Hyperspectral and filter-based Raman microscopy and imaging: Design and evaluation of practical imaging systems.

摘要

Several hyperspectral and filter-based Raman imaging microscopes were designed and constructed. Microscope performance was evaluated using Raman images of objects with complex or weak spectra.; Filter-based Raman imaging microscopes were built around electronically tunable liquid crystal dual-cell Fabry-Perot interferometers (11-14 cm{dollar}sp{lcub}-1{rcub}{dollar} and 11-13 cm{dollar}sp{lcub}-1{rcub}{dollar} band passes) and used to acquire nearly diffraction limited fluorescence and Raman images. Image acquisition times were reduced to several seconds when using an intensified detector. Raman image contrast through the first-generation filter was limited by its 10{dollar}sp3{dollar} out-of-band rejection while that of the second generation filter was limited by its intrinsic luminescence.; A hyperspectral Raman microscope was constructed around a high efficiency axial transmissive spectrograph. A Powell lens illuminator used in this microscope and designed to generate a uniform line from a Gaussian laser beam overcame limitations of scanning-mirrors, A-O deflectors, and cylindrical optics. Intensity variation along the line was {dollar}pm{dollar}4%. High contrast lateral and axial confocal Raman images were obtained with nearly diffraction-limited spatial resolution.; Factor analysis was applied to large data sets acquired from Raman-monitored serial dilution of saturated sodium dihydrogen phosphate solutions. Spectra obtained from the factor analysis were used to identify dihydrogen phosphate hydrogen-bonded oligomers. Recovered spectra were further used to identify dihydrogen phosphate oligomers formed during freeze concentration of sodium phosphate buffers and allowed visualization of heterogeneities in frozen buffers.; Hyperspectral Raman microscopy combined with factor analysis allowed acquisition of high contrast Raman images from samples with significant luminescence backgrounds and weak Raman signals. An intensified CCD video camera was used with this hyperspectral microscope to monitor diffusion-limited dissolution of potassium nitrate with 68 msec resolution. With this system, 100 x 350 pixel hyperspectral Raman images (35,000 spectra) were acquired in less than 10 sec with 5 cm{dollar}sp{lcub}-1{rcub}{dollar} resolution.; In summary, the hyperspectral Raman microscope performance exceeded that of the described filter-based system. Addition of intensified detection and multivariate analysis significantly expanded the utility of Raman microscopy to "real-world" samples. Filter-based Raman imaging remains limited by available technology.