Depth Profiling with Confocal Raman Microscopy

16 Spectroscopy 19(11) November 2004 art I of this article series (Spectroscopy 19[10], 22–28 [2004]) discussed the distortion of the laser focal volume, but what of the confocal aperture? What is the depth resolution of the detected Raman signal? Initial papers on these topics had an over-simplified treatment of the aperture, concluding that the aperture is remarkably inefficient at improving the depth resolution and that the observed Raman signal originates from a very broad region throughout the laser focus (1, 2). Subsequently, Baldwin and Batchelder (3) presented a more rigorous treatment of the aperture, which showed that while the Raman depth resolution remains degraded, it still is significantly better than the axial blurring of the focused laser beam alone would imply. This explains the monotonic decrease in Raman signal that is obtained in practice on focusing deeper into a transparent sample; the laser profile broadens dramatically but only Raman photons originating within a reduced fraction of the excitation volume can pass through the aperture. Baldwin and Batchelder suggested also that the axial resolution might be improved in some circumstances by choosing a lower numerical aperature (NA) objective. Their treatment still neglected the effects of diffraction, however, and only considered on-axis aberrations.