Microscope: Raman Confocal

Raman confocal microscope

EMSL's Raman confocal microscope is located outside of the EMSL building in a radiological buffer area and can accommodate higher levels of activity than currently allowed within the EMSL building. Onsite users will be required to take Radiological Worker I training to access the Raman spectrometer. Users also have the option of sending samples to the scientific consultant for remote analysis.

The Raman spectrometer is a Dilor XY 800 which is a high-resolution, modular triple spectrometer that can be operated in high-resolution or high throughput modes, with both bulk sample and confocal microscopic capability. This Raman instrument is a research grade triple spectrometer designed with a high degree of flexibility for bulk solid and liquid samples and as well as confocal microscopy for spatially resolved analysis. As a triple spectrometer in subtractive mode with high density gratings it can collect Raman spectra to within 5 cm^-1 of the exciting line as well as stokes-antistokes measurements with a resolution of 0.3 cm^-1. In triple additive mode, the resolution increases to 0.10 cm^-1. As a single spectrometer with a notch filter it can collect data on weakly scattering samples in high-throughput mode. Detection in these configurations is provided by a 800 x 2000 pixel, back-thinned, liquid nitrogen cooled Charge Coupled Device (CCD) detector. The CCD detector has a read-out rate of 0.5 second, as a result some kinetic measurements are possible.

It is anticipated that the greatest demand from EMSL Users will be for confocal Raman microscopy. Confocal microscopy has made significant impact in almost every field of physical and chemical science. In the field of geosciences, this technology allows extremely accurate spatial resolution in the x-y plane as well as depth profiling without distorting spectral resolution. This technology is accomplished by the use of an adjustable pinhole in the scattered light path which limits the contribution of scattered light above and below exact plane of focus. This can be important in when identifying underlying minerals in petrographic thin sections or secondary mineralization in vein fillings. Depth profiling capability is also of critical importance for the analysis of radiological samples since the containment required for these samples often adds an interfering background to the acquired spectrum. The ability to probe "beneath" the containment is of great importance in the analysis of amorphous samples or other weak Raman scatters.

Optical micrograph (100x objective) of the reaction front during thermal decomposition of NH3BH3 at 90C. The numbers refer to the approximate positions of Raman spectra shown in the next figure.

The confocal microscope is equipped with 10X, 50X, and 100X long working distance objectives and also has line-scanning capability which rasters the focused laser beam across the sample in the x-dimension. Operating in this mode, the scattered light is then mapped on to the vertical dimension of the CCD detector providing simultaneous spatially resolved Raman spectra. In this case, the exact sample location where individual spectra are collected is specified using a live video image of the sample and a graphical user interface. This capability can be used for detecting stress gradients or compositional differences at interfaces. The line-scanning function can also be used to reduce the thermal load of the focused laser on highly absorbing homogeneous samples without resorting to reducing the laser power.

The Raman microscope is also outfitted with a computer controlled Linkam THMS600 stage for temperature-dependent Raman measurements between 100 K and 898 K ± 0.1 K. Excitation for Raman spectra is currently provided by a 100 mW, 532 nm CW diode laser and a 30 mW, 632 nm HeNe laser. While both lasers are low powered by most standards, they are adequate for most samples especially if excited under the microscope.

The Raman spectroscopic capability enables reseach in three areas: (1) spatially resolved mineralogic phase identification in heterogeneous samples or on mineral interfaces, (2) microbial mineral surface interactions, and (3) the analysis of radiological samples. On going research in the area of minerologic phase identification is currently supported by advanced time-resolved fluorescence and FTIR resources. Confocal Raman microscopy will compliment these techniques. In particular, Raman analysis can be particularly helpful for identifying phases where the fluorescence spectrum is not distinctive. Since both capabilities have temperature stages, analogous measurements as a function of temperature are possible.

Raman spectra recorded at the position indicated by the numbers in the previous figure (top=1, bottom=5).

Current research in the area of microbial interaction on mineral surfaces is supported by EMSL Bio-AFM imaging resource. Since water is a poor Raman scatterer, spectroscopic probing of cellular membranes of live cells or identification of biogenic mineral phases can be done in the presence of water. These experiments would not be possible using IR spectroscopy because water strongly absorbs at infrared frequencies.

The analysis of radiological samples in the EMSL facility is limited to sealed sources or volumetrically released quantities, which significantly impacts the types of experiments that are possible as well the nuclides that can be investigated. One advantage of the current location of the Raman system in a Radiologic Buffer Area is that significant quantities of radioactive materials and diverse radionuclides can be investigated.