A map is worth a thousand words

Vibrational spectroscopy expert Dr Elizabeth Carter tells us how Raman mapping can help examine the what, where and maybe even the why of complex samples.

I am the facility manager of the Vibrational Spectroscopy Core Facility, one of six core facilities at the University of Sydney. The unit provides access to high-end research and services not only to the university community but also to government, not-for-profit organisations (NFPO), industrial and private companies. The VSCF is one of the largest and most advanced facilities of its kind in the Southern Hemisphere.

The Facility contains a range of vibrational spectroscopy (Raman and FTIR) instruments, each with specialised capabilities and sampling accessories that are used for collecting point spectra and producing chemical/biochemical maps and images.

Complementary characterisation of thin films and the surface layers of bulk materials is provided by x-ray photoelectron spectroscopy (XPS) for elemental composition, chemical and electronic states, and ellipsometry to characterise composition, roughness, thickness (depth) crystalline nature, doping concentration, electrical conductivity and other material properties.

One of my main duties is to provide extensive and specialised training to users from a variety of disciplines such as archaeology, agriculture, geology, dentistry, pharmacy, engineering and medicine together with the more traditional sciences of chemistry, physics and biology. This diversity has strongly influenced my research interests, which include analysis of natural glasses (obsidian, tektites, and fulgurites)¹-?, archaeological materials and museum objects (ceramics, textiles, pigments, and manuscripts)?,? and characterisation of healthy and diseased biological cells and tissues?-¹?.

A Renishaw inVia Raman spectrometer was purchased in 2006. The instrument is used for a wide range of experiments from simple point spectroscopy to more sophisticated experiments using a temperature variable stage, a high throughput screening module and a cell incubator to precisely control the environment of live cells during Raman analysis.

The instrument has multiple lasers offering excitation lines ranging from the visible to the near infrared (488, 514, 633, 785 and 830 nm). This provides us with the flexibility to offer the most suitable configuration for researchers experimental requirements.

It can also be coupled to a FEI Quanta 200 3D Scanning Electron Microscope (SEM) to allow for combined Raman/SEM analysis. This is achieved by using an interface fitted to the SEM column to unite the two instruments to allow morphological, elemental, chemical (Raman), physical, and electronic analysis from the same region of a sample in the SEM chamber.

One of the most popular techniques in the facility is Raman mapping.

Mapping is a specialised sampling technique used to not only identify the type of components within a sample but it can produce a false-colour map that illustrates the size, distribution and spatial location of the various components within a sample.

When the instrument was first installed in 2006 the only type of mapping available was point-by-point mapping. This yielded very interesting information but it was a very time consuming experiment as the spectra are collected sequentially point-by-point over a defined region with a user-defined step size in both the x and y directions.

To achieve ‘optimal sampling’ a step size that is equal to the diameter of the laser spot (~1 micron) is required. By increasing the step size, larger sample areas could be investigated but at the cost of a reduced spatial resolution.

Technology has advanced and upgrades to the instrument have provided the facility with rapid mapping capabilities which allows for the collection of maps approximately 200 times faster than the traditional point-by-point mapping. Also, the addition of a high speed encoded stage then provided the instrument with high-resolution capabilities.

The motorised encoded sample stage has a 100nm step size allowing for oversampling whereby the step size is smaller than the laser spot diameter. This is used to generate Raman maps that are more detailed than using the ‘optimal sampling’ methodology.

APPLICATIONS

Fulgurites belong to a group of naturally occurring glasses formed by a number of different terrestrial phenomena such as lightning strikes, volcanic eruptions, and meteorite impacts. Fulgurites form when a lightning bolt hits the surface of sand, soil, or rock and an exchange of a thermal energy occurs, estimated to be as large as 1GJ¹¹. This is accompanied by temperatures momentarily reaching between 18,000 and 39,000K in the air and a minimum of 2,000K in the superheated matrix¹²,¹³.

The silicaceous components of the sand, soil, or rock fuse to form a fragile hollow tube with a diameter of a few centimeters and a length that can vary from centimeters to meters¹?,¹?. The formation of fulgurites is accompanied by mineralogical and sometimes compositional changes, and may record information about the environment in which they were formed.

Initial research involved the collection of Raman spectra from number of regions within an individual fulgurite sample using point spectroscopy³.

Spectral analysis revealed several forms of crystalline quartz, fused silica, polyaromatic hydrocarbons (PAH), anatase, and Raman spectra similar to that from shocked crystalline quartz.

Quartz plays an important role in the field of geosciences, it is an indicator of extraterrestrial impacts, as well as the principal shock barometer in the terrestrial environment. The discovery of the presence of shocked quartz in fulgurites¹? suggests that this material is not exclusive to impacts. Some impacts have been identified almost solely on the presence of shocked quartz¹?.

To locate the shocked quartz regions and to understand the nature and spatial relationship of the various inorganic and organic components the fulgurite sample was then mapped, see Figures 1 and 2¹. Mapping the entire sample allowed numerous regions containing anatase and polyaromatic hydrocarbons to be located, which would have been impossible to achieve using point spectroscopy.

[caption id="attachment_51966" align="alignnone" width="200"] Figure 1: Optical image of a fulgurite from Greensboro, North Carolina, USA.[/caption]

[caption id="attachment_51967" align="alignnone" width="141"] Figure 2: Raman maps illustrating the: a distribution of crystalline quartz (blue) and anatase (magenta) and b position of the quartz band (464cm-1, A1 mode)[/caption]

Combined analysis raman/SEM

Raman spectrometers have been interfaced with a number of alternate technologies to produce an integrated system that enables multiple analyses of the same sample region, under the same conditions, in a single instrument. One such system in our facility is a combined SEM/Raman.

The structural and chemical analyser (SCA) unites the SEM and Raman spectrometer to allow morphological, elemental, chemical, physical, and electronic analysis. A fibreoptic cable delivers the incident laser light to the SCA where it is then directed along a retractable tube, which contains the collection optics. This optical transfer tube is inserted into the SEM vacuum chamber and sits between the objective lens and the sample. A video system and white light illumination unit housed within the interface are used to locate the area of the sample to be analysed.

The Raman scattered light is then returned via fibre–optic cable to the spectrometer for detection. One of the first opportunities to test the versatility of this combined system was part of a multi-disciplinary geoarchaeological investigation of the ‘underwater road’ crossing the Tonle Sap Lake in Cambodia?.

The medieval city of Angkor, Cambodia, is surrounded by many myths.

One such persistent myth, sustained by generations of local Khmer fisherman, is of an ancient road leading from the Angkorian ‘port’ at Phnom Krom across the vast Tonle Sap Lake to the productive rice lands in the Battambang province and the temples situated in the city of Battambang 70km southwest of the capital. This ‘road’ supposedly acted as a critical transportation route during the dry season when lake levels were low.

An exceptional drought in Cambodia (2003-2004) provided the opportunity to finally resolve the issue of Tonle Sap ‘road’. Sub-samples from a 1.366 kg sample were made and analysis was performed using SEM/Raman spectroscopy, energy dispersive X-ray spectroscopy (EDS), particle size and accelerator mass spectrometer (AMS) radiocarbon dating.

Figure 3, illustrates the advantage of using a combined SEM/Raman system with the analyses allowing for in situ identification of the cementing matrix, which was siderite, iron carbonate (FeCO3). The EDS spectra in Figure 4 acquired from sand-sized mineral clasts indicated that there were siderite spherulites ~125-300 ?m in diameter. Collectively the data conclusively demonstrated that the consolidated surface of the Tonle Sap ‘road’ was a naturally occurring siderite concretion, which probably formed approximately 5500 years ago.

[caption id="attachment_51968" align="alignnone" width="200"] Figure 3: Scanning electron microscope (SEM) image of a small sample of the Tonle Lake ‘road’ and corresponding Raman spectra collected from three different areas of the cementing matrix.[/caption]

[caption id="attachment_51969" align="alignnone" width="200"] Figure 4: Energy dispersive X-ray spectroscopy (EDS) spectra collected from similar sample region from where the Raman spectra were measured confirming an elemental composition attributable to siderite.[/caption]

Exploring nature - 3D volume mapping

Recent technological developments now allow for samples to be investigated using 3D volume mapping and our initial efforts have focused on understanding the limitations of the equipment and optimising experimental methodologies for a range of biological, archeological and geological samples. Figure 5 presents a 3D Raman volume map collected from a glioma cell clearly showing the location and size of the cell body, nucleus and lipids within the cell.

[caption id="attachment_51970" align="alignnone" width="200"] Figure 5: Section from a 3D Raman volume map of a glioma cell.[/caption]

Our most recent system upgrade was the purchase of a cell incubator this year which will be used to control the environment of live cells during Raman analysis. A large component of research in the facility is within the area of biospectroscopy. By studying the changes in the biochemical content and distribution between healthy and diseased cells and tissues significant new insights into the biochemical mechanisms of disease pathogenesis can be made.

One of the major issues in biospectroscopy is the understanding of the effect that sampling has on distorting the biochemistry of the system.

We have demonstrated using FTIR spectroscopy and PIXE elemental mapping that amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl- and K+, leach from tissue cross-sections into the aqueous fixative medium during formalin fixation and significant lipid peroxidation/oxidation can also occur¹?. Therefore, for cell studies, it is optimal to conduct experiments directly on live cells under culture conditions.

This requires a non-destructive technique capable of rapid acquisition times. Our 2D and 3D mapping capabilities, coupled with the live cell incubator will allow us to map an entire cell that will illustrate the biomolecule content. This accessory will enable researchers to study time dependent processes, such as sugar metabolism, the effects of oxidative stress or drugs.

Raman mapping is no longer the new kid on the spectroscopic block – in fact, these days mapping is probably regarded to be more of a standard sampling technique than a specialised one. However, technology just keeps growing and evolving to push limits and challenge spectroscopists.

It is an exciting time to be in science – sometimes it is overwhelming and daunting trying to keep on top of such a rapidly changing technique and the amount of data and its subsequent meaningful analysis.

Author

Dr Elizabeth Carter is Facility Manager of  the Vibrational Spectroscopy Core Facility at the University of Sydney

References

1 E. Carter, M. Pasek, T. Smith, T. Kee, P. Hines, H. Edwards, 397 (2010) 2647-2658. 2 E.A. Carter, M.D. Hargreaves, N. Kononenko, I. Graham, H.G.M. Edwards, B. Swarbrick, R. Torrence, Vib. Spec., 50 (2009) 116-124. 3 E.A. Carter, M.D. Hargreaves, T.P. Kee, M.A. Pasek, H.G.M. Edwards, Phil. Trans. R. Soc. A, 368 (2010) 3087-3097. 4 S.J. Kelloway, N. Kononenko, R. Torrence, E.A. Carter, Vib. Spectrosc., 53 (2010) 88-96. 5 C. Pottier, D. Penny, M. Hendrickson, E.A. Carter, 39 (2012) 2604-2611. 6 H.G.M. Edwards, J. Montgomery, N.D. Melton, M.D. Hargreaves, A.S. Wilson, E.A. Carter, 41 (2010) 1243-1246. 7 J.B. Aitken, E.A. Carter, H. Eastgate, M.J. Hackett, H.H. Harris, A. Levina, Y.-C. Lee, C.-I. Chen, B. Lai, S. Vogt, P.A. Lay, 79 (2009) 176-184. 8 E.A. Carter, C.P. Marshall, M.H.M. Ali, R. Ganendren, T.C. Sorrell, L. Wright, P.A. Lay, in: New Approaches in Biomedical Spectroscopy,Vol. 963, Chap. 6, ACS, 2007. 9 K.L. Munro, K.R. Bambery, E.A. Carter, L. Puskar, M.J. Tobin, B.R. Wood, C.T. Dillon, 53 (2010) 39-44. 10 J.Z. Zhang, N.S. Bryce, R. Siegele, E.A. Carter, D. Paterson, J.M.D. de, D.L. Howard, C.G. Ryan, T.W. Hambley, 4 (2012) 1072-1080. 11 C. Price, J. Penner, M. Prather, J. Geophys. Res., 102 (1997) 5943-5951. 12 E.P. Krider, G.A. Dawson, J. Geophys. Res., 73 (1968) 3335-3339. 13 M.A. Uman, J. Atmos. Terr. Phys., 26 (1964) 123-128. 14 K. Pye, J. Sediment. Res., 52 (1982) 991-998. 15 E.J. Essene, D.C. Fisher, Science, 234 (1986) 189-193. 16 J. Garcia-Guinea, M. Furio, M. Fernandez-Hernan, M.A. Bustillo, E. Crespo-Feo, V. Correcher, L. Sanchez-Munoz, E. Matesanz, in: Mainz (Germany), 2009, pp 34-35. 17 A.K. Clymer, D.M. Bice, A. Montanari, Geology, 24 (1996) 483-486. 18 M.J. Hackett, J.A. McQuillan, F. El-Assaad, J.B. Aitken, A. Levina, D.D. Cohen, R. Siegele, E.A. Carter, G.E. Grau, N.H. Hunt, P.A. Lay, (2011).