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A Simple Guide For Preparing Samples For Sem Imaging

July 3, 2023Guides & Resources, SEM & Imaging - Guidewebprofits

Scanning electron microscopes (SEMs) are versatile instruments and they can do much more than you would expect. An SEM can provide key information such as structure, morphology and elemental composition about the surface or near-surface region of a sample. For this reason, it has become the tool of choice for several fields from material science to forensics, battery and additive manufacturing and more. Desktop SEMs have now been personalised enabling faster, easier to use, on-site SEM imaging and analysis.

Good sample preparation is a critical step when a high-quality SEM image is needed. Some samples can be quite challenging to image particularly if they are non-conducting. This guide will provide users with a few helpful tips and tricks when preparing samples for imaging. Meant for those who are approaching scanning electron microscopy for the first time, or are relatively new to it, this guide will ensure you obtain good results and get the highest detailed information from your samples. The content is valid for small to larger sample sizes of various compositions. For more detailed information on specific kinds of samples, please contact us.

Every SEM is equipped with a sample holder or a loading chamber where the sample can be inserted.

To load a sample in a SEM, the use of aluminium stubs is recommended. These come in different, standard sizes and are readily available on a commercial basis.

Sample adhesion to the surface of the stub is crucial before placing it in the sample holder or stage. This will prevent pieces of sample being dislodged under vacuum and contaminating the SEM column which can affect the final image quality. It may also damage the SEM imaging system which can be expensive to repair.

TIP 1: Stick the sample securely to the pin stub, by using:

Double-sided carbon sticker
Conductive paint
Conductive tape
Special clamps
A combination of the above.

TIP 2 : Remove all loose particles from your sample after adhering the sample to the pin stub by:

Holding the aluminium stub with tweezers, tilt it by 90° and gently tapping it on its side.
Spraying dry air on the sample.

TIP 3: Use tweezers when handing the pin stub

This should be done in order to prevent contamination.

TIP 4: Make sure that the mounting procedure is solid

This is so that you do not introduce mechanical vibrations due to incorrect mounting.

TIP 5: DO NOT spray dry air in the direction of any electronics

Or a scanning electron microscope, because it might be flammable.

TIP 6: Make sure there is no condensed liquid in your spray air straw

You can do this by first spraying away from your sample.

These precautions will help to reduce the risk of contamination of your system and sample holder and guarantee better performance over time. Below we discuss best practice sample preparation techniques for 5 common sample types which include: Non-conductive samples; Magnetic samples; Beam sensitive samples; Powders and particles and Samples containing moist or outgassing samples.

Non-Conductive samples

When a non-conductive material like a biological sample is imaged, the electrons fired onto the sample surface don’t have a path to the ground potential, causing them to accumulate on the surface. The image will become increasingly bright or entirely white until details are no longer visible. Mild movement can also be detected, caused by the mutual interaction of the electrons. This will cause blurriness in the collected image.

Several solutions are widely used:

Conductive tapes or paints

By covering part of the sample with a piece of conductive tape (e.g. copper tape) or some conductive paint, a bridge to the surface of the aluminum stub is created. SEM image of sugar cube charging. SEM image of sugar cane in low vacuum. This will allow the sample to partially discharge and is enough to image mildly non-conductive samples when imaging areas close to the tape edge.

Low vacuum

Introducing an atmosphere in the sample chamber allows beam interaction with air molecules. Positive ions are generated and attracted by the large number of electrons on the sample

surface. The ions will further interact with the electrons, discharging the sample. While this technique adds some noise to the final image, you can analyse the sample faster and at lower cost without further processing.

Thermo Scientific Phenom Charge Reduction Sample Holder

Designed to eliminate additional sample preparation of non-conductive samples, it allows samples such as paper, polymers, organic materials, ceramics, glass, and coatings to be imaged in their original state. The charge reduction sample holder contains a pressure limiting aperture which allows a controlled amount of air into the sample chamber to raise the pressure around the sample. The leakage rate is designed for optimal charge reduction while maintaining a high vacuum in the column for stable system operation. Compared to standard holders, the charge reduction sample holder can be used to obtain significantly higher magnification images from non-conductive materials.

Sputter coating

By using a sputter coater such as the LUXOR series, it is possible to create a thin layer of a conductive material on the sample surface. This creates a connection between the surface of the aluminum pin and the ground potential. The choice of coating material is strongly dependent on the kind of analysis to be performed on the sample. Gold and platinum are ideal materials for high-resolution images because both have extremely high conductivity. Lighter elements, like carbon, can be used when Energy Dispersive Spectroscopy (EDS) analysis on non-organic samples is required. An alloy of indium oxide and titanium oxide (ITO) can create transparent, conductive layers, to be used on optical glasses to make them suitable for SEM.

However, there are disadvantages to using a sputter coater: Additional instrumentation is required, the analysis becomes more time consuming, and the samples undergo more pumping cycles. Also, any advantage of using a backscatter electron detector (BSD) to image the sample is lost, as the contrast becomes very homogeneous and there is no difference in gray intensity for different elements. The option for EDS analysis for elemental analysis is also lost.

Magnetic samples

Samples that generate a magnetic field can interfere with the accuracy of the electron beam, reshaping it and producing deformed, blurry images, usually elongated along one axis.

This problem is known as stigmation alteration and consists of an increase in the eccentricity (a measure of how circular the curve is) of the beam cross section. Bigger eccentricities are less curved.

Stigmation correction

All SEMs offer the chance to tune the stigmation. Certain instruments require the user to fix stigmation values every time, while others can store standard values that are valid for most samples.

The procedure alters the magnetic field of the lenses, which reshapes the beam. When the shape is circular again, the best image can be produced. When changing the stigmation, it might be necessary to finetune the focus again.

Beam-sensitive samples

Delicate samples, like thin polymeric foils or biological samples, can be damaged by the electron beam due to the heat generated in the interaction area or the rupture of chemical bonds.

This will result in either a hole in the surface or a progressive deformation of the scanned area.

Beam settings

The easiest way to reduce this effect is to use lower values for voltage and current. In these cases, the smallest possible values are recommended.

Sputter coating

In the worst cases, a thin coating layer can be applied to the sample to shield the sensitive surface. Increased conduction will also improve image resolution.

Cooling

Thermal effects can be reduced by using a temperature controlled device. Removing the heat generated by the beam will protect the sample from thermal-induced surface modifications.

Time

Spending a long time on a specific spot will cause damage to the sample, over time. Being quick during the analysis will prevent excessive alterations, but might not produce the best results in terms of image quality.

Magnification

Zooming in implies having the same number of electrons shot on a smaller area. The thermal drift is increased and the deformation effects will become more evident. When possible, low magnification is recommended.

Powders and particles

When imaging particles, information like particle size or shape are important in the design of the process flow. The easiest way to prepare a powder or particles sample is to collect a small amount of sample with a spoon and let it fall on a carbon double-sided sticker, then using spray air to remove the excess particles.

Unfortunately, this method will cause many particles to overlap, hiding important features, or to be blown off, inducing errors in particle counting routines.

Particles disperser

The best way to prepare a powder sample is by using a particle disperser unit such as our Nebula. This will allow an even distribution of the sample on the sticker, reducing the incidence of overlapping particles and generating a pattern that can be used to study granulometry. Operational parameters, such us the vacuum level and the amount of sample needed, depend largely on the nature of the powder. Factors to consider:

Fine powders require a smaller amount of sample.
Delicate samples might break due to strong pressure outburst.
Hydrophilic samples might need a higher vacuum burst to be separated.

Samples containing moist or outgassing samples

When electron microscopes operate in high vacuum levels, every wet sample that is loaded in the imaging chamber will immediately start to outgas.

Certain samples have microstructures that will resist the phase change, providing excellent results without major concerns.

A typical example is a fresh leaf. A sample without a rigid structure can be imaged if force drying or critical point drying is used to prepare it.

Force drying

To verify whether the sample will resist the vacuum, the use of another instrument, such as a desiccator or a sputter coater, is recommended. Eventual changes in the sample should be immediately noticeable.

Critical point drying

Also known as supercritical drying, this technique forces the liquids in the sample to evaporate, maintaining a low temperature. The evaporation is driven by the pressure level, which is broughtbelow the vapor tension of the liquid in the sample. During this process, the liquids will create fractures in the sample, causing modifications in the structure.

Cooling

This is an alternative to drying techniques that will preserve the structure of the sample completely intact by freezing the sample. If the phase change is quick enough, the liquids in the sample will not form crystals and the structure will be perfectly preserved. It is important to consider that the phase change is not permanent and a prolonged exposure to a high vacuum will increase the evaporation rate.

Low vacuum

If the sample does not have a particularly high moisture content, using a small amount of sample at a reduced vacuum level can be enough to collect images. The overall image quality will be lower, but the sample can be imaged in its original state.

Small amount of sample

Using a small quantity of sample is sometimes enough to contain the effects of vacuum and evaporation. The sample can be collected with a toothpick and a veil of it can be deposited on the stub. This technique is particularly effective with gels and emulsions.

Sample preparation is just the beginning for faster and better analysis. Learn how to improve your process even more by speaking with an SEM expert. Contact us today.

Reference: https://www.thermofisher.com/au/en/home/global/forms/industrial/sem-sample-preparation-e-guide.html

3 Factors to Consider for Automated Live-Cell Imaging

May 29, 2023Guides & Resourceswebprofits

Live-cell imaging is important for many applications however limitations of conventional methods have constrained its routine use. Reliable live cell imaging requires an environment that keeps the cells functioning during the experiment while also being able to ensure the experimental method is not perturbing the cells and affecting the interpretation of the results. Here we discuss the growing popularity of automated live-cell imaging systems and highlight some key features to look for when selecting a live cell imaging system.

What is live-cell imaging?

Live-cell imaging is a microscopy-based technique used to examine living cells in real-time. It offers deeper insights into dynamic cellular processes such as migration, confluency and signaling and can reveal findings that might otherwise have been overlooked. Both brightfield and fluorescence-based live-cell imaging modalities support a range of different analysis needs.

How is live-cell imaging used?

Applications of live-cell imaging span basic research through to biopharmaceutical manufacturing. In a research setting, live-cell imaging can be used during cell culture to help define the best time for harvest, determine the senescence status of cells, assess drug treatments for cytotoxicity or detect and monitor phagocytosis. For example, Phagocytosis, is a process by which certain live cells, called phagocytes internalise foreign matter. This defensive reaction against infection is key in the study of immunology and plays an important role in immune responses, tissue homeostasis, and continuous clearance of apoptotic cells. Generally, phagocytotic activity is assayed using flow cytometry. However, this process only provides quantitative data and does not provide the means to monitor phagocytosis in real time. Performing fluorescence-based assays using the CELENA® X High Content Imaging System with pH-sensitive fluorescent particles, like pHrodo™ Green, can be an effective and efficient system for quantifying and monitoring apoptosis activity. For biopharmaceutical manufacturing, live-cell imaging has broad utility for process development and control throughout the production of biologic drugs and vaccines.

Limitations of conventional live-cell imaging methods?

Historically, live-cell imaging has involved manually monitoring cells by culturing them in a CO2 incubator. The culture vessel is removed several times to take images of cells over time using a digital microscope. This approach is labor-intensive and highly prone to human error, largely because it offers no means of finding the same position in the culture vessel. Fluctuating environmental conditions can also cause cellular stresses, which can compromise results. While benchtop imaging systems improve on this method, they are bulky and cumbersome, and often struggle to maintain a stable environment.

3 Factors to consider when choosing an automated live-cell imaging system

Automated live-cell imaging systems like the CELENA® X offer a flexible design that is smaller, faster and easier to use to meet both the demands of the drug discovery industry and the basic research needs of the smaller laboratory.

Multiple imaging modes that are affordable

Live cell imaging systems that offer both brightfield and fluorescence options for either time-lapse or real-time monitoring offer maximum flexibility. Celena X integrates an automated fluorescence microscope with quantitative image analysis software to process large datasets at an affordable cost. Its interface allows a user to run multi-well or multi-spectral experiments with capacity for multi-point imaging with only a few clicks. The microscope provides for a multitude of fluorescence cell imaging possibilities by supporting all objective magnification from 1.25x to 100x, both brightfield and phase contrast illumination, and with LED filter cubes.

Stable scanning performance and compatibility

The system is compatible with a wide range of cell culture vessels such as multi-well plates, dishes, flasks and slides to cover a wide variety of assay types. Depending upon the application, either image-based or laser-based autofocus methods can be used. The CELENA® X can be used for image confluency in McCoy cells seeded in 96-well plates over 48 hours with brightfield image-based autofocusing, demonstrating how the system can be modified and applied as a high throughput method for various cell-based assays. With laser-based autofocusing and multiple filter cubes, this method utilised the CELENA® X to image the dose-dependent effects of anti-cancer drugs throughout the cell cycle in HeLa cells seeded in 96-well plates, demonstrating how the system can be used in a multivariate drug screening process.

User friendly interface and 3D modeling

An analysis of high content images with large datasets can cause problems with most types of analytical software. Each new assay requires the creation of new modules, which can be challenging for lab staff and often involves IT staff. The CELENA® X provides an easy-to-use modular-design analysis software based on a powerful CellProfiler engine. Tens of thousands of images can be analysed automatically to obtain quantitative information with a complex software setting or optimisation.

Three-dimensional (3D) cell models can provide a more accurate representation of real cell environments than 2D cell culture systems but requires a different strategy of imaging and analysis compared to 2D cell culture. Organoids, for example, are organ-specific 3D cell models derived from human stem cells, designed to mimic the functionality and structure of human organs while 3D spheroids can represent a gradient of nutrients and oxygen between cells located in both outer and inner layers which is more relevant to physiological environments. These 3D models are notably useful for studying various types of cancers.

The challenge when imaging organoid and spheroid assays comes from organoids having multiple focal planes making it difficult to acquire in-focused images for multiple organoids. For live/dead cell viability of the single organoid, a different analysis strategy is required since individual cells in an organoid do not exist as a single live/dead status. To address this issue, CELENA X employs MergeFocus software module after acquiring Z-stack images from multi-channel fluorescence.

We invite you to use the Celena X automated live cell imaging system today and compare it for yourself.

ATA Scientific Pty Ltd
+61 2 9541 3500
enquiries@atascientific.com.au
www.atascientific.com.au

Product page link: – https://www.atascientific.com.au/products/celena-x-high-content-auto-cell-imaging-system/

5 Step Guide to Improving the Accuracy of Automated Cell Counting

May 15, 2023Guides & Resourceswebprofits

Cell counting underpins numerous applications, spanning basic research through to the development and production of cell therapies. In recent years, manual cell counting methods have been replaced by the use of automated cell counters, which are both faster and more accurate, especially for complex sample types. Here we discuss 5 factors to consider when choosing an automated cell counter and share useful tips for instrument use to ensure accurate results.

1. Match the instrument specifications to the cell type

While cell-based research has traditionally relied on immortalised cell lines, it is increasingly common for more complex sample types to be used. These include primary cells, peripheral blood cells, stem cells, dissociated tumor cells, and even engineered T cells, which vary in terms of size, shape, and aggregation properties. To improve cell counting accuracy, many users have switched from manual, haemocytometer-based methods to counting cells with automated platforms. However, automated cell counters can only provide accurate results if they feature the right specifications for the cell type in question. Although an automated cell counter equipped with brightfield microscopy optics and a low-magnification objective represents a budget-friendly option for counting well-isolated, homogenous cell lines, an instrument capable of measuring fluorescence is often a better choice for counting clumpy samples or smaller cell types such as peripheral blood mononuclear cells (PBMCs).

2. Determine an appropriate sample dilution range

So, the question is, what factors should you consider when using an automated cell counter? A primary concern is the optimal dilution range, typically provided as cells/mL. This differs among instruments based on the size of the field of view (FOV), the magnification and the image sensor size. For counting accuracy, the sample should be diluted such that it falls within this range—if too dilute, counts will be inaccurate; if too concentrated, the instrument software will struggle to distinguish individual cells.

3. Consider using fluorescence staining for viability measurements

Most automated cell counters allow viability measurements to be performed using trypan blue staining and brightfield imaging. But, while this approach provides accurate results for homogenous samples like cancer cell lines, it is less reliable for more diverse sample types. For example, primary cells are often contaminated with large numbers of red blood cells, which will be mistakenly classified as dead cells after staining with trypan blue. Cellular debris and non-cellular particles can also be misidentified in this way, leading to data being artificially skewed. Fluorescence-based methods such as acridine orange/propidium iodide (AO/PI) staining provide greater accuracy than trypan blue, regardless of cell type, and are fast becoming a preferred method for measuring cell viability.

4. Maximise the counting volume

Once samples have been prepared for counting, they are loaded onto a chamber slide; this functions to provide a fixed volume measurement based on the chamber height. Because the chamber height is set by the slide manufacturer, scanning multiple FOVs is the best way of increasing cell counting volume to achieve greater accuracy and precision. Modern instruments equipped with an automated scanning stage can count volumes of up to 5.1 µL, which is 10-fold higher than a conventional haemocytometer measurement.

5. Optimise the counting protocol

The most common error when counting cells is incorrect focusing, which, for brightfield microscopy, can lead to poor discrimination of live cells from dead cells. If an automated cell counter offers only manual focusing, it is recommended that users obtain a bright spot at the center of each live cell for brightfield imaging to produce an accurate count. Fortunately, most modern automated cell counters feature an autofocusing function, which can minimise focus-related issues when running in brightfield mode. Alternatively, since fluorescence imaging is relatively insensitive to focus, using a fluorescence cell counter with an autofocus function will eliminate this problem.

Another widespread mistake is failure to optimise the cell counting algorithm, also known as the counting protocol. This is proprietary to each automated cell counter and is used for image preprocessing, object finding, and object classification. As it would be impossible for a single counting protocol to cover every cell type, most algorithms allow the end user to perform further optimisation. Factors to consider here include the cell size, spot brightness, cell detection sensitivity, roundness, noise reduction, and fluorescence intensity, all of which should be carefully tailored to the diversity of cell types within the sample.

Introducing the LUNA™ Automated Cell Counter

The LUNA™ automated cell counter family from Logos Biosystems provides fast, accurate counting for a broad range of cell types, including PBMC and CAR T cells. Dilution of highly concentrated samples and replicate counts at low cell concentrations are no longer necessary when using the LUNA series of Automated cell counters.

The LUNA-FX7 is the newest member of the LUNA Automated Cell Counter family that provides unmatched cell counting accuracy, dual fluorescent and brightfield detection, advanced declustering algorithm, precision autofocus and 21 CFR PART 11 compliance. It has built-in quality control features and precise validation slides for monitoring QC and bioprocesses. LUNA-FX7 meets a broad range of counting applications from confirming cell quantities for single-cell sequencing to highly accurate cell therapy dose.

Key benefits:

Larger counting volumes, up to 5.1 µL for high accuracy (1% CV)
High throughput using either 8- or 3- channel slides.
Bioprocess package that can monitor individual and specific batches.
Innovative CountWire™ package for 21 CFR Part 11 compliance.

Higher throughput. Offering a variety of slide options, the LUNA-FX7™ utilises a counting volume of up to 5.1µL, lowering error and CV for each count.

We invite you to use the LUNA-FX7 Automated Cell Counter today and compare it for yourself.

ATA Scientific Pty Ltd
+61 2 9541 3500
enquiries@atascientific.com.au
www.atascientific.com.au

Product page link:
https://www.atascientific.com.au/products/luna-fx7-automated-cell-counter/

The Essential Tools That Are Driving Advanced Additive Manufacturing

September 5, 2022Guides & Resourceswebprofits

Australia’s Additive manufacturing (AM) industry is off and running, transforming the way we produce and distribute goods. Parts which required multiple components to assemble manually can now be produced more viably using AM in a one-step build process. It removes the need for complex shipping arrangements to move instruments from place to place, relying instead on digital files to print products on-demand. In the heat of the COVID-19 pandemic, 3D printing stepped up to become a vital technology to provide solutions to severe disruptions in supply chains ranging from personal protective equipment (PPE) to emergency dwellings to isolate patients. From aerospace to automotive engineering, and from the medical to the dental industry, AM is an evolving technology revolutionising industries across the country.1

Backed by the Australian Government ‘Modern Manufacturing Strategy’ as well as the funding available for the sector, has enabled new manufacturing-focused research facilities that work alongside industry.2 With new opportunities to deliver cutting-edge R&D in AM and materials processing, highly complex or previously unachievable products can be created quickly and efficiently for the global market. CSIRO established Lab22 with a vision to grow a new manufacturing industry as Australia’s Centre for Additive Innovation citing a recent focus on critical mineral and hybrid manufacturing.3 The University of Sydney and GE Additive have also joined forces to collaborate on R&D topics and demonstrate AM technology via the new Sydney Manufacturing Hub.4 These and many other AM research facilities in Australia underpin a growing AM industry helping to build sovereign capabilities. Instead of sending processed ores overseas and importing them back as powders, Australia can forge on finding ways to turn minerals into new AM innovations.

But as exciting as the possibilities are in AM, the process itself is not without its challenges. Problems with final product consistency and a narrow range of expensive raw materials are some of the biggest obstacles to the widespread adoption of AM. In processes that use powder as the raw material, for example, just one particle could contaminate the rest of the material, impacting the overall quality of the end product. To maintain consistent high quality in these components, producers need to ensure that their input materials are carefully monitored and optimised.

Why particle characterisation is critical

Key to developing and manufacturing high quality materials with the required functionality and performance is understanding the relationship between material structure and material properties. From metals and polymers to composites and ceramics, monitoring the particle size and shape is important to ensure the powder supply is consistent and meets specifications. Beyond quality control, it also plays a vital role when investigating novel alloys or composites or developing a new AM process.

Below are two key analytical tools that support additive manufacturers with material characterisation.

The use of laser diffraction for particle size distribution

Particle size distribution is critical for powder bed AM processes since it affects powder bed packing and flowability which in-turn impacts on build quality and final component properties. The Malvern Mastersizer 3000 uses laser diffraction, an established technique for measuring the particle size distribution of metal, ceramic and polymer powders for additive manufacturing, and is employed by powder producers, component manufacturers and machine manufacturers worldwide to qualify and optimise powder properties. A complete high-resolution particle size distribution is provided in a matter of minutes (from 10 nm to 3.5 mm) using either wet or dry dispersion. The technique can also be integrated into a process line to provide real-time particle sizing.5

Automated Image Analysis for particle shape and composition

Powder bed density and powder flowability are influenced by particle size and shape. Particle morphology is therefore, another important metric for powder bed additive manufacturing, with smooth, regular-shaped particles preferable as they can flow and pack more easily than those with a rough surface and irregular shape. The Malvern Morphologi 4-ID provides automated optical image analysis to classify and quantify the size and shape of metal, ceramic and polymer powders. The fully integrated Raman spectrometer also enables component-specific morphological descriptions of chemical species.

The Phenom ParticleX AM is a specialised high-resolution desktop scanning electron microscope (SEM) dedicated to optimising AM metal powders and final product quality. By combining an imaging resolution of <8nm and magnifications up to 200,000x together with X-ray analysis (EDS) for elemental composition, properties such as structural integrity, print resolution, surface uniformity, phases and the presence of impurities or defects can be determined to contribute unique insights not possible with other systems. A scanning area of 100x100mm, grants a large degree of freedom to image and assess the size and shape of whole parts or sections of a larger component simultaneously. This fully integrated system is simple to operate and eliminates the need for outsourcing for quality checks, speeding up time-to-market.

We can go further together

At ATA Scientific, we don’t just sell our instruments – through collaboration with a broad range of industries and academic institutions, we play a key role in the AM ecosystem. We support our customers by providing optimal material characterisation techniques used in AM together with key insights into the application, measurements and analysis to fully understand material behaviour.

ATA Scientific Pty Ltd
+61 2 9541 3500
enquiries@atascientific.com.au
www.atascientific.com.au

References

Additive manufacturing: could it drive global success for Australian businesses?
https://www.materials-talks.com/how-additive-manufacturing-can-take-your-industry-to-new-heights/
Additive manufacturing and critical minerals come together at CSIRO’s Lab22 – CSIRO
https://www.exportfinance.gov.au/resources/article/additive-manufacturing-could-it-drive-global-success-for-australian-businesses
Additive manufacturing and critical minerals come together at CSIRO’s Lab22 – CSIRO
https://www.csiro.au/en/work-with-us/industries/mining-resources/Resourceful-magazine/Issue-27/Lab22-and-critical-minerals
$25M Sydney manufacturing hub launches to drive state wide innovation
https://www.3dprintingmedia.network/25m-sydney-manufacturing-hub-launches-to-drive-statewide-innovation/
Characterising material properties for powder additive manufacturing
https://www.materials-talks.com/characterizing-material-properties-for-powder-additive-manufacturing/

The Importance of RNA Research in Australia

August 5, 2022Guides & Resourceswebprofits

As RNA science continues to make headlines around the world as the new way of making safer, more targeted medicines, it has sparked a wave a new studies that offer significant potential not only as broad-spectrum vaccines but also treatments for cancer, genetic and autoimmune diseases. This is quite extraordinary, given prior to the COVID-19 outbreak, few people outside the RNA research community were aware that this technology even existed. That Australia is still unable to manufacture mRNA vaccines – despite their proven success against the SARS-CoV-2 virus – presents a huge opportunity.

The COVID-19 pandemic brought together world-leading researchers in a mass response from across many fields, diverting resources to deliver a vaccine that could help save millions of lives. Scientists stepped up and worked tirelessly despite the myriad of challenges, the biggest one of which was the challenge of vaccine supply. Recognising this challenge and the need for building local capability, led to a humble yet exceptionally talented scientist from Iceland to step up and run the first RNA institute in Australia [1].

Australia’s RNA capability strengthens as UNSW RNA Institute opens

Professor Pall (Palli) Thordarson, an award-winning researcher and chemistry professor at UNSW Science, is set to kickstart RNA capabilities here in NSW and finally launch our Genetic Medicine Ecosystem. Fuelled by his interest in the interface between chemistry and biology, Palli has always been fascinated by the role of RNA and now, this molecule is the focal point of his work. RNA science seems like an overnight success, but it has been decades in the making and holds huge potential for making critical contributions toward advancing human health. The facility will allow scientists to connect and network with industry partners and other collaborators to meet research and manufacturing needs.

The importance of driving onshore advances in RNA research and therapies

Today, the NSW Government together with 14 universities that constitute the NSW RNA Bioscience Alliance and the dozen research organisations within the NSW RNA Production Research Network are now involved [2]. The goal to create a national Genetic Medicine manufacturing facility in Australia, was captured during an early meeting of the Australian RNA Production Consortium (ARPC). It is inspiring to see so much occurring around the nation knowing each of the original members of the ARPC are tirelessly working to build this, in a fashion that is not simply replicating facilities in each state, but to build a collaborative web of skills and infrastructure. These people have changed the future for scientific research in Australia – thank goodness for this cognisant few!

It seemed puzzling to find a chemistry expert amongst so many RNA giants however, as time passed, it had become clear the sheer value someone like Palli had in such a forum. Palli’s fascination with RNA began to emerge early in his PhD, “I remember looking at RNA and RNA-based systems biology as a PhD student and thinking, ‘These are the most fascinating chemical machines in the world’.” Palli has long tried to connect the dots between the pure chemistry world and the biological sciences and just how that chemical machine results in a biological translation.

As we spend inordinate efforts to educate and develop a constant crop of scientists, from high school to early career researchers, we hear the same things from PhD students today as we did back in the 1980’s and I suspect before- “we can’t find work as we are either too inexperienced or we are over-qualified!” Such enormous investment into this ecosystem can only help resolve much of this.

Whilst there is a great deal of investment in Victoria and some in QLD, there are synergies between the research institutes and many collaborations. The global nature of science has throughout history proven there are no boundaries, no borders and only one tribe – the science tribe – we see beyond nationality – and we travel the planet in a quest for knowledge, going to where the research is. To be part of this global community Australia needs vision such as shown by Palli. Without this, we could never attract the best of the best to come to Australia, nor would we retain our global giants.

Next generation technologies will nurture a wide range of genetic medicines

Being privy to much of the research and seeing technologies you supply have material impact in the future lives of potentially millions of people is inspirational. Consider this technological revolution answers the question we didn’t even know existed – to solve a problem we didn’t know we had. As Palli often states- the missing link to all this manufacturing ecosystem is the humble Lipid Nanoparticle. Now humble should not elicit the thought it is simple, far from it. However, it has been made a great deal simpler by the Micropore Technologies Range [3]. These technologies are unique. They create very small particles, quickly and repeatably. They encapsulate a payload, such as mRNA, a peptide, protein, or small molecule for the delivery to the cell by stealth. Imagine you are a cancer researcher and you have figured out the mechanism of how the cancer cells are replicating and where they stem from. Using a Nobel prize winning technique called CRISPR you edit the RNA – how do you do this? Hitch a ride inside a Lipid Nanoparticle, slip into the tumour and stop it in its tracks! This is elite science happening right here in Sydney with team collaborations across the nation. This is the key – we can do wonderous science that will save lives – this is what drives the scientist. The government sees the billions in revenue and the possibility of jobs, jobs, and jobs. It makes ethical and economic sense!

The benefits of RNA research are far-reaching

For those who assume it is just medical, think again. The Agricultural industry is set to benefit from multi millions in investment with the help of Palli to aid in biosecurity, disease prevention and control – think lumpy skin or foot and mouth disease in cattle. It is hard to see any downside to what Palli has orchestrated. It is visionary and all power to UNSW backing him to run down this path. Nice to see Palli back on the farm.

ATA Scientific collaborates with thousands of scientists throughout Australia and New Zealand providing solutions for scientific challenges. We are constantly adding novel technologies that help pose the obscure questions needed to advance science. Collaborate with us today www.atascientific.com.au

References

1) https://newsroom.unsw.edu.au/news/science-tech/australias-rna-capability-strengthens-unsw-rna-institute-opens Website Accessed 19 July 2022
2) https://newsroom.unsw.edu.au/news/general/unsw-celebrates-119m-funding-support-rna-research-across-14-universities Website accessed 19 July 2022
3) https://microporetech.com/applications/lnps-and-liposomes Website accessed 2nd May 2025

An Introduction to Battery Research and Manufacturing

May 2, 2022Guides & Resourceswebprofits

The need to build new energy storage solutions to address the increasing global demand has helped drive a power revolution in battery research and technology. Lithium-ion (Li-ion) batteries are predicted to play a key role in the trend toward renewable and sustainable industrial electrification solutions. As fossil fuels are phased out and CO2 regulations become more stringent, the increase in demand to provide ever more lightweight, low-cost, safe, high-power and fast-charging batteries has accelerated advances in battery technology.

Access to the right tools and technologies can help optimise R&D and production cycles, investigate causes of battery failure, improve safety, and speed up time-to-market, to keep technological progress moving in sync with modern global demands. Here we discuss a complementary set of physical, chemical, and structural analysis solutions designed to enable rapid, high-precision analysis of particle size and shape distribution plus elemental composition of battery materials for the entire process from research through to production.

With the Mastersizer 3000, particle size can be rapidly analyzed with ease, while the Morphologi 4 can image and classify thousands of particles automatically with high statistical accuracy. Our Zetasizer can analyze the zeta potential of a dispersion and also the size and agglomerate state of nanosized materials. Phenom XL G2 scanning electron microscope (SEM) is an unrivalled technique that allows users to observe the 3D structure of powders and electrodes and also identify elements and the presence of contaminants.

The importance of particle size measurement

The performance of a battery can be characterised according to the amount of energy that it can store or the amount of power that it can produce. The maximum battery power can be increased by decreasing the particle size of the electrode material and increasing the surface area. Battery power is determined by the rate of reaction between the electrodes and the electrolyte, while storage capacity is a function of the volume of electrolyte within the cell. These properties are intrinsically linked to the intercalation structure and particle size of the electrode particles, which determine how well the mobile ions are taken up and released by the electrode. Particle size distribution and particle shape influence particle packing, hence the volume of electrolyte that can be accommodated within the interstitial voids of the electrode, which affects storage capacity. As a result, a mixture of coarse and fine particles is often used in the electrodes to increase surface area, whilst also controlling the overall packing fraction of the electrode material to allow good contact between the electrode and the electrolyte.

Particle sizing of electrode materials is commonly performed using the Mastersizer 3000 which uses automated laser diffraction technology. With a measurement range that runs from 0.01 to 3500 µm, the Mastersizer is the particle sizing technology of choice for most battery manufacturing applications – starting from precursor to the final milled electrode materials.

Figure 1 Particle size distribution of three batches of NCM cathode materials synthesized with different processing parameters

Figure 2 Particle size distribution of three batches of synthetic graphite synthesized with different heating conditions

The Malvern Insitec online process systems deliver real-time monitoring of particle size for automated process control. These can be used for either the monitoring of particle size evolution in precursor slurry or in the control of electrode material size right after the mill. Smaller particles in electrode slurry production can be prone to agglomeration and/or flocculation, resulting in uneven electrode coatings and ultimately compromising the electrochemical performance. Aggregation and stability can be monitored by measuring zeta potential (particle charge) using the Malvern Zetasizer Ultra. A low zeta potential will indicate particles likely to aggregate whereas a high zeta potential will form a stable dispersion. The Malvern Zetasizer Ultra builds on the legacy of the industry-leading Zetasizer Nano Series adding high-resolution sizing (Multi-Angle Dynamic Light Scattering) and particle concentration capabilities.

The importance of measuring porosity

Porosity is an important parameter both for the separator and for the electrolyte to transport lithium-ions between the anode and cathode. By controlling porosity, higher intra-electrode conductivity can be achieved to ensure adequate electron exchange as well as sufficient void space for electrolyte access/transport of lithium-ions for intercalation of the cathode. Higher porosity means less heat generated in the cell and greater energy density. However, excessive porosity hinders the ability of the pores to close, which is vital to allow the separator to shut down an overheating battery. Therefore understanding the porosity of the electrode materials is important to guarantee the right ion accessibility and charging speed.

Recognised as the most advanced instrument in the field for material surface characterisation the Micromeritics 3Flex has become a crucial tool for the battery industry. The 3Flex is a high-performance adsorption analyser designed for measuring surface area, pore size, and pore volume of powders and particulate materials. Analysis of BET surface area, pore volume and pore size distribution helps to optimise battery components.

The Micromeritics AutoPore V Series utilises Mercury Porosimetry, a technique based on the intrusion of mercury into a porous structure under controlled pressures, to calculate pore size distributions, total pore volume, total pore surface area, median pore diameter and sample densities.

The importance of measuring surface area

Increasing the surface area of the electrode improves the efficiency of the electrochemical reaction and facilitates the ion exchange between electrode and electrolyte. Lower surface area materials are better suited for improved cycling performance of the cell resulting in longer battery life. High surface area presents some limitations due to the degradation interaction of the electrolyte at the surface and resultant capacity loss along with thermal stability. Nanoparticles hold promise to increase surface area without capacity loss by permitting shorter diffusion paths for lithium-ions between the graphite particles which facilitates fast charge and more efficient discharge rates and improves the capacity of the battery.

The Micromeritics TriStar II Plus is an automated, three-station, surface area and porosity analyser. MicroActive software allows the user to overlay a mercury porosimetry pore size distribution with a pore size distribution calculated from gas adsorption isotherms to rapidly view micropore, mesopore, and macropore distributions in one easy-to-use application.

The importance of measuring particle shape

Shape will affect the electrode coating in terms of packing density, porosity and uniformity. Spherical shaped particles will pack more densely than fibrous or flake shaped particles. The average strain energy density stored in a particle increases with the increasing sphericality. Fibrous and flake shaped particles are expected to have a lower tendency for mechanical degradation than spherical-shaped particles. Automated imaging using the Malvern Morphologi 4 is commonly employed for particle shape analysis of electrode materials but can also be coupled with Raman spectroscopy to give particle-specific structural and chemical information.

The importance of analysing chemical composition

Deviations in chemical composition or impurities in electrode materials can significantly affect final battery performance. For this reason, chemical composition and elemental impurity analysis are an integral part of the battery manufacturing process. Simple to operate and fast to learn, the Phenom XL G2 scanning electron microscope (SEM) is an unrivalled technique that allows users to observe the 3D structure of electrodes after production; the size and granulometry of raw powders; the size of pores and fibres in insulating membranes and the response of materials to electrical or thermal solicitations. Using fully integrated X-Ray analysis (Energy Dispersive Spectrometer, EDS) the distribution and identity of elements including the presence of contaminants in the battery sublayer can quickly be revealed.

The Phenom XL G2 is the only SEM that can be placed within an argon-filled glovebox, allowing users to perform research on air sensitive lithium battery samples.

The future of batteries

Driven by our need to reduce greenhouse gases with renewable energy and portable communication devices, the Li-ion battery market is growing at 14% Compound Annual Growth Rate (CAGR). Deutsche Bank forecasts lithium-ion batteries will account for 97 percent of battery use in energy storage alone by 2025. Most automotive companies are now investing in batteries and are in a race to patent critical next-generation battery technologies and battery management systems. Companies related to fossil energy or mining are also entering the battery value chain.

Australia is well positioned to capitalise on the significant opportunities presented, having the world’s third-largest reserves of lithium and is the largest producer of spodumene (mineral source of Lithium). Australia currently produces nine of the 10 mineral elements required to produce most lithium-ion battery anodes and cathodes and has commercial reserves of graphite – the remaining element. Australia has secure access not only to all the chemicals required for lithium-ion battery production including precursor, anode, cathode, electrolyte materials but also the knowhow through various research groups at our universities and institutions such as the CSIRO, meaning more advanced batteries can be manufactured locally. However, as demand for lithium batteries continues to increase, it will eventually outstrip supply, so we need to think beyond Lithium to other technologies that can deliver safer, more abundant and cheaper materials (such as sodium, zinc or vanadium) to store renewable energy.

At the University of Wollongong , the Smart Sodium Storage Solution (S4) Project aims to develop sodium-ion batteries for renewable energy storage. This ARENA-funded project builds upon previous research undertaken at the University of Wollongong and involves three key battery manufacturing companies in China. Gelion, a spin-off company from the University of Sydney, is developing gel-based zinc-bromine batteries. The technology uses a unique gel electrode that transforms zinc-bromide technology into a high-efficiency non-flow battery.

Energise your battery research with ATA Scientific

Whether you are a battery component manufacturer looking for greater process efficiency and better quality control, or a researcher striving to determine the performance parameters of newly emerging battery materials, our solutions will offer you the new levels of insight and control needed to power the production of superior quality batteries. Contact us via phone (+61 2 9541 3500), or through our website for a demonstration or quote today!

Characterising Lipid Nanoparticles for Vaccine Development

December 14, 2021Guides & Resources, Particle Science - Guidewebprofits

Lipid nanoparticles (LNPs) either loaded with nucleic acids or as liposomes containing an aqueous core, have received great interest from pharma as delivery vehicles for different therapeutic treatments for many different reasons.LNPs offer improved stability and delivery efficiency by protecting drug molecules from degradation by the body’s natural immune processes. Moreover, the LNP can be specifically targeted using customised ligands attached to its surface.

The breakthrough of mRNA-based vaccines

The fast pace of progress in mRNA vaccines (e.g. for COVID-19) would not have been possible without major recent advances in RNA encapsulation and delivery methods. Recent breakthroughs with mRNA-based highlight the potential of lipid-based particles as powerful and versatile delivery vectors for vaccines and gene therapies, to treat previously untreatable diseases. Extensive basic research into RNA and lipid and polymer biochemistry has made it possible to translate mRNA vaccines into clinical trials and has led to an astonishing pace of global vaccination.

LNPs have been found to be the most effective mRNA formulation/delivery approach and function to protect the mRNA from degradation when injected into the patient and to promote entry of the mRNA into cells. LNPs typically consist of four components: an ionizable cationic lipid, which promotes self-assembly into virus-sized (~100 nm) particles and enables endosomal release of mRNA to the cytoplasm; lipid-linked polyethylene glycol (PEG), which increases the half-life of formulations; cholesterol, a stabilising agent; and naturally occurring phospholipids, which support the lipid bilayer structure.Inactive ingredients such as salts, sugars, and stabilizing acids are added to achieve formulation stability during transport and storage.

Analytical characterisation of these nanoparticles is critical to drug design, formulation development, understanding in vivo performance, as well as quality control during formulation and manufacture. The use of ever-more structurally complex molecules warrants a growing requirement for complementary and orthogonal analytics to ensure data quality and the reliability of research.

How does Malvern Panalytical contribute to the characterisation of lipid nanoparticles

When developing LNP drug candidates, several critical quality attributes (CQAs) need to be addressed. Nanoparticle characterisation CQAs for particle size, particle size distribution and concentration can be measured using Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA). These techniques are both orthogonal and complimentary in addressing this. Surface charge, another CQA can be probed using Electrophoretic Light Scattering (ELS), a measure of particles colloidal stability CQA.

Particle size and stability using light scattering techniques

Light scattering techniques are used extensively in the characterisation of lipid nanoparticle and liposome research to measure particle size, stability, zeta potential and particle concentration. The Zetasizer range of light scattering instruments can be used to optimise lipid-based formulations and process conditions, such as monitoring stability, understanding surface modification, and developing formulations. Non-invasive backscatter (NIBS) optics enable reliable measurements of concentrated, turbid samples without the need for dilution. Delivering data in a short time frame, the Zetasizer allows users to implement this technique throughout the development pipeline.

Aggregation/ encapsulation efficiency using Nanoparticle Tracking Analysis (NTA)

NTA allows you to visualise and size individual particles in the preparation, generating important information about nano-particle content. For instance, the presence of larger particles could represent either non-viral cell debris from the cell culture process or aggregates of virus particles containing many individual virions. In either case, such aggregates/contaminants represent a possible problem to the manufacturer. NanoSight helps vaccine developers devise a solution.

Biomolecular interactions using Isothermal Titration (ITC) and Differential Scanning Calorimetry (DSC)

Detailed characterisation of protein structure enables understanding of protein function. It is therefore among the activities central to academic and industrial research and development. ITC is used in quantitative studies of a range of biomolecular interactions, that work by directly measuring heat that is either released or absorbed during a biomolecular binding event. By providing a complete thermodynamic profile of the molecular interaction, ITC can explain mechanisms’ underlying interactions, and enable more confident decision-making in hit selection and lead optimisation.

DSC is used to characterise stability of a protein or other biomolecules directly in its native form and achieves this by measuring heat change associated with the molecule’s thermal denaturation. Precise and high-quality data obtained from DSC provides vital information on protein stability in process development, and in the formulation of potential therapeutic candidates. The ‘first principle’ nature and high resolution of DSC makes it a well-established technique for extended structural characterisation and stability profiling of biomolecules and viruses in solution. Due to its direct readout, broad temperature range and sensitivity to thermally-induced unfolding, DSC is also used as the gold standard technique for validation of data from higher throughput thermal stability assays.

How does the RedShiftBio (MMS technology) contribute to the characterisation of lipid nanoparticles

The nature and composition of a vaccine makes them inherently difficult to characterise. The active ingredient such as a toxoid or protein subunit is often in very low concentration. The adjuvant, such as alum, can often be in quite high concentration relative to the biologic, and its particle size can range from nanometers, to microns. Adding preservatives and antibiotics to the mix, the result is a material with such diverse properties that many typical analytical tools struggle to measure the formulated product.

Microfluidic Modulation Spectroscopy (MMS) combines mid-infrared laser spectroscopy with microfluidics to overcome many of the limitations of traditional spectroscopy-based technologies. Its ability to provide multiple attribute data reduces or eliminates the need for performing separate measurements across different tools. MMS offers ultrasensitive, highly reproducible, automated structural measurements of protein aggregation, quantitation, stability, structure and similarity – measurements that underpin drug safety and efficacy. This novel method can be used to examine protein secondary structure for a wide range of applications from mAb-based biotherapeutics to robust measurements of ADCs, AAVs, and mRNA. It provides direct, label-free measurements over the concentration range <0.1 mg/ml to >200 mg/ml. Real-time background subtraction eliminates need to dialyse samples. Even at low concentrations the AQS3Pro system allows detection of <2% change in secondary structure.

We can help manufacture your success

ATA Scientific provides a range of physicochemical characterisation tools that are used from the initial characterisation of biological materials through to final manufacturing and quality control, and which deliver information essential to ensuring the stability and efficacy of the vaccine product.

Contact us for more information on the latest analytical technologies from Malvern Panalytical (DLS, NTA, ITC/DSC) and Redshiftbio (MMS technology).

Changing the Way We Measure ‘Undetectable’ Differences in Protein Structure

October 27, 2021Guides & Resourceswebprofits

How do you resolve an issue in a product when your analytical methods fail to indicate any difference between batches? Consider the ramifications should your pharmaceutical product cause harm despite your best efforts of your procedures, not to mention the need to analyse in non-native conditions. Most basic assessments cannot delineate conformational effects from colloidal effects .

Making complex processes simple

Biophysical characterisation of proteins can be a daunting task, given the number of methods required to elucidate the results for Aggregation, Quantitation, Stability, Similarity and Structure. RedShift Bio’s AQS³ pro, powered by Microfluidic Modulation Spectroscopy (MMS), can perform these 5 measurements in a single analysis which is impressive. Sensitivity is improved up to 30 times that of conventional FT/IR instrumentation with a desirable concentration range of 0.1mg/ml to over 200mg/ml and with protein analytics that is equally enviable to the operator. Savings in sample testing time can be more than 80% thanks to the fully automated multi-sample capability.

Often the buffers used in a formulation are not compatible for the analytical method, as seen with spectropolarimetry. The AQS³ pro experiences no interference from excipients in the buffer. This is a game changer, to measure at concentration, and in the final drug conditions, removing the guesswork from formulation and de-risking many steps.

What is the magic behind the AQS³ Pro system?

There are 3 key components in the AQS³ pro that allow the system to achieve all this and separates it from all other systems,

a mid IR tuneable quantum cascade laser
a thermal, electrically cooled detector
a Y shaped microfluidic transmission cell.

The Tuneable laser provides an optical signal almost 100X brighter than the conventional light source used in FT/IR allowing the use of a simple thermal electrically cooled detector without the need for liquid nitrogen cooling. Given the intensity of the laser, the system is amenable to low concentration samples as low 0.1mg/ml. The measurement differs from the conventional as well. The sample and the reference stream are injected alternately through the Y shaped microfluidic cell passing through the observation zone. Alternating at a rate of 1 – 5hz, the absorbance of the reference and sample are measured almost simultaneously allowing the reference absorbance to be subtracted from the sample absorbance in real time resulting in the collection of reference corrected absorbance spectra. Such a real time buffer subtraction and auto-referencing greatly enhances the sensitivity method and produces an almost drift free signal.

These innovations create the 30X sensitivity boost. The speed of the AQS³ pro impresses. Where it may take 30 mins per sample for the FT/IR, the AQS³Pro system with MMS technology can measure samples in less than 1.5min. The AQS³ delta analytics packages are refreshingly simple and intuitive, applying advanced analytical tools enabling its use in a vast range of applications across the industry.

What does the AQS³ pro actually measure?

This is an infrared system that measures the Amide 1 band of a protein which is in the wavenumber range of about 1580 – 1720. It takes about 32 data points across the range alternating between buffer and sample, plotting a differential absorbance which is interpolated into a spectrum, which in turn is converted to a second derivative affording fine detail changes from spectrum to spectrum to be elucidated. Flipping the spectra shows the baselines and area of overlap plot enables you to look at similarity and trends between spectra. This can then be deconvoluted using a normal gaussian deconvolution, giving rise to secondary structure motifs, the percentage of the components such as α-Helix, β-Sheet, and Anti-parallel β-Sheet Higher Order Structures (HOS).

Case study: complexity of a vaccine.

The diverse composition of a vaccine makes biophysical characterisation challenging. Not only does a vaccine have antigens and antibodies, they can also have an array of excipients such as preservatives, stabilisers, and buffers plus they can contain antibiotics. The concentrations of the components vary by orders of magnitude and the particle size distribution of a final formulation can range from nano to micron.

The additional ingredients can introduce protein and non-protein, organic and inorganic materials. If you add up the numbers of tests and instrumentation required to perform these tests it can be incredibly diverse. MMS is a single technology that provides unique insights into many of the parameters required to fully understand these biophysical properties of the sample, from looking at interaction effects of antigen and adjuvant or the effect of looking at varying stabiliser concentrations or altering the buffer pH. All these steps can be performed to understand their effect on the protein secondary structure.

The total characterisation of a protein-based vaccine should include:

Biophysical Characterisation to understand Protein antigen properties such as pH, ionic strength, HOS, and aggregation propensity.

Stabiliser Screening These convey stability by addition of amino acids, surfactants, proteins, sugars and antioxidants to improve shelf life.

Adjuvant Screening Adjuvant surface chemistry (eg alum) as well as adjuvant-antigen interactions should be characterised.

Process Design & Control Process design and vaccine-surface interactions of process equipment should be understood – ie protein loss on filtration substrates as well as denaturation of protein samples.

Stability Studies Real time and accelerated stability studies ensure antigen is in chemically and physically stable state

With this in mind, let’s look at the tools and parameters needed for such biophysical characterisation of protein antigens; from molecular weight in a chromatography system to surface charge on an electrophoretic light scattering system. A few are listed below.

LC – HPLC and SEC Liquid Chromatography in the form of reverse phase, ion exchange, size exclusion can be used to assess chemical and physical stability.

DSC- Differential scanning Calorimetry Thermal stability properties of antigen for insights into formulation conditions.

MALDI – Maldi TOF High resolution molecular weight providing information about primary structure and post translational modifications.

ELS – Electrophoretic Light Scattering – Surface charge of pure adjuvant versus adjuvant in formulation will identify protein adjuvant interactions.

DLS – Dynamic Light Scattering Particle size distribution of antigen, adjuvant and complex mixtures, Colloidal stability parameters KD and B22

It is important to note – all these parameters need to be measured at some point during development of a protein vaccine.

Where does MMS fit into the development of vaccines?

Given MMS measures protein secondary structure, we can leverage this capability to measure in final formulation conditions to assess HOS as stabilisers are varied to determine if they impart a stabilising effect and prevent aggregation processes. The AQS³ pro could be used for adjuvant screening, investigating the interaction and to determine if it is damaging to the antigen. Process design can be studied to establish, for example, if a filtration membrane causes a negative effect on the structure of the antigen, or to understand the stability of the formulation using long-term stability studies. It is now clear that MMS can be applied across the entire development pipeline with a wide range of measurements to help understand the full properties of protein-based vaccines.

De-Risk Drug Development

MMS’s sensitive, accurate measurements coupled with a robust data analysis package provides simple, accessible, and reliable results to de-risk and accelerate drug development workflows. The AQS³ pro can identify at-risk candidates far earlier than traditional methods, clearly saving time and resources.

ATA Scientific are proud to have the RedShiftBio AQS³ pro within our suite of instrumentation. Should you require further information on the AQS³ pro or indeed many of the techniques cited above, please do not hesitate to contact us.

Do you need Size Exclusion chromatography (SEC) & Gel Permeation Chromatography (GPC) in Additive Manufacturing?

April 16, 2021Particle Science - Guidewebprofits

Gel Permeation Chromatography (GPC) is a widely used technique that separates macromolecules such as proteins and polymers based on size. As researchers and additive manufacturers continue to push the boundaries of performance and demand more complex material properties, the evolution of the technique has enabled a better understanding of the key indicators that lead to a high performance printed product.

What is Additive Manufacturing?

Additive manufacturing (AM) is a technology that uses three-dimensional computer models to print parts by building the component layer by layer. For metallic products, the traditional method used for manufacturing metal parts is subtractive, whereby milling machines form the part from a solid block of metal.

In the AM process, high-precision electron beams or lasers move at high speeds to selectively melt layer upon layer of metal, tens of microns thick. Highly complex parts are rapidly formed with novel functionality using less material than other methods. Multiple fields use AM, including construction, prototyping, biomechanical, and others, to produce prostheses individually adapted to humans and animals.

Types of Additive Manufacturing Processes

Powder Bed Fusion (PBF), like Selective Laser Sintering (SLS), uses a laser to selectively fuse thin layers of powder particles (usually metal, polymer, or ceramic). Thermoplastic polymers such as nylon are well suited for use in PBF as they are processed reliably due to their semi-crystalline nature, which provides a distinct melting point. The wide temperature working window between melting (during heating) and subsequent crystallisation (via cooling) makes nylon the choice polymer.

Stereolithography is one of the first additive manufacturing or 3D printing technologies developed. Initially, parts manufacturers used the process to create polymeric prototypes, but now it is also used in final-part production. In stereolithography, a large tank or vat of photopolymer resin (composed of oligomers, monomers, and photoinitiators) undergoes cross-linking upon exposure to Ultraviolet (UV) or Visible (Vis) light. A support platform moves the cured object upward or downward layer by layer to form the final product. The tensile stiffness and elasticity of the solid product are essential for additive manufacturers to analyse and ensure consistent quality. Controlling the oligomers’ molecular weight distribution, structure, and proportion of photoinitiator used achieves optimisation. These properties also affect the photopolymer formulation’s rheology and viscosity.

Fused Deposition Modelling (FDM) uses a material-extrusion technique where a thermoplastic filament is drawn through a nozzle, heated to its melting (or glass transition) point, and then deposited layer by layer to cool and harden, repeating the process until the 3D structure is complete. Assessing the melt characteristics and determining the structure of the polymers (i.e. molecular weight distribution, molecular density, and degree of branching) is critical in developing novel feedstock material with unique mechanical properties that are also printable.

Material/binder jetting uses a liquid binding agent to join the metal, ceramic, or polymer powder particles rather than melting or fusing with a laser or electron beam used in PBF. This process forms a green part removed from the printer with solidiﬁcation via a secondary de-binding or sintering step. Accurate determination of molecular weight and structure of polymeric powders and binders is required to optimise final component properties.

What are the main challenges of Additive Manufacturing techniques?

The leading challenge additive manufacturers face relates to the quality of the final product made, which is highly dependent on understanding the quality of the feed material. Selecting high-quality metal or polymer powders highly spherical and free from satellites or deformed/ agglomerated particles can reduce variation and prevent cracking, distortion, weakness, and poor surface finishes of final products. However, high-quality materials are relatively expensive, which contribute to high build costs. Although the ability to recycle the unused material can save on costs, reusing the polymer powder can age it and cause unfavourable structural changes. By accurately characterising the molecular properties, such as the molecular weight, molecular size, and size distribution of the bulk polymer and polymeric structure (branching, crystallinity), manufacturers can optimise specific AM processes and prevent processability issues that impact the quality of the final component.

Why is particle size and structure important for 3D printing?

Understanding key properties such as particle shape, structure, particle size, and particle size distribution in the powders is essential. These properties can impact the powder’s packing density, flowability, and compressibility. Each characteristic ensures uniformity and must be optimised to create a product free from defects such as pores, cracks, inclusions, residual stresses, and unwanted surface roughness. Irregularly shaped particles tend to increase interparticle friction and decrease ﬂowability while the preferred smoother, more regular-shaped particles ﬂow more easily. As particle size decreases, the forces of attraction between particles increases. Optimisation of flowability and packing density occurs as ﬁner particles increase density by ﬁlling the gaps left by larger ones. Therefore, measuring particle shape and size distribution impacts the powder material properties; it is vital for ensuring the feed material is suitable for an application.

One of the primary characterisation techniques for analysing polymers used in additive manufacturing is size exclusion/gel permeation chromatography (SEC/GPC). This technique enables a better understanding of macromolecular characteristics, such as particle size and structure of the feed material, the effects on powder reusability, and ultimately, the final product’s quality.

What is Size Exclusion Chromatography (SEC) or Gel Permeation Chromatography (GPC)?

SEC or GPC is a liquid chromatography technique that separates polymers according to their size (hydrodynamic volume) to measure molecular size and structure. GPC or SEC involves separating the sample as it passes through a porous chromatography column. Larger molecules unable to penetrate the pores are excluded and thus travel through the column faster than smaller molecules, allowing separation based on size.

GPC or SEC can be used to measure molecular weight (MW), molecular weight distribution, intrinsic viscosity, and the hydrodynamic size of macromolecules. The inherent viscosity measurements combined with the molecular weight identify structural differences between samples.

What is absolute Molecular Weight (MW)?

The MW of a polymer is the sum of the atomic weights of the individual atoms that comprise a molecule. It indicates the average length of the bulk resin’s polymer chains. There are different kinds of molecular weight: Number average molecular weight (Mn), weight average (Mw), and z-average molecular weight (Mz). Various techniques can measure each MW moment (Mn, Mw, Mz). For instance, osmotic pressure calculates the number of molecules present and provides average molecular weight regardless of their shape or polymers’ size. In comparison, SEC or GPC in a single measurement provides complete and accurate MW distribution characterisation while also providing structural information. GPC or SEC determines the polymer or biopolymers’ absolute molecular weight and branching degree by measuring light scattering at various angles as a concentration function.

The molecular weight (MW) and molecular size play a key role in determining the mechanical, bulk, and solution properties, determining how the polymer material will behave during processing as a final product. For AM, selecting the correct polymer MW is a balance between printing ease and final-product performance. Low MW polymers exhibit low viscosity and offer better flow properties with fewer stresses. As MW and cross-links increase, so do polymer strength, brittleness, melt temperature, and viscosity, but solubility decreases.

Why use a multi-detection SEC or GPC system?

A conventional GPC or SEC system setup usually consists of only an isocratic pump and a detector, either Refractive Index (RI) or Ultraviolet (UV). This setup provides only a concentration profile of the size-separated sample and relative MW. The calibration standards contain a polymer mixture of known MW correlated against the RI traces in the calibration process. However, this calibration plot is accurate only if the standards’ intrinsic viscosity is identical to that of the sample. Only polymers of the same MW with equivalent intrinsic viscosity will elute at the same rate, a significant limitation when gathering precise data for the detailed comparison of relatively similar polymers when the calibration standards are sub-optimal for the polymers of interest.

In contrast, the Malvern Omnisec GPC/SEC system employs the universal calibration technique to address this limitation. It uses highly informative multiple detection regimes to directly and accurately measure MW. This process includes a concentration detector (RI or UV-Vis), a multi-angle light-scattering detector (RALS/LALS/MALS), plus a self-balancing viscometer that enables the measurement of structural features such as branching or conformation. Multiple detectors provide additional information about a sample when simultaneously evaluating a single injection. This information includes Absolute MW and MW moments; Intrinsic Viscosity (IV), hydrodynamic radius (Rh), the radius of gyration (Rg), dn/dc calculated value, sample concentration, and recovery, to name a few. The Rh of a sample is the radius of a sphere with the same mass and density of the sample based upon molecular weight and intrinsic viscosity. Rg represents the root mean square distance of the molecule’s components from the molecule’s mass centre. Both provide valuable molecular size information. Plotting the MW measured directly from the light scattering detector against the IV measured from the viscometer detector produces a Mark-Houwink plot to illustrate the relationship between molecular structure and molecular weight.

The pioneering work from Viscotek, a market leader in GPC, led to the Omnisec system from Malvern. For the last two decades, the system has continued to evolve. Today, it is the most advanced GPC system for measuring absolute molecular weight, molecular size, intrinsic viscosity, branching, and other structural parameters.

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What is the power of multi-detection GPC/SEC?

In a complete GPC/SEC system, OMNISEC integrates the separations unit and all four detectors with inter-detector tubing temperature control.

RI detector: This is the most common detector for any GPC or SEC system. RI detectors are referred to as concentration detectors because the difference in refractive index between the sample solution and the solvent is proportional to the sample’s concentration. It provides a dn/dc value, the refractive index increment, which is essential because it is the link that translates the raw RI signal to sample concentration. Knowing the concentration allows the calculation of all molecular parameters, including absolute molecular weight and IV.

UV-VIS PDA detector: UV-VIS detectors are also concentration detectors but require the sample to have a chromophore and absorb light at a detectable wavelength between 190 – 900nm.

Capillary differential viscometer: First invented by Max Hanley in 1984, this unique viscometer measures the changing solution viscosity to calculate the sample’s intrinsic viscosity (structure). The viscometer detector uses four capillaries, a delay column, and two transducers (DP and IP). It has a user-replaceable self-balancing bridge that helps to reduce downtime and maintenance requirements. The viscometer works by directly measuring the specific viscosity by subtracting the solvent’s contribution in a balanced capillary bridge. When used with a concentration detector, it will measure the IV distribution of any polymer.

Static Light Scattering (SLS) detectors: Light scattering occurs when a photon from an incident beam is absorbed by a macromolecule and re-emitted in all directions. The intensity of light scattering measures MW and Rg described by the Rayleigh theory. Small molecules less than 10 – 15nm in radius will scatter light evenly in all directions and are known as isotropic scatterers. Large molecules with an Rg of more than 15nm (radius) and high MW are anisotropic scatters. They have multiple scattering points and tend to scatter more light in different directions with different intensities. A Debye plot models this angular dependence of samples scattering and is used to determine the MW andRg at every data slice within the chromatogram using multi-angle light scattering.

There are four types of SLS instruments:

Low Angle (LALS) measures the intensity of light scattering very close to the Zimm plot’s axis or very close to 0°. The calculated MW will be very close to the actual MW therefore ideal for anisotropic scatterers such as large polymers.
Right Angle (RALS) measures the intensity of light scattering at 90° and with sample concentration provides the measurement of MW for molecules, <15nm (radius) in size, ideal for proteins. Low molecular weight polymers are isotropic scatterers. The resulting partial Zimm plot is flat with a zero slope; therefore, it is unsuitable for these smaller materials. Isotropic scatterers, smaller than 10 – 15nm in radius, will scatter light evenly in all directions, enabling only the MW measurement.
For large polymers with an Rg >15nm that exhibit angular dependency in the light they scatter, a Multi-Angle (MALS) detector makes it possible to determine molecular size Rg in addition to MW. A conformation plot (plot of Rg against MW) allows the measurement of any structural differences between the samples.

The LS detectors’ high sensitivity enables molecular weights measurements as low as 200 Da or injection masses as low as 100 ng of material. This sensitivity measures low molecular weight samples, novel polymers with low dn/dc, or tiny amounts of precious samples. An RI detector combined with light scattering and viscometer detectors provides the sample’s exact concentration at each data slice using the sample’s dn/dc value to calculate the absolute molecular weight intrinsic viscosity.

By housing all the detectors together in a single compartment, the distances between them can be kept to an absolute minimum, reducing the level of band-broadening and tailing. Additionally, the use of a single temperature-controlled compartment for detectors and all connecting tubing means the temperature can be elevated for polymer applications to reduce the viscosity of certain mobile phases such as DMSO (dimethyl sulphoxide). Combining all of these factors makes Malvern Omnisec Reveal the most-advanced multi-detection platform for analysing natural and synthetic polymers.

Whether you’re looking for the Malvern Omnisec Reveal or another scientific instrument to assist your addictive manufacturing, our team has the expertise to match your research scope to the right analytical instruments. Contact us for more information.

References

How to Efficiently Transport Live Cell Cultures Without Freezing Them

November 16, 2020Biomolecular Science - Guidewebprofits

Ross Harrison first attempted cell culture in the early 1900s. Fast forward 100 years and 3D in-vitro cell culture is routine, and an organ on a chip is a reality. Cells are fundamental to all bodily functions; they encompass a multitude of components with interactions that depend on the cell’s purpose. The awe-inspiring knowledge is they originate from stem cells that differentiate to become a particular cell — i.e., nose, ear, blood, etc. Billions of replications and the body generally works for life spans sometimes eclipsing a century. Years ago, cellular research had to be content with cells on a slide, stained to expose their secrets, and labs had to start from scratch to emulate the procedure.

How are cells transported today?

Today, researchers utilise specific cell lines created by facilities around the globe. Whether novel or challenging to isolate, these specimens are often sought after and must be transported from lab to lab. Shipping is required to accelerate research or to ensure treatment of the precise cell. This need has created a quandary — live cells are photosensitive; they also require 37oC and a 5% CO2 atmosphere. This atmosphere is hard to achieve. Cryopreservation has been the best way to meet such requirements.

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What happens when you freeze cells?

Cryopreservation abates the disadvantages of freezing cells. Most living organisms die when frozen due to cryoinjury, the formation of intracellular crystals when cells are rapidly frozen. Slowing this process unearths further challenges such as extracellular ice formation that cause osmotic stress and mechanical damage. Epigenetic modifications may result from incomplete cryopreservation amongst other batch-to-batch variations. Using an antifreeze such as dimethyl sulfoxide (DMSO), a cryoprotective agent, gives rise to other issues – particularly effects on natural cell behaviour.

An entire industry has emerged in culture media with a focus on cell growth, 3D structures such as support for spheroids and organoids, regenerative medicines, cell expansion, and bioprinting. Nanocellulose structures provide exceptional growing conditions by forming a hydrogel that is easily enzymatically cleared to release the cells for clinical use.

As research begins to reject processes that fail to emulate a natural environment, cryopreservation will take a back seat. The procedures and techniques that fail the natural behaviour test reveal that cells being studied are in an altered state, encouraging further scientific scrutiny.

Be prepared to ask yourself questions such as:

Are you exposed to foreign genetic material in your media?
Have you cryopreserved?
Have you used fluorescent tags in the treatment of these cells?

Variations may be seen as cells are exposed to foreign genetic material or when fluorescently tagged. Whether occurring locally or internationally, the need for a reliable method to transport fragile and valuable cells has led to the development of a portable CO2 incubator.

Can live cells be transported without freezing?

To put it simply, the answer is If cells are simply frozen to ship, their cellular processes become compromised. If a natural route is chosen – such as keeping the cells warm with lots of media to sustain them for their journey – it’s likely they will die due to a lack of CO2 and a disruption to their constant temperature, highly probably for long distance trips. Consider a 10-minute walk across a University campus from the animal house to the lab in an insulated container. On a cold day, mouse embryos may perish. Such is their dependence on precise thermal regulation. Sympathetic to this outcome, the term ‘Live Cell Shipping’ could be categorised as a misnomer.

What are the alternatives to freezing?

You can lob your cells into an esky and cross your fingers, hoping for the best. Some cargo just doesn’t survive a few minutes out of 37oC and 5% CO2. If the journey is long, there is little hope the cells will arrive at their destination alive. The best-known method to transport not only fragile cells but the whole gamut of living tissue is the Cellbox.

The Cellbox is a live-cell shipper, designed by the Fraunhöfer Institute from the ground up to solve the cell-transport conundrum. It is the first portable CO2 incubator intended to transport cells by road, rail, or air. The Cellbox is a significant game-changer. Prepared and packed under UN 3373, you need not defrost biological material; they are ready to go as soon as they arrive. There are no toxic substances added and no freeze-thaw loss, which saves cells and time.

Choosing Cellbox with ATA Scientific

A well considered live cell research plan has accounted for how the cell lines will be transported to the dedicated lab and facilities thereafter

The Cellbox incubator is a technological advancement. With it, you can track the events of the entire journey, then download to your smartphone to correlate the conditions to the cellular activity. This insight is instrumental if you are shipping clinical samples such as blood from the blood bank, embryos, cord blood, cell cultures, and tissue engineering.

To evaluate the Cellbox’s benefits to your facility, call or email ATA Scientific today. This one call may transform your capacity to align your research methods with more natural behaviour. Speak to ATA Scientific for a demo or trial of the Cellbox in your facility.

TIP 1: Stick the sample securely to the pin stub, by using:

TIP 2 : Remove all loose particles from your sample after adhering the sample to the pin stub by:

TIP 3: Use tweezers when handing the pin stub

TIP 4: Make sure that the mounting procedure is solid

TIP 5: DO NOT spray dry air in the direction of any electronics

TIP 6: Make sure there is no condensed liquid in your spray air straw

Non-Conductive samples

Magnetic samples

Stigmation correction

Beam-sensitive samples

Beam settings

Sputter coating

Cooling

Time

Magnification

Powders and particles

Particles disperser

Samples containing moist or outgassing samples

Force drying

Critical point drying

Cooling

Low vacuum

Small amount of sample

What is live-cell imaging?

How is live-cell imaging used?

Limitations of conventional live-cell imaging methods?

3 Factors to consider when choosing an automated live-cell imaging system

Multiple imaging modes that are affordable

Stable scanning performance and compatibility

User friendly interface and 3D modeling

1. Match the instrument specifications to the cell type

2. Determine an appropriate sample dilution range

3. Consider using fluorescence staining for viability measurements

4. Maximise the counting volume

5. Optimise the counting protocol

Introducing the LUNA™ Automated Cell Counter

Why particle characterisation is critical

The use of laser diffraction for particle size distribution

Automated Image Analysis for particle shape and composition

We can go further together

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The importance of driving onshore advances in RNA research and therapies

Next generation technologies will nurture a wide range of genetic medicines

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The importance of particle size measurement

The importance of measuring porosity

The importance of measuring surface area

The importance of measuring particle shape

The importance of analysing chemical composition

The future of batteries

Energise your battery research with ATA Scientific

The breakthrough of mRNA-based vaccines

How does Malvern Panalytical contribute to the characterisation of lipid nanoparticles

Particle size and stability using light scattering techniques

Aggregation/ encapsulation efficiency using Nanoparticle Tracking Analysis (NTA)

Biomolecular interactions using Isothermal Titration (ITC) and Differential Scanning Calorimetry (DSC)

How does the RedShiftBio (MMS technology) contribute to the characterisation of lipid nanoparticles

We can help manufacture your success

Making complex processes simple

What is the magic behind the AQS3 Pro system?

What does the AQS3 pro actually measure?

Case study: complexity of a vaccine.

Where does MMS fit into the development of vaccines?

De-Risk Drug Development

What is Additive Manufacturing?

Types of Additive Manufacturing Processes

What are the main challenges of Additive Manufacturing techniques?

Why is particle size and structure important for 3D printing?

What is Size Exclusion Chromatography (SEC) or Gel Permeation Chromatography (GPC)?

What is absolute Molecular Weight (MW)?

Looking for the perfect analytics instrument for YOUR next big discovery?

What is the power of multi-detection GPC/SEC?

How are cells transported today?

Looking for the perfect analytics instrument for YOUR next big discovery?

What happens when you freeze cells?

Can live cells be transported without freezing?

What are the alternatives to freezing?

Choosing Cellbox with ATA Scientific

What is the magic behind the AQS³ Pro system?

What does the AQS³ pro actually measure?