CONFOCAL 3-DIMENSIONAL SCANNING LASER RAMAN-SERS-FLUORESCENCE MICROPROBE - SPECTRAL IMAGING AND HIGH-RESOLUTION APPLICATIONS

A multi-purpose instrument which allows the recording of confocal micr o-Raman, micro-SERS and microfluorescence spectral images of sample ar eas from 5 x 5 to 150 x 150 mum with a lateral resolution of ca. 0.3 m um and an axial resolution of ca. 1 mum was developed. The instrument is a combination of two spectrometers both coupled to the same microsc ope, motorized sample stage, confocal entrance chamber, macro-sample c hamber and CCD detector. The first spectrometer includes a double mono chromator coupled with a spectrograph and exhibits the properties of a typical high-resolution Raman instrument permitting measurements of t he low-wavenumber regions of the spectra. The second spectrometer incl udes a Notch filter and a spectrograph equipped with two interchangeab le low-dispersion gratings and exhibits the properties of a high-lumin osity spectrometer, suitable for low resolution, over a wide spectral range and highly sensitive micro-Raman and microfluorescence measureme nts. The choice of spectrometer most suitable for a particular applica tion can be made automatically without additional prealignment of the system. The system of optical scanners operating in the confocal mode and two-dimensional CCD detection allow the accumulation of spectra fr om hundreds of points of the sample under the microscope simultaneousl y. A computer-controlled scanning sample stage and a system using a 's canning line' of the laser beam allow fast recording of well resolved confocal spectral images (CSI) without sample degradation. Conventiona l images of species could be recorded with a TV-CCD camera through the microscope optics. The software supports all stages of CSI recording and allows the combined treatment of conventional and spectral images including their spatial calibration and conversion of spectral image i nto a conventional image, to assign point-by-point the spectral data t o the conventional image. The applicability of the instrumentation and techniques to the study of polymeric materials, industrial samples an d fluid inclusions in minerals was demonstrated. Spectral images based on the micro-SERS analysis of an antitumour drug adsorbed on the hydr osol were recorded and are discussed in terms of their application to the micro-SERS imaging studies of living cells.