© 1999 Robert A. Freitas Jr. All Rights Reserved.

Robert A. Freitas Jr., Nanomedicine, Volume I: Basic Capabilities, Landes Bioscience, Georgetown, TX, 1999

4.8.4 Near-Field Optical Nanoimaging

Electromagnetic waves of optical wavelength l that interact with an object are diffracted into two components, called "far-field" and "near-field." The propagation of electromagnetic radiation over distances z > l acts as a spatial filter of finite bandwidth, resulting in the familiar diffraction-limited resolution ~l/2.⁴⁹² Classical optics is concerned with this far-field regime with low spatial frequencies < 2/l, and conventional optical imaging will be difficult at the cellular level in vivo (Section 4.9.4). (Short-wave X-rays will damage living biological cells.) Information about the high spatial frequency components of the diffracted waves is lost in the far-field regime, so information about sub-wavelength features of the object cannot be retrieved in classical microscopy.

However, for propagation over distances z << l, far higher spatial frequencies can be detected because their amplitudes are then of the same order as the sample (z = 0). This second diffraction component is the "near-field" evanescent waves with high spatial frequencies >2/l. Evanescent waves are confined to subwavelength distances from the object. Thus a localized optical probe, such as a subwavelength aperture in an opaque screen, can be scanned raster fashion in this regime to generate an image with a resolution on the order of the probe size.

The original Near-field Scanning Optical Microscope (NSOM) surpassed the classical diffraction limit by operating an optical probe at close proximity to the object. The NSOM probe uses an aluminum-coated "light funnel" scanned over the sample. Visible light emanates from the narrow end (~20 nm in diameter) of the light funnel and either reflects off the sample or travels through the sample into a detector, producing a visible light image of the surface with ~12 nm resolution at l = 514.5 nm⁴⁹² provided the distance between light source and sample is very short, about 5 nm, with signal intensities up to 10¹¹ photons/sec (~50 nanowatts). This represents a resolution of ~l/40. The near-field acoustic equivalent is found in the medical stethoscope, which exhibits a resolution of ~l/100.⁵⁷⁶

Applications include dynamical studies at video scanning rates, low-noise high-resolution spectroscopy, and differential absorption measurements. Optical imaging of individual dye molecules has already been demonstrated,^3195,3196 with the ability to determine the orientation and depth of each target molecule located within ~30 nm of the scanned surface.⁴⁹³ Molecules with nanometer-scale packing densities have been resolved to ~0.4-nm diameters using STMs to create photon emission maps⁴⁹⁴ (an electric current generates photons in the sample), and laser interferometric NSOMs have produced clear optical images of dispersed oil drops on mica to ~1 nm resolution.⁴⁹⁵ Live specimen 250-nm optical sectioning for three-dimensional dynamic imaging has also been demonstrated.⁴⁹⁶

Submicron laser emitters have been available since the late 1980s.⁴⁹⁷ It should be possible to use NSOM-like nanoprobes to optically scan the surfaces of cells or organelles to <~1 nm resolution, mapping their topography and spectroscopic characteristics to depths of tens of nanometers without penetrating the surface. However, a proper membrane-sealing invasive light funnel ~20 nm in diameter might not seriously disrupt some cellular or organelle membranes and thus could be inserted into the interiors of these bodies or through the cytoskeletal interstices to permit deeper volumetric scanning. The thermal conductivity of water at 310 K is 0.623 watts/m-K and the energy per 500-nm photon is 400 zJ, so for 1 micron³ of watery tissue the maximum scan rate is ~35 micron³/sec-K, if e^SNR photons are used to image each 1 nm³ voxel with SNR = 2. Thus a 1-sec volumetric optical scan of an aqueous ~1-micron³ sample volume to 1 nm³ resolution requires a ~1 nanowatt scanner running at ~GHz bit rates, raising sample volume temperature by ~0.03 K.

NSOM permits the determination of five of the six degrees of freedom for each molecule, lacking only the optically inactive rotation around the dipole axis.⁴⁹³ In principle, it should be possible to produce <~1 nm resolution near-field optical scans of in situ protein molecules, since with atomically precise fabrication and single lines of atoms as conductors a minimum light guide (metal-dielectric-metal) is 3 atoms wide. Given a molecular laser⁹⁹⁰ and adequate collimation, photons passing through folded proteins will scatter according to the molecular structure. Detection of sufficient photons comprising these scattering patterns should allow the noninvasive determination of protein structure; polarized photons provide information on chirality. Absorption and fluorescent signals will be visible from phenyl rings, tryptophan, and bound cofactors such as ATP and adenine (which is fluorescent). Positions of monoclonal antibodies on virus surfaces are now identifiable experimentally using NSOM;¹²⁵⁸ it should be possible to map binding sites on virus and cell surfaces using fluorescently labelled antibodies. Single molecule detection has been proposed as a tool for rapid base-sequencing of DNA^991,992 (Chapter 20).

Other optically-based cellular imaging techniques must be distinguished. Optical Coherence Tomography (OCT) uses Michelson interferometry to achieve ~10 micron spatial resolutions over tissue depths of 2-3 mm in nontransparent tissue in the near infrared.⁷³⁶ However, OCT requires numerous physical components not easily implemented on a micron-size detector (e.g., beam splitter, lens-grating pair, galvanometer mirror, optical prism), femtosecond pulse shaping, and illumination power levels of ~10⁷ watts/m² >> 100 watts/m² "safe" continuous limit in tissue (Section 6.4.2). Bioluminescence techniques in which the light source is placed inside the tissue (e.g., a transgenic mouse with a luciferase gene in every cell of its body) has a spatial resolution limited to ~10% of the depth, or ~10 microns resolution at a ~100 micron depth.⁷³⁷ Coherent anti-Stokes Raman scattering (CARS) already permits organelle imaging in living cells and can in theory create a point-by-point chemical map of a cell using two intersecting lasers.³²³⁹

Three-dimensional observation of microscopic biological nonliving structures by means of X-ray holography requires a high degree of spatial coherence and good contrast between target and surroundings. Good contrast may be achieved in the wavelength range between the K absorption edges of carbon (l = 4.37 nm) and oxygen (l =2.33 nm), the "water window" where carbon-containing biological objects absorb radiation efficiently but water is relatively transparent.⁹⁸⁸ A 50-micron diameter emitter has been tested that uses near-IR 5-femtosecond laser pulses impinging upon a helium gas target to create a well-collimated (<1 milliradian) beam of coherent soft X-rays at a 1 KHz repetition rate producing a brightness of 5 x 10⁸ photons/mm²-milliradian²-sec, a peak X-ray intensity of >10¹⁰ watts/m² on the propagation axis behind the He target.⁹⁸⁸

Last updated on 17 February 2003