Research Institute, University of Twente, PO Box 217, 7500AE Enschede, The Netherlands

View larger version (74K): [in a new window]   Fig. 1. The principle of scanning probe microscopy. In AFM and NSOM, a sharp probe is used to map the topographic features on the sample surface accurately. This is done by physically scanning the probe over the surface while maintaining a constant probe-sample distance by force feedback.

View larger version (21K): [in a new window]   Fig. 2. Schematic lay out of a near-field scanning optical microscope. The NSOM probe is a tapered optical fiber (Fig. 3A). Laser light is coupled into the fiber and is used to excite fluorophores as the probe scans the sample surface. The probe-sample distance is maintained constant at <10 nm during scanning by shear-force-based distance detection in combination with an electronic feedback system controlling the piezoelectric scan stage. Fluorescence is collected by a conventional inverted microscope. Dual-channel optical detection allows wavelength and/or polarization discrimination.

View larger version (71K): [in a new window]   Fig. 3. The near-field optical probe. (A) An optical fiber is pulled to a final diameter of 20-120 nm and subsequently coated with aluminum. This coating serves to confine the light to the tip region. A subsequent etching step results in a flat and circular endpoint and aperture. The aperture functions as a miniature light source, and its diameter primarily determines the optical resolution of the microscope. (B) The principle of surface-specific excitation. The optical near-field generated at the aperture has significant intensity only in a layer of <100 nm from the aperture; lower lying fluorophores are therefore not excited. Hence, background fluorescence is effectively suppressed. This forms the basis for the high optical detection sensitivity of this technique.

View larger version (55K): [in a new window]   Fig. 4. (A) Single molecule detection on cells by NSOM. This figure shows a 40 nm optical resolution near-field ‘zoom-in’ on the indicated area (3.2 x 3.2 µm2) in the bright-field image of a fibroblast expressing LFA-1-GFP. GFP excitation is accomplished using 488 nm light (Ar-Kr laser line) linearly polarized along 90°. Fluorescence is collected by a 1.3 numerical aperture oil-immersion objective in combination with standard optical filters. A polarizing beam-splitter cube (Newport, Fountain Valley, CA) is used to split the fluorescence signal into two perpendicular polarized components (compare with Fig. 2). Both signals are then detected by photon-counting avalanche photodiode detectors (APD, SPCM-100, EG&G Electro optics, Quebec). The red/green color-coding of the signals reflects the orientation of the GFP molecules in the plane of the sample. Examples of clustered molecules (arrows) as well as examples showing clear single-molecule detection sensitivity are indicated (circles and squares). The squares show the fast-blinking behavior typical of single molecule GFP fluorescence. The circles present demonstrations of discrete photodissociation phenomena. (B) Estimation of the resolution in the near-field image. This figure shows a line trace through the feature marked with the hexagon in the near-field image. The full width at half maximum (FWHM; arrows) of such traces can be used to obtain an estimate for the maximal resolution (half the FWHM) in the near-field image. On this basis, we estimate the resolution in the near-field image to be 40 nm.