Quorum Technologies Ltd has recently installed a PP3010 Cryo preparation system at the NMI Natural and Medical Sciences Institute in Reutlingen, Germany. The PP3010 was fitted on to their Zeiss 550 Crossbeam to help with the laboratory’s research projects into Materials and Life Sciences.

Quorum Technologies, the market leader in coating and cryo-prep solutions for electron microscopy (EM), is set to launch its latest product range in August. The Q Plus Series is comprised of a range of versatile coaters that provide sputtering and carbon evaporation, with the ability to deliver both in a single combined system. The flagship Q150V Plus offers users the ability to coat samples at pressures in the 10-6 mbar range. The Q150V Plus will make its debut at the Microscopy & Microanalysis (M&M) 2018 show, in Baltimore.

The University of Bristol prepared samples of fossilised skin for SEM imaging as part of the characterisation of a sea dragon skeleton aged more than 200 million years, which was featured in the BBC documentary, Attenborough and the Sea Dragon.

David Attenborough has been fascinated by fossils since he was a boy. It was not therefore surprising to find him down on the Dorset coast of England to film the latest discovery of fellow experienced fossil collector, Chris Moore. The full story is told in a wonderful BBC documentary called Attenborough and the Sea Dragon.

The seas in Jurassic times, around 200 million years ago were ruled by large dolphin-like creatures called ichthyosaurs. Not unique in terms of being found as fossils, the recent discovery in the limestone cliffs west of the village of Lyme Regis turned into a very special find. The story began with the careful removal of the skeleton embedded in the rocks. Back in Moore’s laboratory, it was very carefully cleaned with all rock removed to reveal the bones of the ichthyosaur. However, it was not fully characterised. The final shape and size of the creature, known also as a sea dragon, had to be predicted from various scientific analyses. The skeleton was quite large in terms of 3-4 metres (in multiple pieces). Initial studies were made using computerised tomography at the University of Southampton and the National Veterinary College. The data was analysed at the University of Bristol from which it was possible to construct a complete ichthyosaur. Further work on the bones revealed more details of the life and death of this skeleton.

One very interesting aspect of the characterisation process was made after further removal of rock debris around individual bones of the skeleton. Samples of skin were carefully scraped away and were analysed at the University of Bristol in the Schools of Earth and Biological Sciences. Researcher Fiann Smithwick takes up the story. “Once we had the skin separated, we wanted to image it using scanning electron microscopy (SEM) to find out about the sub-micron features and try to identify components such as pigments as these would indicate how light or dark the skin of the ichthyosaur was 200 million years ago.

However, before we could image the specimen, we had to make it conductive. Fossil samples are normally insulators and are hard to image in an SEM without coating due to a build-up of charge on the surface, which distorts the images. To prevent this, a thin, uniform gold coating was applied to dozens of samples (Quorum Q150R ES rotary pumped sputter coater). We were then able to identify 0.5 micrometre structures called melanosomes.

These are organelles found in animal cells and are the site for synthesis, storage and transport of melanin, the most common light-absorbing pigment found in the animal kingdom. By looking at samples taken from the top and bottom of the ichthyosaur,we could see that it had lots of pigment on the top, but very little on the underside, suggesting that it had a dark back and light belly.

This is a colour pattern called countershading seen today in animals like sharks and dolphins but the first time it has been reported for an ichthyosaur. Countershading is thought to act as camouflage in living animals but may also provide protection against UV light and to help regulate body temperature.” This contrast is well shown here in this cgi reconstruction of the ichthyosaur.

As the final reconstruction of a 3D image of the ichthyosaur was completed, it became clear this particular specimen was a previously unknown species having larger front paddles than others. Overall, the “new” ichthyosaur would have measured nearly 4½ metres. This shows the reconstructed image of the ichthyosaur’s head.1

It is amazing how much can be discovered from a single fossil. Digital reconstruction has enabled the research team at Bristol to build an image of this animal to see it looked and moved 200 million years ago. The SEM imaging revealed countershading for the first time. Science has added to the story to characterise the life of an ichthyosaur, filling a further gap in the palaeontology jigsaw. As Dr Attenborough says, “Fossils give you an extraordinary vivid insight into what the world looked like millions of years before human beings even appeared on this planet.”

The challenge of imaging using scanning electron microscopy (SEM) is the ability to keep samples in their natural state, hence the requirement to employ cryo sample prep and the selection of Quorum’s PP3000T system for the task.

Ir Davy Van de Walle is a senior researcher in the Laboratory of Food Technology & Engineering at the University of Ghent. He works for the head of the laboratory, Professor Koen Dewettinck. Their mission is to design foods on the nano- and microscale, which provide the answer to the consumer demand for new products that are tasty, satisfying, healthy, convenient and inexpensive to produce. Moreover, this may create added value to by-products and contribute to overall socio-economic welfare. Put more simply, they want to understand how ingredient interactions and processing affect the quality attributes (texture, mouthfeel and taste among others) of foods (dairy products, margarines/shortenings and chocolate products in particular).

Mr Van de Walle describes the background and execution of the imaging process in detail: “Because a field emission gun (FEG) is used in high resolution SEM, it needs to be placed in an ultra-high vacuum of 10-7 to 10-8 Pa. Consequently, these extreme vacuum conditions require that the specimen must not release any gas or vapour when inserted into the SEM. Except for powdered foods such as flour, sugar or milk powder, most foods contain water. Scanning electron microscopes at lower vacuum conditions (Low-Vacuum SEM, Variable Pressure SEM and Environmental SEM) can be used to examine wet, oily and non-conductive samples in their natural state. In this way, there is no need to dry or freeze the specimen. This is particularly useful with fragile food samples. However, cryo-SEM is the most appropriate technique in food research because of its extremely high resolution.

The goal of the cryo-SEM is to vitrify the liquid phase with all the constituents, i.e. macromolecules, thus preserving them in their natural and original state. This process is achieved with the use of the Quorum PP3000T cryo sample preparation system. The initial rapid cooling of the sample is the most critical part in the use of the cryo-SEM system. A slush of liquid N2 (-210 °C) is used for the fast freezing to minimize any damage ice crystals may cause. Following, the sample is transferred under vacuum to the cold stage of the preparation chamber, which is mounted on the SEM chamber. The sample can be fractured to expose internal microstructure, etched to reveal greater detail and coated with metal (e.g. Pt) by sputtering. Finally, the sample is transferred into the SEM chamber where it is mounted on a cold stage specifically tailored to the SEM.”

Describing the background to choosing the Quorum sample preparation system, Mr Van de Walle said the process followed a tender procedure involving several FEG-SEM vendors. “Several companies (e.g. Jeol and Hitachi) offered their FEG-SEM in combination with the Quorum preparation chamber. During our meetings with these suppliers, they recommended Quorum as being the best performing. We have been very satisfied with our choice and the group has started to publish papers where the cryo prep system has been invaluable. One example is the PhD research1 of Dr Phuong Diem Tran in which the applicability of cryo-SEM for storage stability testing of pralines is reported. These pralines suffered from fat and sugar bloom. These phenomena are illustrated in the images below. Further work including the fat bloom examples have been published in a paper in the European Journal for Lipid Science Technology.2”

Reference

1.    P D Tran, (2017). Novel strategies to develop filled chocolates for the tropics – PhD thesis, Ghent University, Belgium, 258p.

2.    Controlling the stability of chocolates through the incorporation of soft and hard StOSt-rich fats; P D Tran et al, Eur. J. Lipid Sci. Technol. 2015, 117, 0000–0000, DOI: 10.1002/ejlt.201400584.

Quorum Technologies is pleased to announce the opening of a new demonstration laboratory at Nanjing Agricultural University, China, in association with distributors, Nanjing Tansi Technology Company.

With a wide range of samples to analyse and imaging requirements from the micron to the nanometre level, a reproducible and solid coating system was an essential requirement.