The use of fluorescence microscopy for investigating the three-dimensional structure of cells and tissue is of growing importance in cell biology, biophysics and biomedicine. Three-dimensional data are obtained by recording a series of images of the specimen as it is stepped through the focal plane of the microscope. Whether by direct imaging or by confocal scanning, diffraction effects and noise generally limit axial resolution to about 0.5 microns. Here we describe a fluorescence microscope in which axial resolution is increased to better than 0.05 microns by using the principle of standing-wave excitation of fluorescence. Standing waves formed by interference in laser illumination create an excitation field with closely spaced nodes and antinodes, allowing optical sectioning of the specimen at very high resolution. We use this technique to obtain images of actin fibres and filaments in fixed cells, actin single filaments in vitro and myosin II in a living cell.

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Standing-wave fluorescence microscopy (SWFM), a method which utilizes interference to create a periodic excitation pattern along the optical axis, has been shown to provide improved axial resolution in thin, fluorescently labeled specimens. In each plane of focus, a complete standing wave data set is obtained by acquiring an image at each of three distinct positions of the interference fringes. Thicker specimens require through-focus data consisting of three images per plane. In this report we describe the recovery of information from this data using 3-D image processing.The effective optical transfer function (OTF) of the standing wave microscope consists of the conventional OTF and two sidebands which are copies of the conventional OTF shifted axially by the spatial frequency of the interference fringes. The large gaps between the central band and the sidebands lead to significant ringing in the 3-D reconstruction if linear deconvolution methods are employed.The use of non-linear, constrained image processing techniques has been shown to allow accurate extrapolation outside the OTF band limit. We demonstrate the extent to which the sidebands enhance recovery of information in the gaps, and provide a comparison between deconvolution using inverse-filtering and maximum-likelihood estimation. EXPERIMENTAL APPARATUSThe analysis and simulations in this paper complement the optical system and data format of the basic standing-wave microscope in our laboratory. 2 'fl instrument is based on a Zeiss inverted microscope (1M35), originally configured for epifluorescence. Specimens are mounted in an index matching medium between two coverglasses, and illuminated by counterpropagating mutually coherent laser beams of wavelength 0.5 145 micron. Under these conditions, the resulting standing-wave intensity field is periodic along the optical axis, with a period of 0.1715 micron. The "phase" of the standing-wave intensity with respect to the object is controlled by changing the optical path of one of the illumination beams. Images are acquired through one of the objectives at 590 nm with additional magnification of 3.2x onto a Photometrics 512 x 512 cooled CCD camera with 19 micron square elements. The effective transverse sampling in object space is therefore 0.060 micron/pixel. Three-dimensional data sets are obtained by stepping through focus in increments of 0.25 micron and collecting three images: q, r and 5, per focal plane at three phases of standing-wave illumination. The phase of the standing wave is held stationary with respect to the specimen for each q, r and s image set respectively. The relative phase between the q and r sets and between the r and 5 sets is ic/2 radians. The phase of the standing-wave with respect to the in-focus plane of the microscope is not measured. THEORY OF STANDING-WAVE flUORESCENCE MICROSCOPYIn the standing-wave microscope, axial modulation of the illumination acts as a spatial weighting factor on the object o(r). The modulated object is convolved with the point spread function (PSF), p(r...

Axial resolution in fluorescence microscopy can be improved significantly by using standing wave illumination to selectively excite planes within the depth of field of the microscope. When the specimen is thinner than 0. 1 8 .un, an estimate of its three-dimensional structure may be determined from three images within the same focal plane without re-focusing . Thicker objects require a combination of multi-focal-plane data and/or a priori knowledge.