We describe a holographic microscope with a spatial resolution approaching the diffraction limit. The instrument uses a tiny drop of glycerol as a lens to create the spherically diverging reference illumination necessary for Fourier-transform holography. Measurement of the point-spread function, which is obtained by imaging a knife edge in dark-field illumination, indicates a transverse resolution of 1.4 microm with wavelength lambda = 514.5 nm. Longitudinal resolution is obtained from the holograms by the numerical equivalent of optical sectioning. We describe the method of reconstruction and demonstrate the microscope's capability with selected biological specimens. The instrument offers two unique capabilities: (1) it can collect three-dimensional information in a single pulse of light, avoiding specimen damage and bleaching; and (2) it can record three-dimensional motion pictures from a series of light pulses. The conceptual design is applicable to a broad range of wavelengths and we discuss extension to the x-ray regime.

Ultrahigh-resolution three-dimensional images of a microscopic test object were made with soft x-rays collected with a scanning transmission x-ray microscope. The test object consisted of two different patterns of gold bars on silicon nitride windows that were separated by approximately 5 micrometers. Depth resolution comparable to the transverse resolution was achieved by recording nine two-dimensional images of the object at angles between -50 and +55 degrees with respect to the beam axis. The projections were then combined tomographically to form a three-dimensional image by means of an algorithm using an algebraic reconstruction technique. A transverse resolution of approximately 1000 angstroms was observed. Artifacts in the reconstruction limited the overall depth resolution to approximately 6000 angstroms; however, some features were clearly reconstructed with a depth resolution of approximately 1000 angstroms.

No abstract

Scanning soft x-ray microtomography was used to obtain high-resolution three-dimensional images of a microfabricated test object. Using a special rotation stage mounted on the scanning transmission x-ray microscope at the XlA beamline at the National Synchrotron Light Source, we recorded nine two-dimensional projections of the 3D test object over an angular range of -50" to +55". The x-ray wavelength was 3.6 nm and the radiation dose to the object per projection was approximately 2X 10" Gy. The object consisted of two gold patterns supported on transparent silicon nitride membranes, separated by 4.75 ,um, with lOO-to 300-nm-wide and 65-nm-thick features. We reconstructed a volumetric data set of the test object from the two-dimensional projections using an algebraic reconstruction technique algorithm. Features of the test object were resolved to -100 nm in transverse and longitudinal extent with low artifact in three-dimensional images rendered from the volumetric set. Q 1995 American Institute of Physics.

No abstract