Experimental Evaluation of the Depth Resolution of a Raman Microscope

Abstract

Raman microscopy has been attractive because of its ability to characterize materials on a spatial scale commensurate with optical microscopy. Typically the lateral spatial resolution is quoted as determined by the Airy disc[1] which is 1.22λ/NA where λ is the wavelength of the illuminating light, and NA is the numerical aperture which is equal to nsinθ, where n is the index of refraction of the medium (1.0 in the case of air) and  is the angle subtended by the optics. However, the Airy disc description cannot be correct for a Raman microscope. The Airy disc assumes uniform illumination of the focusing optic, and the laser profile is anything but. In addition, in some instruments the Gaussian laser profile is not well matched to the aperture of the focusing objective. At any rate, this article is going to concentrate on the depth resolution of the Raman microscope. Optical calculations for depth resolution of an optical microscope state that the it is proportional to λ/(NA) 2 . The essential point to recognize is that the spatial resolution of any Raman microscope depends on the detection optics as well as the focusing optics. How effectively does the optical system collect the Raman signal excited in the laser focal spot, and reject the signal from the surrounding volume that is illuminated by the laser but not in focus?Then the essential question becomes how to evaluate the depth resolution experimentally. Historically people have used a piece of a polished Si wafer for these tests. But this material was originally chosen more for the repeatability of any measurement of its signal rather than its appropriateness for answering the questions of depth resolution. The problem with using silicon is that when performing a depth profile, as the sample surface is moved away from the focal plane, the laser-illuminated area is increased; the final signal is a convolution of the losses because the laser illuminated area is not passed efficiently through the confocal hole, and the increase in signal because of the increased excitation volume. In addition, the depth of penetration of the laser into the crystal is not necessarily negligible. At 633nm, it will penetrate 3µm, at 785nm 12 µm. In trying to determine a better way to determine the confocal properties of a Raman system microscopic polymer beads were selected. With such a sample, when the sample is defocused, the Raman volume cannot be larger than the volume of the bead. Comparison of depth profiles of 2µm and 0.5µm beads and silicon will be shown to provide insight into the confocal behavior of the Raman microscope. These measurements are done using the 532nm excitation wavelength whose depth of penetration into silicon is about 0.7µm. This avoids the complications of volume effects. Figure 1 shows depth profiles of the 2µm and 0.5µm spheres of polystyrene recorded while varying the confocal hole. As the confocal hole is increased, the signal strength increased because light from more of the bead volume is transmitted. However, the full 360

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