With the aim to perform spectroscopic studies and spectral images inside living cells, a microspectrofluorometer has been designed for two-dimensional spectral imaging in the visible and in the near-UV region. The main advantage of the device relies on its ability to scan the laser beam along one direction of the sample. This scanning is optically coupled with one direction of a bidimensional detector, allowing an instantaneous recording of a one-dimensional spectral image. The overall scanning of the sample is achieved by means of submicrometric displacements of the stage in the perpendicular direction. The main characteristics and performances of the microspectrofluorometer in terms of sensitivity (detection of a few molecules), spatial resolution (0.5 x 0.5 x 1 microm), and spectral resolution (1 nm) are presented. Finally, applications of this new apparatus concerning in situ localization and spectral characterization of two dyes are shown with Drosophila salivary glands (ethidium bromide) and T47D tumor cells (Hoechst 33342).

Confocal Raman spectral imaging is discussed in terms of its application to studies within living cells. The main difficulty which arises, keeping in the mind that cell viability should be respected, is the low sensitivity of Raman spectroscopy. We describe here some possible solutions to this problem: optimisation of the instrumental conditions as well as use of surface-enhanced Raman scattering (SERS). SERS imaging is dependent on topology of the metal surface which is necessary to observe this effect. The advantages and limitations of different types of the SERS-active substrates when used for confocal Raman imaging of cells are analysed using examples. We describe here the procedures and precautions we took when investigating metal-cell interfaces. The confocal Raman-SERS images presented demonstrate the applicability of SERS technique to selective analysis of intracellular distribution and molecular interactions of antitumor drugs.

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.

With classical fluorescence microscopes, the sample is illuminated by a monochromatic source and the image is recorded through a bandpass filter that selects a portion of the emitted fluorescence. Recently, the spatial resolution and sensitivity of these devices have been considerably increased with the introduction of confocality and large 2D CCD array detectors and it is now possible to perform even 3D mapping of fluorescent markers in single living cells. Unfortunately, small changes occurring in fluorescence spectra cannot be mapped so easily. A set of colored filters is hardly enough even to separate slightly overlapping fluorescence contributions from multiple marking. The number of biological studies requiring a detailed description of fluorescence spectrum is growing continuously. Spectral changes are induced, for example, by environment (hydrophilic/hydrophobic binding), pH, oxygenation, ion concentration (Ca, Mg, Na), conformation of bound macromolecule (A/B DNA, protein folding), metabolism, dimerization of the probe, etc. . . . The classical approach to these problems is to couple a spectrograph to a microscope and obtain, point after point, a set of significative fluorescence spectra from the sample. This is a long procedure which gives incomplete information. We report in this paper on two methods we have developed to quickly record an array of spectra over the sample and to map spectral features such as bandwidth, maxima shifts, or decomposition in multiple overlapping components. We distinguish between the confocal scanning method and the global illumination (non confocal) method.

A multi-purpose instrument which allows the recording of confocal micro-Raman, micro-SERS and microfluorescence spectral images of sample areas from 5 x 5 to 150 x 150 mum with a lateral resolution of ca. 0.3 mum and an axial resolution of ca. 1 mum was developed. The instrument is a combination of two spectrometers both coupled to the same microscope, motorized sample stage, confocal entrance chamber, macro-sample chamber and CCD detector. The first spectrometer includes a double monochromator coupled with a spectrograph and exhibits the properties of a typical high-resolution Raman instrument permitting measurements of the low-wavenumber regions of the spectra. The second spectrometer includes a Notch filter and a spectrograph equipped with two interchangeable low-dispersion gratings and exhibits the properties of a high-luminosity spectrometer, suitable for low resolution, over a wide spectral range and highly sensitive micro-Raman and microfluorescence measurements. The choice of spectrometer most suitable for a particular application 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 from hundreds of points of the sample under the microscope simultaneously. A computer-controlled scanning sample stage and a system using a 'scanning line' of the laser beam allow fast recording of well resolved confocal spectral images (CSI) without sample degradation. Conventional 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 into a conventional image, to assign point-by-point the spectral data to the conventional image. The applicability of the instrumentation and techniques to the study of polymeric materials, industrial samples and fluid inclusions in minerals was demonstrated. Spectral images based on the micro-SERS analysis of an antitumour drug adsorbed on the hydrosol were recorded and are discussed in terms of their application to the micro-SERS imaging studies of living cells.