Microscopic spectral imaging using a video camera

Abstract

A technique is described which permits the simultaneous acquisition of multiple fluorescent emission and/or absorption spectra from discrete regions of a specimen under microscopic observation. The instrument consists of a modified inverted microscope, an optical diffraction grating, a silicon intensified target (SIT) camera, and a digital video image processor. Observation of the zero diffraction order of the grating with the SIT camera permits an optimal slice of the specimen to be selected by positioning the region of interest over the entrance slit of the grating housing. To obtain the spectral characteristics of this optical slice, the grating is rotated to impinge the first order diffraction on the camera. The video image of this first order diffraction maintains spatial integrity along the slit''s long axis and provides spectral dispersion on the perpendicular axis. Thus, each of the horizontal video lines along the long axis of the slit represents a spectral analysis of the corresponding spatial location within the specimen. The spectral resolution (0.2 nm/channel) of each video line is determined by the resolution of the camera system in conjunction with the resolution of the grating. The image processing system acquires and processes all 500 spectra in 33 ms and permits the accurate localization of the source of each spectrum in the slice. This type of topological spectral analysis permits the determination of both spatial and spectral characteristics of intrinsic and extrinsic chromophores within the specimen. In addition, this technique permits the detection of and the possible correction for photobleaching, light scattering and image plane effects. The application of this technique to the study of single cells is discussed and an example of the technique in determining the fluorescence spectra of acridine orange within the nucleus of an intact mammalian blastocyst is described.