Control of Out-of-Focus Light Intensity in Confocal Raman Microscopy Using Optical Preprocessing

Abstract

Optical preprocessing, the manipulation of imaging-forming light prior to detection, is used in many forms of microscopy to enhance image information and interpretation. The rejection of out-of-focus light by a pinhole in confocal microscopy is an example of optical preprocessing, where spatial filtering is carried out in a plane conjugate to the object focal plane. The rejection efficiency afforded by this arrangement is, however, sometimes insufficient. Insufficient rejection of out-of-focus light intensity can lead to incorrect interpretations of confocal Raman depth and line maps. An alternative approach is to reject out-of-focus light by implementing spatial filtering in one of the several planes conjugate to the pupil plane. This paper shows that mapping enhanced by structured pupils (MESP) provides substantial additional rejection of out-of-focus intensity relative to traditional confocal microscopy, yielding a potential solution to the problem of misleading Raman maps. In addition, lossless MESP is proposed, wherein simple phase masks simultaneously direct in-focus and out-of-focus light intensity transmitted by confocal optics to different regions of a charge-coupled device (CCD) detector.