Laser-scan fluorescence microscope with annular excitation optics

Abstract

We discuss the three-dimensional imaging characteristics of a laser-scan fluorescence microscope with an annular pupil in the excitation optics. As is well known, the use of an annular pupil in a conventional incoherent imaging system increases the depth of focus and the lateral resolution as compared with the use of a circular pupil.1,2 However, an annular pupil has not been practically used for fluorescence microscopy because it stops a large amount of the fluorescent light arriving at the objective lens. In the case of a laser-scan fluorescence microscope, we may place an annular pupil in the excitation optics rather than in the imaging optics. In this case, we do not waste fluorescent light because the objective-lens pupil is fully open.