Dark Field Raman Microscopy

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• 作者：Matthew V. Schulmerich ; Rohith Reddy ; Anil K. Kodali ; Laura Jane Elgass ; Krishnarao Tangella ; Rohit Bhargava
• 刊名：Analytical Chemistry
• 出版年：2010
• 出版时间：July 15, 2010
• 年：2010
• 卷：82
• 期：14
• 页码：6273-6280
• 全文大小：454K
• 年卷期：v.82,no.14(July 15, 2010)
• ISSN：1520-6882

文摘

Confocal Raman microscopy is often used for optical sectioning but is problematic when the sample plane of interest has a weak Raman cross-section/signal relative to areas that are out-of-focus. This is especially true for clinical samples in pathology, which consist of a thin tissue (5 μm) sample placed on a thick glass slide. Here, we recognize that the problem is the result of the extent of the illumination at the confocal plane being larger than the size of the sample and propose a dark field illumination scheme to efficiently reject substrate signals. The ability of several optical configurations in rejecting out-of-plane signal is investigated for two model systems: SU-8 photo resist over Teflon and SU-8 photo resist over polystyrene. The proposed reflective dark field approach, in which excitation converged to a focal point slightly above the focal plane of the collection optics, was found to be most effective in recording data from the sample. The proposed approach is validated by the rejection of substrate response (fluorescence) in spectra acquired from 4 μm of breast tissue on a glass microscope slide. The proposed approach is easy to implement on existing confocal systems, has a straightforward optimization in acquiring data, and is not expected to result in loss of lateral resolution in mapping experiments.