Integrated Raman – FIB – SEM

A Correlative Light and Electron Microscopy Study

  • 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.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.
  • 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.
  • 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].
  • Fig. 3: (A) SEM image of multiple graphene flakes, noticeable by the darker color for multilayers. (B) Raman spectrum measured on the position indicated in A, of a representative location on the graphene flake, the D, G, and 2D bands are indicated. (C, D, E) integrated Raman intensity of the D, G, and 2D graphene bands.
  • Fig. 4: (A) SEM image of FIB patterned silicon. (B) 1st order silicon Raman band analysis. (C, D, E) Intensity, spectral width, and peak position of the 1st order silicon band over the FIB patterned region. (F) Raman band of amorphous silicon, indicated on the 450 cm-1 region. (G) Intensity map of amorphous silicon [3].

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.

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 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.

Chemical Specificity with Electron Microscopy
Integrated Raman and electron microscopy enables the correlative analysis of samples with both chemical specific Raman- and nanometer resolution electron microscopy. Applications demonstrating correlative chemical specificity and high resolution are performed on multiple samples. First a sample containing multiple different crystals is analyzed, showing spectral Raman analysis correlated to particle morphologies observed with SEM. Second a sample of graphene flakes, fabricated through chemical deposition, is investigated for potential contaminations and spectral intensity analysis of the D, G, and 2D Raman bands. Correlative chemical and high resolution microscopic analysis is demonstrated in figure 2, where crystal sub-micron morphological and chemical specific analysis is demonstrated.

The samples contain many different structures two calcium carbonate polymorphisms, calcite and vaterite and calcium sulfate crystals, further the photosynthetic bacteria M. aeruginosa is analyzed. Figure 2A, and B shows the correlative SEM and Raman cluster image from the observed region of interest. Specific structures in the analyzed region are indicated in 2C, and the corresponding spectra are presented in figure 2D, E, and F. Calcium sulfate crystals are identified by their 1006 cm-1 Raman band, and a needle like shape is observed in the SEM analysis, the polymorphisms calcite and vaterite are identified by the Raman band positions at 1086 cm-1 and 1088 cm-1 respectively.

Correlative Raman Electron Microscopy of Graphene
Correlative Raman micro-spectroscopy with electron microscopy is performed on a sample containing graphene flakes (fig. 3). The sample is fabricated with a chemical vapor deposition method on a nickel substrate. The process fabricates single- and multi-layer graphene, with multi-layers visible as darker areas in SEM analysis. The graphene structure quality on nickel is investigated with Raman micro-spectroscopy. The known Raman bands for graphene the 2D, G and D bands are visible in the spectra. The graphene D band is often an indication of disorder in the graphene structure. Performing a Raman microscopic image reveals an overall high quality sample, low D-band intensity, specific the locations with high D-band intensity can potentially be further investigated at higher resolution using correlative electron microscopy. Further the Raman intensity maps of the G- and 2D- band are provided in figure 3D and E, showing increased Raman activity for multi-layer graphene on nickel. The use of Raman spectroscopy for detection of single or multiple layers of graphene and analysis of the thickness uniformity and spatial distribution of the flakes is demonstrated, additional correlative SEM enables the high resolution analysis of interesting sample features or potential defects. Furthermore electron microscopy enables the fast localization of regions of interest (ROI) for chemical specific Raman analysis. Further applications, for example, using correlative Raman analysis after FIB modification of graphene are within reach using the correlative FIB-SEM-Raman microscope [3].

Correlative Raman Analysis with FIB Ablation
Raman spectroscopy is a promising tool for correlative analysis in combination with FIB sample modification. Using the FIB for material ablation enables micromachining of samples, by ablation of sample surface material with a high energy ion beam. This method potentially leaves the sample vulnerable for contamination through redeposition of removed material, and for sample damage, and molecular defects through ion penetration into the sample. Raman analysis of a FIB patterned sample is demonstrated on a silicon waver sample (fig. 4). The FIB is used to pattern an easily recognizable structure, which is subsequently analyzed with Raman microscopy. The analysis reveals changes in the 1st order silicon crystal Raman band on regions where FIB patterning is performed. Rigorous analysis enables the detection of peak shifts and band broadening with 0.1 cm-1 accuracy. Further a broad Raman band at 450 cm-1 is indicative for amorphous and micro-crystalline silicon [4] which is accurately detected after FIB treatment.

The compact commercial Raman microscope is integrated as an add-on module to the FEI Nova Nanolab 600 FIB-SEM. The integration has placed on limitation on operation of the FIB, SEM or Raman microscope, thus FIB modification and SEM or Raman microscopic analysis of large samples e.g. 6 inch wavers is possible. Correlative Raman microscopy in-situ in the vacuum chamber is enabled and demonstrated on multiple samples in combination with both FIB and SEM. The combination of Raman chemical specificity with FIB and SEM, as part of the broader field of iCLEM, promises exciting new opportunities in both biological and materials sciences.

[1] Pascal de Boer, Jacob P. Hoogenboom, and Ben N. G. Giepmans: Correlated light and electron microscopy: ultrastructure lights up! Nat. Methods, 12, 503-513 (2015) DOI 10.1038/nmeth.3400
[2] Frank J. Timmermans, and Cees Otto: Review of integrated correlative light and electron microscopy, Rev. Sci. Instrum. 86, 011501-1-13 (2015) DOI 10.1063/1.4905434
[3] Nick F. W. Thissen, R. H. J. Vervuurt, J. J. L. Mulders, J. W. Weber, W. M. M. Kessels, and A. A. Bol: The effect of residual gas scattering on Ga ion beam patterning of graphene, Appl. Phys. Lett. 107, 213101-1-5 (2015) DOI 10.1063/1.4936334
[4] Thomas Wermelinger and Ralph Spolenak: Correlating Raman peak shifts with phase transformation and defect densities: a comprehensive TEM and Raman study on silicon, J. Raman Spectrosc. 40, 679-686 (2009) DOI 10.1002/jrs.2181

Frank Timmermans
Medical Cell Biophysics group
MIRA institute
University of Twente
Enschede, The Netherlands
Tel.: +31 53 489 4160

Frank Timmermans,*a Barbara Liszka,a Derya Ataç,b Aufried Lenferink,a Henk van Wolferen,c Cees Ottoa

Medical Cell Biophysics group,
MIRA institute
University of Twente,
PO Box 217 7500 AE Enschede
The Netherlands

NanoElectronics group
MESA+ institute
University of Twente, Enschede
The Netherlands

Transducers Science and Technology
MESA+ institute
University of Twente, Enschede
The Netherlands

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