Resolution improvement in optical microscopy by use of multi-beam interferometric illumination

Abstract

In conventional optical microscopy, the numerical aperture (NA) of the objective lens intrinsically links the resolution, depth of field (DOF), working distance (WD) and field of view (FOV). In a diffraction-limited system, changing the NA to improve resolution is not possible without degrading the other optical performances. This linkage, which represents an important limitation in conventional microscopy, has now been broken. In the new method, a target is illuminated by a sequence of finely textured light patterns generated by interference of multiple coherent beams that converge in a cone. The corresponding sequence of brightness values, measured by a single photodetector (e.g., a single pixel of a CCD), encodes the target's sub-pixel contrast pattern. Fourier domain components at spatial frequencies contained in the probing illumination patterns can be recovered from the pixel brightness sequence by solving a set of over-determined linear equations. For a given wavelength, the resolution of the reconstructed image is primarily determined by the NA of the cone of beams, rather than the NA of the microscope objective. A low NA objective, with large DOF, long WD, and large FOV, can be used without compromising resolution. A cone of 31 coherent beams with NA of 0.98 is produced from a single source beam ([lambda] = 488 nm) using an acousto-optic deflector (AOD) and an all-reflective beam delivery system. The target is placed where the beams overlap. Fluoresced light from the target is collected with a 0.2 NA objective for 930 different interference patterns. Brightness sequences are decoded to reconstruct an image with resolution comparable to what would be obtained

using a conventional system with a 0.98 NA objective. Further, changing the NA of the actual objective from 0.2 to 0.1 causes negligible change in resolution, demonstrating that resolution is primarily determined by the illumination produced by the cone of beams. Another restriction of conventional systems is removed by the use of reflective elements. The illumination system, which determines resolution, can be constructed using only reflective elements. Ultraviolet or x-ray illumination can be used with fluorescing targets to obtain resolution beyond what is possible using visible light. The well-known problems of refractive optics at short wavelengths can be avoided.