Confocal Raman microscopy: Why the depth resolution and spatial accuracy can be much worse than you think

This paper describes continuing studies on the effect of refraction on the depth resolution and spatial accuracy of confocal Raman microscopy. Previous work showed how the apparent position of interfaces, and their breadth, was grossly in error when "optical sectioning" was carried out with a high-power metallurgical objective (the usual configuration for commercial confocal Raman instruments). Simple equations were presented which model these effects given only the numerical aperture of the objective and sample refractive index. This paper extends these studies to the measurement of the position and thickness of buried structures and shows how the refraction theory provides a good basis for interpreting Raman intensity-depth profiles that have been acquired by optical sectioning of complex structures. To summarize the magnitude of the problem, it is typical for the apparent thickness and positional depth of a buried layer to be about half of the true values-hence, correcting for refraction is critical when interpreting intensity-depth profiles. In order to minimize the effects it is possible to use a specialized objective (e.g., an oil immersion objective) to reduce refraction at the sample surface. Data are presented which show that the apparent position and thickness of structures obtained with such an objective are much closer to the actual values, even when the sample index is not perfectly matched by the coupling fluid. The use of immersion objectives is highly recommended if depth profiling by optical sectioning is to be attempted.