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Standing-Wave and Interference Techniques

Standing-wave and interference (InM) techniques employ axially structured illumination to spatially modulate the excitation light in a widefield instrument configuration. In standing-wave microscopy, the excitation light consists of two counter-propagating, non-focused laser beams that interfere to form a standing wave, which creates an excitation intensity that varies rapidly in the axial direction. The interference techniques use opposed objectives and recombine signals into the same light path for detection. Due to the fact that the two light paths are equal in length, interference of the signals generates an interference pattern. Excitation patterns for interference microscopy often contain nodes and anti-nodes within the focal plane where the beams are constructively interfering.

Egner, A. and Hell, S. W.

Fluorescence microscopy with super-resolved optical sections.  Trends in Cell Biology 15: 207-215 (2005).  An excellent review of the principles and applications surrounding an emerging family of fluorescence techniques, including 4Pi instruments, which improve axial resolution by almost an order of magnitude. Noninvasive axial sections of 80 to 160-nanometer thickness deliver improved three-dimensional images of subcellular features.

Gustafsson, M. G. L.

Extended resolution fluorescence microscopy.  Current Opinion in Structural Biology 9: 627-628 (1999).  Dr. Gustafsson discusses concepts of several new and innovative techniques that have been introduced to exceed the diffraction limits of the classical microscope. Included are a synopsis on standing-wave microscopy, 4Pi confocal microscopy, I5M, and structured illumination microscopy.

Gustafsson, M. G. L., Agard, D. A. and Sedat, J. W.

I5M: 3D widefield light microscopy with better than 100 nm axial resolution.  Journal of Microscopy 195: 10-16 (1999).  The inventors of structured illumination microscopy introduce a novel interferometric technique that uses opposed objectives to increase the axial resolution by sevenfold. This report confirms resolution improvement using complex biological specimens.

Bailey, B., Farkas, D. L., Taylor, L. and Lanni, F.

Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation.  Nature 366: 44-48 (1993).  The authors describe a fluorescence microscope configuration in which axial resolution is increased to greater than 50 nanometers using standing-wave excitation. This principle allows for the creation of an excitation field with closely spaced nodes and antinodes that enables optical sectioning at a very high resolution.

Bewersdorf, J., Schmidt, R. and Hell, S. W.

Comparison of I5M and 4Pi-microscopy.  Journal of Microscopy 222: 105-117 (2006).  A comprehensive experimental comparison of imaging using these complementary standing-wave superresolution techniques. The authors contrast differences in the optical transfer functions and data analysis to determine the effects of sidelobe artifacts on image reconstruction.

Blanca, C. M., Bewersdorf, J. and Hell, S. W.

Single sharp spot in fluorescence microscopy of two opposing lenses.  Applied Physics Letters 79: 2321-2323 (2001).  Professor Hell and associates demonstrate theoretically, experimentally, and in an imaging application the possibility of generating a single sharp diffraction maximum in the effective point-spread function of a fluorescence microscope that uses two opposing lenses.

Gustafsson, M. G. L., Agard, D. A. and Sedat, J. W.

Sevenfold improvement of axial resolution in 3D wide-field microscopy using two objective lenses.  Proceedings of SPIE 2412: 147-155 (1995).  One of the first reports on I5M microscopy using opposed objectives in a widefield configuration to achieve substantially improved axial resolution using lasers and arc-discharge lamps.

Lee, S. and Gweon, D.

Improvement of the axial resolution in confocal microscopy by the use of heterodyne interference.  Measurement Science and Technology 19: 105502-9 (2008).  A technique for improving the axial resolution of confocal microscopy is proposed and demonstrated in this article. The authors show that by creating a region of illumination generated by heterodyne interference, the point-spread function (PSF) of the confocal microscope is made sharper.

Nagorni, M. and Hell, S. W.

Coherent use of opposing lenses for axial resolution increase. II. Power and limitation of nonlinear image restoration.  Journal of the Optical Society of America A 18: 49-54 (2001).  An analysis of the ability of nonlinear image restoration to remove interference artifacts in microscopes that employ opposed objectives for imaging. The authors calculated images produced by confocal, standing-wave, incoherent illumination interference image interference, and 4Pi confocal microscopy.

Shao, L., Isaac, B., Uzawa, S., Agard, D. A., Sedat, J. W. and Gustafsson, M. G. L.

I5S: Wide-field light microscopy with 100-nm-scale resolution in three dimensions.  Biophysical Journal 94: 4971-4983 (2008).  The authors describe an innovative instrument that is capable of 100-nanometer spatial resolution in both the lateral and axial directions. Experimental images of biological specimens confirm that near-isotropic resolution can be achieved by using structured illumination in a microscope that has two opposing objective lenses.