Biological applications of near-field optical microscopy

Presents several biological applications of near field optical microscopy, in combination with force microscopy. Aperture near field scanning optical microscopy (NSOM) with fluorescence detection gives (bio)chemical specificity and orientational information, in addition to the simultaneously acquired force image. This technique has large potential for DNA sequencing, molecular organization in monolayers, and study of the role of the cytoskeleton in cellular mobility in cell growth, cell migration, formation of protrusions, etc. Fluorescence NSOM gives high resolution on flat, not too deep surfaces. Fluorescence NSOM induces virtually no bleaching, as opposed to confocal fluorescence microscopy. Bright field NSOM in transmission generally yields a complicated contrast, caused by a mixture of dielectric and topographic contributions. Shear force feedback is essential in aperture NSOM operation with fibers, and operates on soft surfaces of cells and chromosomes. Ultimately, aperture NSOM is limited by low efficiency with a source brightness of typically 100 pW to 10 nW. Thus, in spectroscopic applications (fluorescence, Raman, etc.) photon noise will be a fundamental limit in the speed of imaging. Photon tunneling in combination with force microscopy allows routine scanning with a high optical lateral resolution. However, interference effects can be dominant on surfaces which display extensive scattering. As such, the application potential of PSTM to biological surfaces is rather limited. Clearly, the virtues of optics, non-invasiveness, high spectral resolution, and high time resolution all apply to the near field optical domain with its high spatial resolution, which adds extensively to the potential of scanning probe microscopy.