A NEW CONFOCAL STIGMATIC SPECTROMETER FOR MICRO-RAMAN AND MICROFLUORESCENCE SPECTRAL IMAGING ANALYSIS - DESIGN AND APPLICATIONS

A new instrument was developed permitting confocal spectral imaging technique to be carried out with a lateral resolution ∼0.3 μm and an axial resolution ∼1.0 μm for specimen areas ranging from 5×5 to 150×150 μm. The modular Raman/fluorescence spectrometer was equipped with a CCD camera, microscope, motorized sample stage, and the confocal entrance chamber. A system of galvanometer controlled mirror scanners equipped the confocal entrance chamber, allowing spectra to be accumulated up to several hundreds of points of sample in parallel, with adjustable spectral and spatial resolution. Stray light rejection property of the Raman spectrometer provides the possibility of the Raman spectral image to be recorded in the low frequency domain. A software was developed to control image accumulation, creation, and treatment. The methods of spectral analysis being applied to a multidimensional set of spectra permit the multiform spectral images to be created. In order to create these images, the different spectral parameters and their combination can be used, namely: intensity of characteristic bands and their ratios, width of spectral bands, and shift of band frequencies. The decomposition algorithms can be applied to two-dimensional (or three-dimensional) images to deconvolute the overlapped spectra of sample components or to analyze the subtle spectral differences. The three-dimensional performance of the instrument was analyzed in the particular examples of microfluorescence study of matrices for piezoelectric elements production, micro-Raman investigation of fluid inclusions in mineral, micro-Raman mapping of polymeric materials, and microfluorescence analysis of drug interaction with living cancer cells. It is shown that the spectrometer is suitable for a wide range of applications in mineralogy, material research, solid state physics, biophysics, cellular biology, and medicine. © 1995 American Institute of Physics.