Preparation of tooth enamel wafers for Transverse Microradiography (TMR) using the WELL Diamond Wire Saw

Modus  Laboratories based in Reading, UK,  is  a  contract  research  company  for  the  oral  healthcare  industry. Here’s an interesting application note for how they use the WELL diamond wire saw.

Formulations for use in treating or preventing tooth decay in tooth enamel and dentine have been a subject of detailed research since the 18th Century. Since then, modern science has seen many advancements in technology directed towards the investigation of biological systems. In the oral healthcare industry, research on tooth decay and enamel erosion is carried out by highly specialised equipment and techniques. As often is the case, analytical methods used to study and quantify changes in biological materials must be performed with the greatest care to eliminate or to at least recognise the observer effect. The preparation of specimens that are used in an analytical study of biological material, such as tooth enamel, is of fundamental importance to the interpretation of the results.

One example where sample preparation is critical to the results of a study is Transverse Micro Radiography (TMR) and was first reported by Angmar et al in 1963. TMR is an analytical method used to measure the mineral density in tooth enamel or dentine. The general approach is to use a block of enamel a few millimeters wide and 5-10 mm long. The blocks are then subjected to a procedure to form artificial lesions within the enamel block to mimic the condition of caries lesions, the onset of tooth decay. The blocks can then be treated in various ways to investigate the potential of experimental formulations to restore the enamel minerals or even to protect the enamel from the lesion forming.

During the treatment the enamel lesions can be analysed by TMR to gain information about changes in mineral density of lesions and so the effectiveness of treatments. The procedure is to section a thin wafer of enamel transversely through the enamel block and lesion. The section is then x-rayed to produce a digital negative which is then computer transformed into a map of the mineral density of the enamel through the lesion. This then gives highly detailed quantitative information about the mineral content in the lesion and surrounding material.

A crucial aspect of TMR analysis is the sectioning of the thin enamel wafers. For precise measurements the wafers are required to be plano-parallel and thin enough to allow x-rays to pass through (80-100um) the sections and to be absorbed quantitatively by the enamel minerals. The wafers are extraordinarily fragile and the cutting action must be slow and smooth so as not to disturb the lesion. Any changes to the lesion caused by the cutting action may lead to artifacts in the final results.

The saw used for cutting wafers at Modus Laboratories is a WELL 3400 Precision Horizontal Diamond Wire Saw with 100um diamond wire, 60g tension and low speed. This gives a perfectly flat cut surface and wafers of high quality for TMR analysis.

Figure 1: The WELL 3400 Precision Horizontal Diamond Wire Saw.

Figure 2: The position of the enamel block on the wire saw.

Figure 3: Shows the wire saw cutting the first section through the enamel block.

Figure 4: The diamond wire is moved horizontally by 200um using the Vernier scale, then a second section is made.

Figure 5: The result is a thin wafer section through the enamel lesion and enamel block.

Figure 6: Shows the wafer on its side between the tweezers. The lesion is towards the top of the wafer. The wafer is ready for polishing down to 100um before mounting on a slide for TMR analysis.

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mmc2019: a roundup in pictures

A big thank you to everybody who came to visit our stand at mmc2019 at the beginning of July. We hope that you all got a bacteria buddy and that they are all enjoying their new homes now!

Here’s our roundup of the conference…

Joined by experts from Lam Plan Specimen Preparation and WELL Diamond Wire Saws, we were able to showcase a variety of products, including;

We also presented three workshops covering different sample preparation techniques over the course of the conference.

The first was presented by Field Service Executive Ben Hayllar, describing the significant time savings using the BioWave microwave tissue processor.

The second workshop was presented by our Technical Director, Paul Balas, along with Daniel Ebner from WELL. They covered Diamond Wire Saw sectioning for microscopy applications in their presentation.

The third and final workshop was presented by Guillaume Lardon and William Magnin from Lam Plan, about sample preparation using grinders/polishers. Their presentation was illustrated with examples from the aeronautic industry.

Last but not least… bacteria buddies! They were very popular again this year, and we loved how happy they make everyone! Here are some pictures of people enjoying their bacteria buddies…

Thank you again to everybody who came to visit us at our stand. We’ll see you in 2021!

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Agar Scientific acquires London Resin Company

Agar Scientific is pleased to announce the successful purchase of the EM resin manufacturer London Resin Company Ltd. London Resin has developed and supplied acrylic resins for microscopists since 1980, and this acquisition will ensure continued manufacture and supply of the complete product range. 

London Resin’s specially formulated, high quality microscopy resins combine low viscosity, low toxicity and ease of use, and are considered the industry standard for many applications. Production of the LR White, LR Gold and Histocryl resins has now transferred to newly refurbished chemistry labs at Agar Scientific’s Stansted premises – using exactly the same formulations and manufacturing processes – to minimise the impact on customers and ensure supply chain security.

Darren Likely, Managing Director of Agar Scientific, commented: “We are delighted to have successfully completed this acquisition, which will ensure uninterrupted supply of the complete range of high quality London Resins. We have purpose-built a laboratory to meet current and future demand for these products, as well as expanding our chemistry capabilities to allow further developments in the future.”

Dr Brian Causton, founder of London Resin, commented: “I’m very happy that Agar Scientific is taking on the London Resin range. The company has a great team, and I have full confidence that production of these high quality resins is safe in their hands. In the 40 years since I founded London Resin, supporting customers with their unique requirements has always been the best part of the job, and I will continue to advise Agar and answer user questions directly, ensuring a smooth transfer of knowledge.”  

For more information on London Resin’s product range, visit www.agarscientific.com/londonresin

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MMC2019 Workshops – Sample Preparation Techniques

During the first week of July from Tuesday 2nd July until Thursday 4th July we will be attending mmc2019 – we’ll be at stand 710!

Over the course of mmc, we will be holding three interesting workshops discussing various sample preparation techniques, covering Microwave Tissue Processing, sectioning with Diamond Wire Saws and Grinding and Polishing. Here are the extracts for each of the workshops and we look forward to seeing you there! 

Workshop 1 – Microwave Tissue Processing Using the BioWave Pro+

Tuesday 2nd July 11:30 – 12:00, Workshop 1. Presented by Ben Hayllar

Microwave energy itself does not generate heat, but it is the interaction of microwaves with polar molecules and the friction between them that creates heat. 

A common misconception associated with microwave tissue processors is that they use heat to accelerate processing. However, sample heating should be avoided as much as possible to preserve sample physiology.

Conventional benchtop vs microwave processing

In published works, comparing conventional benchtop to microwave processing, the BioWave has been used to show:

  • Better definition of ultrastructure
  • Less shrinkage and extraction
  • Overall preservation is good to excellent (where conventional processing was acceptable to good)
  • Reproducible technique (constant variables)
  • Time and reagent efficient

In this workshop, we will explain how the BioWave can save significant time in preparing tissue specimens.

Diamond Wire Saws for Microscopy Applications

Wednesday 3rd July, 12:30 – 13:00, Workshop 2. Presented by Paul Balas and Daniel Ebner – CEO of WELL Group

Micrometre, Nanometre, Ängstrom – three words we cannot avoid in microscopy today.

As investigations in both material and life sciences become more demanding, it’s important to study samples in their natural state regardless of the microscope performance. In most cases, samples are far too big to be imaged as they are, so cutting and sectioning is the first step for sample preparation.

If you require an instrument that can:

  • Cut any material from the softest to the hardest samples?
  • Cut without over-heating the sample?
  • Produce a cut that is clean, smooth, splinter-free, burr-free and with sharp edges?
  • Cut homogeneous or non-homogeneous materials?
  • Cut without dimensional deformations?
  • Cut without structural modifications?

In which case, WELL Diamond Wire Saws and Agar Scientific can offer the right solution. 

The Future of Sample Preparation

Wednesday 3rd July, 15:00 – 15:30, Workshop 1. Presented by Guillame Lardon and William Magnin from Lam Plan

Lam Plan has been developing, manufacturing and selling lapping and polishing products since 1936. All over the world, the company has been helping its customers reach their objectives by designing efficient machines combined with effect abrasives and polishes. 

We built our expertise initially in the lapping industry, taking care of flatness and surface finishing. Thanks to the knowledge gained in this industry, we began to develop (20+ years ago) our own consumables and machines dedicated to sample preparation.

Nowadays, Lam Plan has become a specialist in various industries, such as scientific, automotive, aeronautic, watchmaking as well as heat treatment. We understand that sample preparation is always expensive, time-consuming and skilful. This is why, with our testing room and laboratory, we are able to provide full support to our customers in order to allow them to reach their objectives, as well as help them in making a more efficient preparation.

This workshop will be a unique opportunity to present you an actual example of sample preparation improvement, with a company working in the aeronautic industry.

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The WELL Diamond Wire Saw in Action

The experimental volcanology and geothermal research group at the University of Liverpool, supervised by Prof. Yan Lavallée, focus on investigating the mechanical and textural properties of magma and rocks at high temperature.

Two of the PhD students in the research group, Josh Weaver and Jenny Schauroth, use a WELL Diamond Wire Saw in their work to section delicate volcanic material. This is what Josh had to say, along with the great image they provided:

“The image shows a sample of obsidian (volcanic glass) that has been heated to a liquid state in a furnace, allowing the dissolved gases in the magma to form bubbles. 

The standard approach to producing these SEM images involved setting the sample in epoxy resin and manually polishing it for several hours. The wire saw reduces this preparation time drastically as the sample face is polished by the cutting, leaving no visible scratches. The sample in the image was placed in the SEM directly after cutting, with no other preparation required. The wire saw also has the ability to cut thin sections in the micrometer range and reduces precious sample waste.”

For more information on the WELL Diamond Wire saws, visit the page on our website.

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‘In conversation’ discussing the easiGlow glow discharge system with Field Sales Engineer Ben Hayllar

What is easiGlow?

The easiGlow Glow Discharge Cleaning System

The easiGlow Glow Discharge Cleaning System is a compact, easy-to-use standalone system. It’s designed to modify the wettability of Carbon film on TEM grids and to clean TEM grids. It’s bench mounted, straightforward to set-up with only one click required to complete a fully automated cycle.

The factory-installed program can be used for most applications where a hydrophilic film is required. This can be run immediately after installation just by selecting ‘Auto Run’ on the touchscreen interface. The operator can change the parameters within the factory-installed program and can create four further protocols with parameters to suit specific applications. The protocols can be stored to facilitate consistent and repeatable glow discharge results. The easiGlow is fully controlled through the touchscreen with three modes; auto, programmed and manual.

A complete system can be supplied, including the vacuum pump, or users can use an existing pumping system as long as it meets the required specifications and can fit the easiGlow vacuum port (a KF16 flange vacuum inlet).

With two independent gas ports, it is possible to create a protocol with two different gasses for the glow discharge cycle and venting the chamber when the cycle ends. The easiGlow offers high versatility and freedom to choose the best conditions for optimum results.

This simplified operation and the minimal training required makes the easiGlow particularly useful in multi-user facilities.

Why is it so useful within EM?

The Carbon support films on TEM grids tend to be hydrophobic. However, a glow discharge treatment will make a Carbon film surface negatively charged (hydrophilic), therefore allowing aqueous solutions to spread easily. In this way the whole TEM grid will be covered with sample and agglomeration will be avoided.

The easiGlow can also be used with different parameters to make the Carbon film positively charged, in case of special applications such as DNA analysis. Also, glow discharge treatment of TEM grids removes adsorbed hydrocarbons, therefore cleaning them while making them hydrophilic.

Consequently, easiGlow allows preparation of a good sample to be imaged and analysed by the electron microscope.

Could you explain how the easiGlow works?

A glow discharge is a kind of plasma. A partially ionized gas, consisting of positive or negative ions, electrons together with a large number of neutral atoms, are created inside a chamber under vacuum by applying a high potential (a high DC voltage) between two electrodes.

The easiGlow uses either air, other gas or a small quantity of liquid which under vacuum will sublimate as a source of ions. The electronics automatically control the voltage applied between the two electrodes and the polarity (which electrode is positive and which is negative) in such a way to have a stable current (mA) of discharge and to have the desired ions deposited on the Carbon film. This will give the carbon film an overall positive or negative charge.

All the parameters that characterise the type of glow discharge are software controlled by setting up the program or protocols via the touchscreen. After the Carbon film treatment is finished, a carefully controlled flow of air is released inside the specimen chamber to avoid displacing the grids inside.

How is it used?

The easiGlow is fully automated and controlled from an intuitive touch screen display. Once the grids are loaded into the vacuum chamber, the selected programme controls the vacuum pump, and once the required vacuum is created it initiates the glow discharge process. For routine grid preparation, just one button instigates the complete cycle: vacuum, glow discharge treatment and flushing.

How long does the process take?

For the most common applications, making TEM support films or grids hydrophilic, the automated process takes less than two minutes, from the time it’s turned on to the end of the cycle.

What if both hydrophilic and hydrophobic treatments are required?

easiGlow dual system

For labs needing both hydrophilic and hydrophobic surface treatments, a dual easiGlow configuration is available to avoid cross-contamination of glow discharge chambers. However, this will only be required when both hydrophilic and hydrophobic surface treatments are required on a regular basis. Having two stand alone easiGlow systems avoids amylamine contaminating not just the chamber but the pumping system as well. Amylamine is added to the easiGlow chamber during the cycle hydrophobic treatment.

Which Glow Discharge Methods are supported?

EasiGlow supports both hydrophilic and hydrophobic treatments with positive and negative charges, as shown in the table below:

Surface StateChargeTreatmentTypical Applications
Hydrophilic Negative AirCarbon-coated TEM grids
Hydrophilic  PositiveAir (with subsequent
magnesium acetate treatment)
Nucleic acid adhesion to carbon films
HydrophobicPositiveAmylamine (Pentylamine)Proteins, antibodies and nucleic acids
HydrophobicNegativeMethanolPositively charged protein molecules, (e.g. ferritin, cytochrome c)

What applications is the easiGlow used in?

The easiGlow is used frequently in Cryo-EM and single particle analysis but also where the application requires the Carbon film on TEM grid to be hydrophilic such as analysis of emulsions.

Nucleic acids (DNA) have a negative charge, therefore will be repelled by negative ions deposited by normal glow discharging. Glow discharge with a post-treatment of magnesium acetate provides hydrophilic films with a positive charge for DNA. Alternatively glow discharge using amylamine also provides a positive charge, but with a hydrophobic film.

Where can we see the easiGlow in action?

We can arrange demos, at your premises or at Stansted, set up a fully operational system and run through the process.

Thanks Ben, that’s all very interesting.

Click here for the full specs for easiGlow.

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Non-Ducted Fume Cabinet FAQs

Meet one of our new products: the Agar Scientific Non-Ducted Fume Cabinets.  Featuring the very latest in air filtration technology, they are perfect for a wide variety of lab applications. Here are some answers to Frequently Asked Questions regarding the fume cabinets:

Are Non-Ducted Fume Cabinets safe?

Non-Ducted Fume Cabinets are as safe as Ducted Fume Cabinets when matched to the appropriate application. Non-Ducted Fume Cabinets are a good choice for laboratories to avoid costly HVAC upgrades and still be able to manage a variety of filtration requirements.

How versatile are Non-Ducted Fume Cabinets?

As requirements and applications change over time, Non-Ducted Fume Cabinets can be adapted to fit new air filtration needs. Filters in are interchangeable and can be customised to protect against a wide variety of chemicals.

Do Non-Ducted Fume Cabinets maintain consistent face velocity?

Non-Ducted Fume Cabinets have high capacity air handling systems to deliver 100 fpm (feet per minute) face velocity. This velocity is sufficient to contain chemical fumes and particulates. Advanced monitoring technology also ensures that this face velocity is maintained consistently, if velocity decrease an audible alarm sounds to warn technicians.

Will a Non-Ducted Fume Cabinet work in my laboratory?

There are certain factors to take into consideration, such as the chemicals you are filtering, how much of each chemical, and whether the environment is caustic or corrosive. Non-Ducted Fume Cabinets are designed for easy installation and can be moved around the laboratory as needed.

How do I monitor my carbon filters?

Each Non-Ducted Fume Cabinets is equipped with a filter saturation alarm that alerts you when the filter needs to be changed, ensuring complete safety for your technicians and equipment.

Are there safety features in the Non-Ducted Fume Cabinets?

Non-Ducted Fume Cabinets can include optional safety filter to offer increased protection across the range of chemicals used in an application. A pre-filter is standard and is coupled with either a customized carbon filter or HEPA / ULPA filtration.

Are Non-Ducted Fume Cabinets as technologically advanced as other types of fume cabinets?

Non-Ducted Fume Cabinets have a variety of built-in alarms to monitor airflow and filter saturation. Manual speed controllers manage fan speed and a variety of control options can be operated independently or tied into a larger, remote controlled monitoring systems.

What type of filtration is available in a Non-Ducted Fume Cabinets?

Carbon filters that protect against a variety of chemicals are available, as are HEPA and ULPA filters. 

How quiet are Non-Ducted Fume Cabinets?

Most Non-Ducted Fume Cabinets have a noise level of less than 55dba at one meter. Some units are even quieter, depending on the fan used and the required airflow inside the hood.

Are Non-Ducted Fume Cabinets economical?

Non-Ducted Fume Cabinets not only save money on extensive HVAC and laboratory utility upgrades for installation, but also cut costs over time by operating more efficiently than other laboratory hoods. Efficient fan motors, low maintenance designs, and complete monitoring systems mean that Non-Ducted Fume Cupboards can cut utility costs and provide a low-cost solution for filtration in a variety of laboratories.

Which industries currently use Non-Ducted Fume Cabinets?

Non-Ducted Fume Cabinets are installed around the world in a variety of industrial and laboratory applications. Non-Ducted Fume Cabinets can be used in the life sciences, pharmaceutical manufacturing, forensics and evidence collection, industrial research, education and environmental sciences.

Can I use Non-Ducted Fume Cabinets and Ducted Fame Cabinets in the same facility?

In most laboratories, a combination of Ducted Fume Cabinets and Non-Ducted Fume Cabinets is an effective way to ensure complete filtration for all chemical applications. Non-Ducted Fume Cabinets can be incorporated to help expand the capabilities of an existing lab, while avoiding additional construction costs and ongoing HVAC and utility expenses. Non-Ducted Fume Cabinets can also be on casters to allow the unit to be moved around within a facility, providing ultimate flexibility of placement.

Do Non-Ducted Fume Cabinets run continuously?

They do not have to run continuously, but can if that is what your application requires. If continuous operation is not required, power switch controls turn off the fan and other monitoring systems. This can help cut utility costs and save on equipment wear and tear.

How do Non-Ducted Fume Cabinets affect future facilities planning?

Non-Ducted Fume Cabinets are invaluable in future facilities planning. If you only have a short-term research contract, are a start-up operation, or are located in a building with no existing HVAC system or where one would be difficult to install, Non-Ducted Fume Cabinets can allow you to get to work immediately. Once installed, you can have complete confidence that your investment will be 100% portable and re-usable.

Filter Guide

FilterSuitable for the removal of
SolventsGeneral organic compounds (iodine, solvents, odours, etc.)
Mineral AcidAcidic compounds (and general organics)
AlkaliAlkali compounds (and general organics)
AmmoniaAmmonia & amine compounds (and general organics)
CyanideCyanide compounds
Diethyl EtherEther compounds
AldehydesAldehydes (and general organics)
SulphursSulphur compounds (and general organics)
CustomSpecial blend of up to 4 of the above carbon types
EducationalTri-layered filter for schools (organic, acid & alkali
removed)

Visit the Agar Scientific Non-Ducted Fume Cabinet webpage for more technical information on the fume cabinets.

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Microwave Sample Preparation: Recipes for Success

BioWave Pro+ Microwave Tissue Processor

We recently attended the Scottish Microscopy Group’s 46th Annual Symposium in Aberdeen. This annual meeting focuses on bringing together scientists using and
developing microscopy and image analysis techniques, and features talks and poster sessions.

One particularly interesting talk was on the BioWave microwave tissue processing system by Kevin Mackenzie from the University of Aberdeen.

Kevin has worked in the microscopy field at the University for over 35 years and is currently Manager of the Microscopy and Histology Facility. The facility offers 18 different microscope systems employing a wide range of techniques for imaging specimens; including light, fluorescence, laser, EM and x-ray.

The University of Aberdeen Microscopy and Histology Core Facility purchased a BioWave from Agar Scientific earlier this year and have had great results with support from Shahriar and Ben from Agar’s sales team.

The BioWave is a sophisticated microwave tissue processing system enabling rapid specimen processing with consistently high quality results. The use of microwaves enhances sample preparation for TEM, immunofluorescence and light microscopy staining.

Kevin, along with his colleagues Gilian Milne, Debbie Wilkinson and Lucy Wight, have produced a great poster explaining the significant time benefits from using BioWave.

The poster describes three examples for TEM, Immunofluorescence and LM, comparing the conventional and microwave process showing significantly reduced sample turnaround time.

Kevin has kindly shared the poster – download a copy here.

See more information on the BioWave Pro+ Microwave Tissue Processor here.




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Introducing… CorrStub™

A new SEM specimen stub for correlative microscopy

When analysing the characterisation of organic and non-organic samples this usually involves the collection of images and data using a variety of instruments and techniques.
In the majority of cases the use of a single analytical approach is unable to provide all of the answers when analysing these specimens.

In an ideal world, determining the complete characterisation and analysis for the same area of interest in a single sample would be achieved through using complementary approaches, for example X-ray diffraction and light microscopy.

With this in mind, Agar Scientific developed a unique range of SEM pin stubs designed specifically for correlative microscopy and forensic gun shot residue (GSR) analysis. We called this range CorrStub.

CorrStub allows the user to determine the precise location of an area of interest through a number of compatible imaging and analysis platforms.

For example, a specific point on the surface of the sample can be analysed through FIB-SEM before or after transferral to an X-ray spectrometer, an X-ray diffraction system or a light microscope.

CorrStub includes the following unique and useful features:

A precise V-notch on an SEM stub. This has many advantages, as it offers a precise X-Y reference point to analyse any sample. The intersection of the X and Y axis of the V-notch also provides a zero coordinate reference point. This means that images acquired from a number of instruments can be overlaid using a crosshair and micrometric stage. Also, the stub surface can be precisely mapped in relation to the V-notch reference point, meaning the stub can be re-visited at a later date or on another imaging platform.

CorrStub can be supplied laser etched with a unique combination of one alphanumerical and four numerical characters for sample identification

CorrStub is available as a standard 12.5mm dia specimen stub to fit LEO/CAMBRIDGE, FEI/PHILIPS, CAMSCAN, TESCAN and ZEISS instruments and can be supplied pre-mounted with either high conductivity Al core carbon tabs or Leit tabs precisely applied and ready loaded in individual plastic tubes or boxes of 12.

CorrStubs can also be supplied with pre-mounted Aluminium core carbon tabs. Compared to a standard carbon tab, these tabs have a pore-free surface which, when combined with the V-notch, makes imaging and analysis easier when adopting a variety of techniques and instruments. Both sides of the carbon tab are covered by aluminium foil and carbon-based adhesive compound, which reduces surface charge during FIB-SEM.

Find out more about CorrStub here:

https://www.agarscientific.com/sem/specimen-stubs-mounts/corrstub-sem-pin-stubs-for-correlative-microscopy




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It’s good to hear from our customers from time to time!

Paul Simpson is the Cryo Electron Microscopy Facility Manager at Imperial College London. Their Biosciences EM Facility has a number of TEM and SEM Microscopes. He has been purchasing products from Agar Scientific regularly. This was some of the feedback he gave us:

“After shopping around to purchase Quantifoil support grids, we chose to use Agar Scientific for a number of reasons; mainly being their website and customer service.

Agar Scientific’s website is easy to navigate. They provide important information such as the price of items without having to request a quote, and more importantly the stock levels and lead times for products that are not in stock. Knowing lead times for products is extremely important to us as it allows us to plan our orders, especially when purchasing a popular product such as Quantifoil support grids.

Their website also gives you the option to buy now, request a quote on different products and also to view your order history so you can keep track of what you have bought previously with Agar Scientific.

Customer service was also key for us; following a poor after-sales experience with an overseas supplier, we cancelled an order with them. We decided from this point we wanted to use a local supplier, with better service and support. Agar Scientific have not disappointed and have proved over the last 10 months to be a reliable supplier, who deliver on time.

Agar’s account managers are in regular communication with us; through both email and in person. They check on our orders and ask for feedback on how to improve their service, and I value this pro-active approach.

I would highly recommend Agar Scientific as a supplier for both TEM and SEM products!”

Thank you, Paul, for the feedback.




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