Single-pinhole confocal imaging of sub-resolution sparse objects using experimental point spread function and image restoration

Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Max imum likelihood image restoration algorithm has been used in conjunction wi th a measure of the experimental point spread function for improving the th ree-dimensional imaging of subresolution sparse objects. The experimental p oint-spread-function profiles have been improved by a factor of 1.95 in lat eral direction and 3.75 in axial direction resulting in full-width half max imum (FWHM) values of 91 +/- 11 nm and 160 +/- 26 mn. This amounts to 1.43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilisi ng a compact confocal architecture for localising punctuate fluorescent obj ects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restora tion approach, and, of course, in the stability of the confocal system bein g used. (C) 2000 Wiley-Liss, Inc.