© (2006) OSA

Holographic microscopy has now been upgraded to subdiffraction-limit imaging. Although such super resolution can already be achieved in two dimensions, using a series of 2D images to generate information in three dimensions can be extremely time-consuming. Scanning holography — in which a phase-encoding excitation pattern is scanned over a 2D raster — provides full 3D information, but the resolution of current holographic techniques is not as competitive as their 2D counterparts. With a simple modification to the standard scanning holography setup, G. Indebetouw et al.1 were able to double the resolution possible.

In conventional scanning holography, the hologram is reconstructed from the interference between a spherical and a plane wave in the entrance pupil objective. The cutoff of the spatial-frequency spectrum, or Rayleigh limit, which determines the hologram resolution, is determined by the numerical aperture of the objective and the wavelength of the radiation. However, by combining several off-axis holograms with different spatial-frequency shifts it is possible to construct an image with a resolution beyond this apparent limit. To realize this mechanism, the team introduced a wedge prism into the scanning holographic microscopy setup. This prism could be rotated to cover various areas of the object’s spatial-frequency spectrum. Preliminary experimental results for an objective with a theoretical Rayleigh limit of 0.9 μm showed that a resolution of 0.6 μm could be achieved. The researchers then combined three off-axis holograms of fluorescent beads and found a transverse resolution gain of nearly a factor of two. With more than three holograms, the resolution could be improved even further.