Confocal Raman Microscopy: Performance, Pitfalls, and Best Practice

Abstract

C onfocal Raman microscopy is an extremely useful technique that permits nondestructive, spatially resolved measurements deep within transparent samples simply by focusing the laser beam at the point of interest. Moving the laser focus allows generation of one-dimensional (1D) depth profiles, and 2D and 3D (volumetric) images. However, in order to correctly interpret the data, it is important to understand exactly where the laser beam is focused and to know the volumetric resolution of the probe beam. These are actually non-trivial questions. The objective of this article is to summarize the critical factors that determine the spatial accuracy, resolution, and sensitivity of confocal Raman microscopy and to highlight the precautions that should be taken to collect high quality, quantitative data. No attempt is made to review the applications of Raman microscopy; these are simply too diverse, spanning topics from art conservation to medical diagnosis. However, the same basic principles must be adhered to, irrespective of the application, if reliable conclusions are to be drawn. Two main topics are considered. The first is the need for properly corrected objectives for depth profiling beneath the surface of transparent samples. If this is not done, the confocal profile will have an incorrect depth scale, degraded depth resolution, and reduced spectral intensity and signal-to-noise ratio (S/N). Even if modeling is used to account for the aberrations and to compute corrected profiles, degraded depth resolution and S/N still occur, which limits the performance. The second key issue is that even with a corrected objective operating with the best attainable resolution, the axial point spread function, which determines the depth resolution, has quite broad wings, so weak signals can be detected from regions quite distant (tens of micrometers) from the point of tightest focus. With thick transparent samples, the integrated signal from these out-of-focus domains can be significant or even dominant, resulting in unusual and counterintuitive observations. This effect is noticeable both for confocal profiling and for lateral scanning over cross-sections; in short, one cannot simply assume that data is acquired with a volumetric resolution of ~1 lm. The effect is especially important when one needs to chemically interpret the spectra rather than just view an image or a profile, since this leads to contamination of spectra with spurious bands. Finally, it is important to note that while some of the effects discussed here seem strange when they are first encountered, most have been known since the early days of confocal micro-spectroscopy. Consequently, few of the results discussed here would necessarily surprise a skilled microscopist. However, it is clear from the literature over the last decade or so that many Raman microscopists (the author included) are gradually re-learning these lessons and, as a result, significant advances have been made in the acquisition and interpretation of confocal Raman data. It therefore seems appropriate and timely to summarize these learning points in a review article.