A Confocal Microscope with Spectrophotometry Detection

Abstract

The Leica TCS SP Confocal Microscope combines spectrophotometric detection with confocal microcopy. The result is a multi-channel confocal imaging spectrophotometer that significantly increases the flexibility and efficiency of the detection system.In conventional point scanning confocal microscopes excitation light from laser(s) is delivered to the scan head via fiber optic, passed through a pinhole, reflected by a primary dichroic to the scanning system, and scanned onto the surface of the specimen. Light emitted from the specimen is descanned and passed to the detection system. The detection pinhole(s) are placed either between the primary dichroics and the detection system as in Leica confocal microscopes or closer to the end of the detection system. In the detection system emitted light is separated using a combination of dichroics, mirrors, and barrier filters before being passed to the photomultiplier detectors.The Leica TCS SP Spectral Confocal microscope has the same beam path as the filtered system up to and including the detection pinhole. The detection system including the secondary dichroics and the barrier filters is replaced by the spectrophotometer detection system (SP). The light emitted from the focal plane that passes through the detection pinhole has both intensity and spectral information. In the SP system this light is passed through a prism. The prism splits the emitted light into a spectrum from 400 to 750 nm. The spectrum is directed at a PMT for detection. The physical dimension of this spectrum is such that the entire spectrum can be imaged onto the window of the PMT.