Design and analysis of multi-color confocal microscopy with a wavelength scanning detector

Abstract

Spectral (or multi-color) microscopy has the ability to detect the fluorescent light of biological specimens with a broad range of wavelengths. Currently, the acousto-optic tunable filter (AOTF) is widely used in spectral microscopy as a substitute for a multiple-dichroic mirror to divide excitation and emission signals while maintaining sufficient light efficiency. In addition, systems which utilize an AOTF have a very fast switching speed and high resolution for wavelength selection. In this paper, confocal-spectral microscopy is proposed with a particular spectrometer design with a wavelength-scanning galvano-mirror. This enables the detection of broadband (480-700 nm) fluorescence signals by a single point detector (photomultiplier tube) instead of a CCD pixel array. For this purpose, a number of optical elements were applicably designed. A prism is used to amplify the dispersion angle, and the design of the relay optics matches the signals to the diameter of the wavelength-scanning galvano-mirror. Also, a birefringent material known as calcite is used to offset the displacement error at the image plane depending on the polarization states. The proposed multi-color confocal microscopy with the unique detection body has many advantages in comparison with commercial devices. In terms of the detection method, it can be easily applied to other imaging modalities. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4717679]