By using a numerical aperture (NA) in the microscope, one can achieve a high lateral resolution in the image. However, the improvement of the resolving power results in a depth-of-focus (DOF) reduction. This trade- off problem between the resolving power and the DOF is common to optical microscopes. When the thickness of the specimen exceeds the depth of focus, only a fraction of the total information content of the specimen is imaged in focus or sharply. In addition, super-position on such a image are blur images from out of focal plane contributions. The result is a low resolution and contrast within the image. Improvement in the depth of focus has been of great interest in all areas of imaging, especially in high resolution microscopy. 3-D imaging in microscopy therefore aims to develop techniques that could provide lateral resolution and at the same time with a large depth of field so that a thick specimen could be observed conveniently. Current 3-D imaging techniques involve the use of critical depth- or z-scanning to acquire 3-D information for thick specimens. Annular pupil increases the depth-of-focus and lateral resolution simultaneously as compared with a circular pupil. However, the use of an annular aperture has a major drawback in that it stops and wastes a large amount of light and, therefore, in particularly has not been used for fluorescence microscopes for the purpose of achieving large depth of focus. The objective of this research is to explore 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 without the critical drawback of stopping and wasting the light. Two laser beams of different Gaussian pupils and different temporal frequencies are first generated. The optical beams are them combined spatially and used to scan the specimen. The scattered light from the object is picked up by a photo- detector whose output consists of a DC and an AC current (due to the optical heterodyning or mixing of the two optical beams). The DC signal is no difference from 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 different size. The AC and the DC signals are subtracted subsequently 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. Since no stopping of the light is used to create the novel annular aperture, the proposed system could prove to be more robust and practical than current available methods in 3-D imaging when extended depth-of-focus is required. In addition, the proposed system does not require z-scanning for 3-D imaging which is important for many practical applications.
