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ELECTRON MICROSCOPY Integrated Raman – FIB – SEM A Correlative Light and Electron Microscopy Study Frank Timmermans 1 ,Barbara Liszka 1 ,Derya Ataç 2 , Aufried Lenferink 1 , Henk van Wolferen 3 , Cees Otto 1 Fig.1: Raman microscope objective integrated in the FIB-SEM vacuum chamber: (Left) Raman microscope objective integrated in the FIB-SEM (FEI NOVA Nanolab 600) vacuum chamber. (Right) Raman microscope added onto the FIB-SEM vacuum chamber. We present an integrated confocal Raman microscope in a FIB - SEM. The integrated system enables correlative chemical specific Raman, and high resolution electron microscopic analysis combined with FIB sample modification on the same sample location. New opportunities in sample analysis using correlative Raman-SEM, and Raman – FIB – SEM are demonstrated on different samples in materials and biological sciences. chamber, with the other components positioned onto and outside the electron microscope, as presented in figure 1. The integration places no limitation on the operation of either the Raman or the FIB-SEM. Figure 1 (left) shows both the Raman objective and integrated 3D XYZ stage, used for sample scanning during optical microscopy. Introduction The field of integrated Correlative Light and Electron Microscopy (iCLEM) has witnessed an enormous growth over the last decade. Different optical microscopes have been integrated in electron microscopes, with the integration performed by both commercial and scientific organizations [1, 2]. In this article a Raman microscope integrated with a focused ion beam (FIB) – scanning electron microscope (SEM) system is presented. The commercial optical Raman microscope, from HybriScan Technologies B.V., is specifically designed for integration in the SEM vacuum chamber. It functions as an add-on module bringing the optical objective into the vacuum Fig. 2: Correlative high resolution SEM (A) and chemical specific Raman (B) analysis of multiple crystals, and crystal polymorphisms. (C) Specific sample structures are identified with SEM, and the corresponding Raman spectra are shown in (D, E, and F). Chemical specific Raman spectroscopy is used for compound identification, showing: calcium sulfate (location 1, 2, 3, 4, 5), the calcium carbonate polymorphism vaterite (location 6), the calcium carbonate polymorphism calcite (7), and multiple fluorescence spectra from the photosynthetic bacteria M. aeruginosa [3]. 34 • G.I.T. Imaging & Microscopy 2/2016
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