We propose a technique for 3-D microscopic imaging with extended depth-of-focus using a novel illumination scheme in a laser scanning optical microscope. The novel illumination scheme creates an effective annular pupil, called the difference-of-Gaussian annular pupil, without the critical drawback of stopping and wasting the light. Two laser beams of different Gaussian pupils with different temporal frequencies are first generated. The laser beams are then combined spatially and used to scan the specimen. The scattered light from the object is picked up by a photodetector whose output consists of a DC and an AC current (due to the optical heterodyning of the two optical beams). The DC signal is no difference from the DC output of a conventional laser scanning microscope with the processing pupil as a Gaussian function, whereas the AC signal is derived from the mixing of the two Gaussian beams and would be given by effectively a Gaussian pupil with a different size than that generated by the DC signal. The AC and the DC signals are then subtracted by electronics and hence the effective pupil function would be given by the difference of the two Gaussian pupil functions. By properly choosing the size of the two Gaussian laser beams, we could realize the difference of the two Gaussian pupils which becomes a new type of annular pupil called the difference-of-Gaussian annular pupil.