Optimizing Depth Resolution in Confocal Raman Microscopy: A Comparison of Metallurgical, Dry Corrected, and Oil Immersion Objectives

Abstract

Spherical aberration is probably the most important factor limiting the practical performance of a confocal Raman microscope. This paper suggests some simple samples that can be readily fabricated in any laboratory to test the performance of a confocal Raman microscope under realistic operating conditions (i.e., a deeply buried interface, rather than the often-selected alternative of a bare silicon wafer or a thin film in air). The samples chosen were silicon wafers buried beneath transparent polymeric or glass overlayers, and a polymer laminate buried beneath a cover glass. These samples were used to compare the performance of three types of objectives (metallurgical, oil immersion, and dry corrected) in terms of depth resolution and signal throughput. The oil immersion objective gave the best depth resolution and intensity, followed by a dry corrected (60x, 0.9 numerical aperture) objective. The 100x metallurgical objective was the worst choice, with degradations of approximately 5x and 8x in the depth resolution and signal from a silicon wafer, comparing a bare wafer with one buried under a 150 microm cover glass. In particular, the high signal level obtained makes the immersion objective an attractive choice. Results from the buried laminate were even more impressive; a 30x improvement in spectral contrast was obtained using the oil immersion objective to analyze a thin (19 microm) coating on a PET substrate, buried beneath a 150 microm cover glass, compared with the metallurgical objective.

Raman Imaging of Lignocellulosic Feedstock

“…Therefore, the highest spatial resolution can be achieved with oil immersion objectives with high NA. Also if depth resolution is important, immersion objectives (oil, water) give better results [109]. Generally the axial resolution is around twice the lateral resolution [110].…”

Confocal Raman Microscopy: Performance, Pitfalls, and Best Practice

“…Assuming a truncated Gaussian laser intensity distribution on the microscope objective (I(m) = I o exp(-2m 2 // 2 )), and neglecting diffraction, the intensity along the z-axis is given by Eq. 8: 16,17 IðzÞ=mIðmÞ dm dz…”

“…In an attempt to rectify the last issue, we carried out a detailed comparison of the performance of two typical metallurgical objectives (1003/ 0.9NA, and 503/0.55NA (long working distance)), an oil immersion objective (1003/1.4NA), and two dry objectives with variable correction collars (603/ 0.9NA and 603/0.7NA). 17 This study considered the signal intensity, the depth scale compression, the depth resolution, and the spectral purity attained for (a) a silicon wafer buried under layers of polymer or glass, (b) polymer laminates buried under glass, and (c) free-standing polymer laminates. Figure 11a shows confocal Raman profiles recorded from a bare silicon wafer (1003 dry objective), and the same wafer buried beneath a~150 lm glass cover slip using each of the objectives listed above.…”

“…It was shown that the resolution of the 503 and 603 objectives was improved by using smaller pinholes (100 and 120 lm, respectively), but the improvement was relatively small, between 14-19%, and at the expense of significant loss of signal (43%-69%). 17 Further insight can be obtained by measuring multilayer structures in which each of the layers has a distinctive spectrum. Figure 11b shows spectra recorded from a PE/PET/PE laminate (nominal thickness~19 lm-100 lm-19 lm) mounted beneath a 150 lm glass cover slip, with the laser focused into the uppermost PE layer so as to maximize the PE/PET signal ratio.…”

The Role of ConfocalRaman Spectroscopy in Food Science

“…However, to further complicate matters, there can be exceptions, e.g., deeply buried thin samples can appear broadened. 38 So, one should consider the type of sample one is dealing with, and then try to minimize the refractive differences in the light path. For a sample that is aqueous based or emulsion like, the different possible setups using different objectives are shown in Figure 9.…”