Abstract
A new method for high-resolution imaging, near-field scanning optical microscopy (NSOM), has been developed. The concepts governing this method are discussed, and the technical challenges encountered in constructing a working NSOM instrument are described. Two distinct methods are presented for the fabrication of well-characterized, highly reproducible, subwavelength apertures. A sample one-dimensional scan is provided and compared to the scanning electron micrograph of a test pattern. From this comparison, a resolution of > 1,500 Å (i.e., ≃λ/3.6) is determined, which represents a significant step towards our eventual goal of 500 Å resolution. Fluorescence has been observed through apertures smaller than 600 Å and signal-to-noise calculations show that fluorescent imaging should be feasible. The application of such imaging is then discussed in reference to specific biological problems. The NSOM method employs nonionizing visible radiation and can be used in air or aqueous environments for nondestructive visualization of functioning biological systems with a resolution comparable to that of scanning electron microscopy.
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- Allen R. D. New observations on cell architecture and dynamics by video-enhanced contrast optical microscopy. Annu Rev Biophys Biophys Chem. 1985;14:265–290. doi: 10.1146/annurev.bb.14.060185.001405. [DOI] [PubMed] [Google Scholar]
- Ash E. A., Nicholls G. Super-resolution aperture scanning microscope. Nature. 1972 Jun 30;237(5357):510–512. doi: 10.1038/237510a0. [DOI] [PubMed] [Google Scholar]
- Barak L. S., Webb W. W. Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts. J Cell Biol. 1981 Sep;90(3):595–604. doi: 10.1083/jcb.90.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fernandez J. M., Neher E., Gomperts B. D. Capacitance measurements reveal stepwise fusion events in degranulating mast cells. 1984 Nov 29-Dec 5Nature. 312(5993):453–455. doi: 10.1038/312453a0. [DOI] [PubMed] [Google Scholar]
- Hui S. W., Parsons D. F. Direct observation of domains in wet lipid bilayers. Science. 1975 Oct 24;190(4212):383–384. doi: 10.1126/science.1179216. [DOI] [PubMed] [Google Scholar]
- Kachar B. Direct visualization of organelle movement along actin filaments dissociated from characean algae. Science. 1985 Mar 15;227(4692):1355–1357. doi: 10.1126/science.4038817. [DOI] [PubMed] [Google Scholar]
- Oi V. T., Glazer A. N., Stryer L. Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J Cell Biol. 1982 Jun;93(3):981–986. doi: 10.1083/jcb.93.3.981. [DOI] [PMC free article] [PubMed] [Google Scholar]