Correlative RISE Microscopy and 3D Reconstruction

Nanocharacterization of Advanced Functional Materials

  • Correlative RISE Microscopy and 3D Reconstruction - Nanocharacterization of Advanced Functional MaterialsCorrelative RISE Microscopy and 3D Reconstruction - Nanocharacterization of Advanced Functional Materials
  • Correlative RISE Microscopy and 3D Reconstruction - Nanocharacterization of Advanced Functional Materials
  • Fig. 1: Secondary electron image of a periodic array of ’nanocorals’ – corrugated polystyrene templates decorated with 20 nm thick nanogapped gold film; inset: tilted view of one nanocoral.
  • Fig. 2: Outputs of RISE microscopy analysis of a periodic nanocoral array: a) SEM image (mixture of secondary and backscattered electron signal) overlaid with a false color SERS map of the identical area, colors from violet to red correspond to different values of SERS intensity (from low to high, respectively) calculated as the area of 1215 cm-1 peak; b) histogram of the integrated SERS intensities; c) corresponding SERS spectra of 4-MP.
  • Fig. 3: Outputs of 3D FIB-SEM analysis: a) Secondary electron image of the periodic composite nanocoral array; b) secondary electron image of the cross-section (nanocomposites embedded in carbon); c) threshold definition for the identification of gold clusters (orange line); d) 3D visualization of the gold nanocoral in ORS Visual software.

Microscopes, in the classical conception, determine the mutual spatial positions of distinguishable pieces of material at high resolution. The rapid development of correlative microscopy techniques is primarily driven by the requirement to link microtopography of the sample to information on the chemical and phase composition. This article illustrates that the concept can be extended further to correlate structural parameters to functional features.

Introduction

One of the current trends in materials science is to develop composites with well-controlled structure and morphology at nanometer level. Such materials, known as nanocomposites or hybrid materials, reveal unprecedented properties and functions, but also make adequate demands on characterization techniques.

Progress in research of hybrid materials is stimulated by the development of modern electron microscopes offering the possibility to image morphology and topology in high-resolution and provide complementary information about chemical composition or 3D structures of the investigated material. Here we present a comprehensive 3D microscopic analysis of metal-dielectric nanocomposites designed for surface-enhanced Raman scattering (SERS), and demonstrate the contribution of correlative electron microscopy – Raman imaging to the understanding of the structure-function relationship in SERS-active substrates.

Surface-Enhanced Raman Scattering (SERS)

Raman spectroscopy is a fast and reliable spectroscopic technique based on inelastic scattering of light, providing spectral “fingerprints” corresponding to molecular vibrational structure. Inherently low efficiencies of Raman scattering can be compensated by significant enhancement at the surface of metal nanostructures, so-called SERS-active substrates.

The rational design of SERS-active substrates is being increasingly employed to generate substrates with optimized sensitivity, optimized frequencies of localized surface plasmon resonance, substrates for the selective detection of certain molecules, or substrates for highly reproducible SERS measurements. Reproducibility, which is a key issue for both technological and elementary SERS studies, is intrinsically ensured at periodic substrates [1].

In figure 1, the regular array of corrugated mesoscale templates (referred to as ‘nanocorals’) decorated with an ultrathin nanogapped and polycrystalline gold film is shown.

The texture similar to a 2D labyrinth increases the surface area in a specific way, and can be viewed as an open form of pores. Due to its mesoscale dimensions, the corrugated particles behave in solutions like porous materials with all consequences for phase equilibria and analyte separation/adsorption. Compounds at trace concentrations can be preconcentrated by sorption and/or capillary-driven deposition during the dry-out process.

Narrow gaps and holes, sharp metal tips and edges, formed at the nanocoral surface, are believed to be responsible for ‘hot spot’ creation resulting in a huge field amplification. Molecules trapped in hot spots can be detected with substantially increased sensitivity, in the extreme reaching single-molecule level.

RISE Microscopy

A nearly ideal approach to effectively develop functional nanomaterials, such as SERS-active substrates, is to perform in situ analysis and obtain data with structure-function correlation. Recently, a novel imaging technique, termed correlative Raman imaging – scanning electron (RISE) microscopy, has been introduced [2]. The integration of confocal Raman microscope into the traditional focused ion beam (FIB)/SEM system enables to acquire Raman images and electron micrographs of the same area within a single instrument. This configuration is well-suited for studying the structure-activity relationship of SERS-active substrates of high morphological complexity.

Nanocorals were prepared by reactive ion etching of close-packed polystyrene microspheres with diameter of 1500 nm and subsequent deposition of a 20 nm thick metal layer (gold or silver) by magnetron sputtering [3]. The metal surface was functionalized with 4-mercaptopyridine (4-MP). This compound is advantageously used as a Raman reporter due to its stability and (high) intensity of signal originating from strong Raman active modes.
Correlative SERS-SEM measurements were performed in RISE microscope (Tescan, Witec), using a 100× objective to focus the probe beam of 532 nm wavelength to a diffraction-limited spot.

SERS spectra were obtained by the automatic mapping of a 6 µm × 6 µm area with the resolution of 60 × 60 px and integration time of 0.5 s for each spectrum. The 1215 cm-1 band in the measured SERS spectra of 4-MP was selected to represent the SERS intensity. A false-color (SERS intensity) map was generated (in Project Plus software) by integrating the intensity of the selected peak for each pixel. Subsequently, SEM image of the same area was acquired and overlaid with the SERS map (fig. 2a).

The correlative image directly links regions with low SERS activity to the metal layer between nanocorals and reveals large differences between SERS intensities detected at different regions of the mesoscale structure. The histogram of SERS intensities (fig. 2b) quantifies the (relative) contribution of highly enhancing ‘hot spots’ to the total SERS activity of the substrate.

Although direct correlation to SEM images yields relevant information about morphological details of highly SERS-active regions, some of the fundamental structure-function features of nanocorals, such as variations in the thickness of the metal layer or topography of the metal grains, remain hidden in the 2D projection.

3D FIB-SEM Analysis

3D FIB-SEM analysis was performed in Tescan GAIA 3 using automated serial sectioning (FIB milling and SEM imaging of each cross-section) in 3 × 3 × 10 nm resolution. Prior to the milling, a 500 nm thick protective layer of carbon was deposited over the SERS-active substrate using electron beam-induced deposition in order to preserve the finest features of metal nanograins and suppress charging effects, as well as ion implantation and thermal damage to the nanocomposite. The acquired data provided sufficient image contrast for semi-automatic demarcation of the polystyrene/metal boundaries during 3D tomographic reconstruction in ORS Visual software. The step-by-step procedure of the 3D reconstruction is illustrated in figure 3.
The reconstructed 3D model (depicted in fig. 3d) contains information on all morphological parameters governing the plasmonic performance of the nanocoral. Variation in thickness of the metal layer, profiles of the sharp tips and size of the gaps between them can be extracted from each cross-section. Moreover, the model can serve to estimate the total surface area of gold exposed to the analyte prior to SERS measurements, determining the maximum number of molecules adsorbed to the surface.

Conclusion

Current techniques of correlated microscopy follow the trends in multimodal characterization of advanced materials conducted in-situ. They reveal structural, compositional and, integrating Raman spectroscopy, even functional (SERS) features at nanoscale level.

Acknowledgements
The authors gratefully acknowledge the collaboration with the group of A. Kromka (IOP ASCR, v.v.i.) on the preparation of nanocorals. This work was supported by the Grant Agency of the Czech Republic, grant No. P205/13/20110S).

References
[1] Cosmin Farcau, Simion Astilean: Periodically nanostructured substrates for surface enhanced Raman spectroscopy, J. Mol. Struct. 1073, 102–111
(2014) http://dx.doi.org/10.1016/j.molstruc.2014.05.016

[2] Jaroslav Jiruše, Martin Haničinec, Miloslav Havelka, Olaf Hollricher, Wolfram Ibach and Peter Spizig: Integrating focused ion beam–scanning electron microscope with confocal Raman microscope into a single instrument, J. Vac. Sci. Technol. B 32, 06FC03 (2014) http://dx.doi.org/10.1116/1.4897502

[3] Lucie Štolcová, Mária Domonkos, Tibor Izak, Jan Proska, Marek Prochazka, and Alexander Kromka: Plasma Treatment as a Versatile Technique for Preparation of Plasmonic Nanoantenna Arrays, PIERS Proceedings, Stockholm, Sweden, Aug. 12-15, 426–430 (2013)

Authors
Lucie Štolcová
(corresponding author via e-mail request)
Dr. Jan Proška
Czech Technical University in Prague
Faculty of Nuclear Sciences and Physical Engineering
Prague, Czech Republic

Rostislav Váňa
Jiří Dluhoš
Tescan Brno, s.r.o.
Brno, Czech Republic

Contact

Technical University in Prague
Brehocva 7
115 19 Prague 1
Czech Republic

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