As we draw towards the end of 2019, we will be attending some exciting conferences in December 2019 and January 2020. Here’s a rundown of what we’ll be attending and when…
47th Scottish Microscopy Symposium: 3rd December 2019
The 47th Scottish Microscopy Symposium will be held on 3rd December 2019 at The Roslin Institute in Edinburgh. This year there’s:
More submitted talks than ever before
A broad spectrum of industry representation to find out about the latest technologies in the field of microscopy
A wide range of talks from across many disciplines and career levels
Poster presentations with best poster prize
Imaging competition with 1st and 2nd place prizes to win
Our field Sales Executive, Jake Panesar, will be attending and will be pleased to meet you there.
SEMT One Day Meeting: 11th December 2019
The annual SEMT One Day meeting will be held at the Natural History Museum. Topics covered by the 2019 meeting include electron microscopy, confocal microscopy and elemental analysis as well as microscope self-build.
The RMS Beginners Competition will also be hosted at the meeting – the beginners competition is specifically aimed at students, interns, apprentices and faculty staff who have given no more than two talks outside their home institution. Entry to this competition is open now and will close on 29th November 2019.
Our Field Sales Executive, Ben Hayllar, will be attending this meeting.
Hosted at the University of Plymouth on the 9th & 10th of January, the EM-UK community meetings are designed to be an open forum for discussion of the latest developments and challenges in the field, suitable for both academic and commercial microscopists.
The meeting will include Techno Bites, talks, discussions about training, an evening meal and plenty of networking opportunities.
Field Service Executive, Ben Hayllar will be attending EM-UK 2020 and will be pleased to meet you there!
Earlier on in 2019, Agar Scientific launched a new direct replacement for Uranyl Acetate – UA-Zero.
UA-Zero is non-radioactive, non-toxic and can be used as a direct replacement to Uranyl Acetate without any changes to standard user protocols.
With the help of Professor Dr. Paul Verde and Dr. Chris Neal from the Wolfson Bioimaging Facility, School of Medical Sciences at the University of Bristol, we have written 4 application notes (so far!) to describe examples of protocols for preparing samples and subsequent imaging using TEM.
This application note is the fixation, staining and processing of HeLA* cell pellets from a culture dish. Uranyl Acetate and UA-Zero were directly compared and the images generated will be shown later on in this blog post.
*HeLa cells are derived from cervical cancer cells taken on February 8th, 1951 from Henrietta Lacks, nowadays being the oldest and most commonly used human cell line in scientific research.
Staining the sample on a TEM grid with a support film requires just a few simple steps before drying, shown in the images below:
Protocol used for TEM sample preparation
All steps within this protocol are to be performed at room temperature unless otherwise stated.
Fixation: 1.5% glutaraldehyde in 0.1M cacodylate buffer (1 hour)
Remove cells to Eppendorf tubes – spin down scraped off cells into cell and supernatant
1.5% glutaraldehyde in 0.1M cacodylate buffer at 4°C (Overnight)
0.1M cacodylate washes (3 x 10 mins)
2% OsO4 (AGR1021) in 0.1M cacodylate buffer (1 hour)
0.1M cacodylate washes (3 x 10 mins)
Deionised water (2 x 5 mins)
Staining: UA-Zero EM Stain in 20% ethanol / 3% Uranyl Acetate in water (1 hour)
Written by Dr Brian Causton, founder of London Resin
The formulation now known as LR White first saw the light of day in 1968. I was studying corrosion in submarine battery grids and needed to prepare metallurgical samples that preserved the fluffy structure of the corrosion product in exact register to the corroding alloy beneath. The existing embedding material was too viscous, and because I was using an early surface analyser called the Cambridge Geoscan the electron beam was ablating my old embedding resin, causing the specimen to disintegrate. So, instead I made a very low viscosity embedding material with a good electron beam stability. In those days we tended to synthesise our own resin components and I based my formula on acrylics because they were less sensitising than epoxies and urethanes. The formulation worked perfectly and I was able to prove the corrosion originated as a result of segregation in the battery grid alloy.
A year later I had seized the opportunity to work with Mike Braden at the London Hospital Medical College in the rapidly growing field of Biomaterials. ‘The London’ was a very inclusive place to work and quickly I was being helped in my research by the advice and sharing of resources of the schools many departments. Foremost among them was Professor ‘Loma’ Miles’ Oral Pathology department. John Linder (JL) was head of the Oral Pathology Laboratory.
It was JL that opened my eyes to the complexity of biological tissue. He greatly increased my knowledge of Light microscopy and would let me use his precious interference microscope to study the propagation of cracks in implants and bio-adhesives. JL was a magnet for struggling young researchers and as a result I met many people in need of solutions to difficult embedding problems.
As the only polymer chemist in the room I was for ever making novel resin system for my colleagues to try as they investigated tissues as diverse as healing burns to old stab wounds to failed hip protheses. However, as a junior academic in the early seventies my most popular formulation was a rust stabiliser, an essential aid when getting ones car through the MOT!
I began to investigate the biofilms that form on implants in the early 70’s. To do this I resurrected my old embedding resin from ’68. The reason being that I needed the help of Wally Goss and his electron microscope to understand the fine detail of the process and didn’t want to use the far more toxic low viscosity epoxy resins on offer at the time. I had joined the RMS by then and had started to try and persuade microscopists to use acrylics as opposed to epoxies for reasons of health and safety. I would slip people bottles of my resin at conferences and RMS regional meetings. Soon I was taking my resin to conferences abroad, I knew no shame in my missionary zeal to convert the microscopy world to acrylics. I was still synthesising more than half the components of the resin, though the growth of soft contact lenses, plastic dental fillings and false nails had made some of the components more available.
At this time, I was starting to get useful feedback on my formulation. It’s use embedding seed coatings and other highly fibrous tissue was proving particularly advantageous. This was to be expected because the formula was designed to enhance the diffusion coefficient of the tissue to the large crosslinking components of the resin by the smaller more agile components of the mix heading the diffusion front and lowering the glass transition temperature of the fibre bundles. The light microscopists, with their large specimen blocks were reporting instances of chattering when cutting thin sections and the electron microscopists with their small blocks reported very little. The cause was the nature of the exothermic curing reaction of acrylics. If the exothermic reaction is allowed to run away this results in a much higher degree of crosslinking in the cured resin. When the resin is cut the crack that grows ahead of the knife blade accelerates away from the blade edge, quickly reaching its terminal velocity in the cured resin. At this point the crack bifurcates and accelerates even more quickly, bifurcating again and again. This is the cause of the chattered surface.
I mention this because I learnt a great deal from this feedback, I learnt that size matters. When using radical polymerised embedding material tailor, the curing instructions to suit the size of your specimen, the larger the specimen the slower the cure should be; lower the oven temperature, reduce the accelerator a little, be watchful of the intensity of light source used in the light-curing of the resin. At this time the other useful feedback evolved around the infiltration of the resin into the block. I mentioned that the resin formula consists of large bulky components and fast diffusing small components. The small components have to be given time to lower the mechanical glass transition temperature of the biomass it is diffusing through. The difference in diffusion coefficient between a mass below it glass transition temperature and one that is above is 1000 times. If the infiltration is rushed and the curing started prior to equilibration, parts of the specimen will only have the smaller components of the resin present, leading to heterogeneous crack propagation during sectioning.
For ten years I gave out small bottles of the resin and built up a feel for how best to use the resin in a wide variety of circumstances. However, during this time there had been an increase in the use of immuno-chemistry in both light and electron microscopy. At the time one of my main fields of study had been the formation of biofilms, and the bodies response to implants. My approach to biofilm formation was to make implant coating that had the structural and charge characteristics of both free and surface perturbed water. At the time I was privileged to give small chats at Julia Polak’s immuno-histology summer school at the Royal Postgraduate Medical School. I immediately saw a link between the need to embed the tissue in such a way that the labelled antibodies could interact with their targets, and my own work on implant biofilm progenesis. So I started slipping bottles of my resin to immuno-cytochemists, modifying the formula to allow the resin to be cured at low temperatures with blue light using Benzil as an initiator. This later became known as LR Gold.
It was Tony Robarts from York that suggested I made the resin in commercial quantities. Ironically, I asked Alan Agar, who had founded a company called Agar Aids if he would like to manufacture the resin, but he said no at the time. So I approached the trustees of the London Hospital.
Back in 1919 an improved form of resorbable catgut suture had been invented in the surgical department. A company had been set up to manufacture the sutures and was known as The London Catgut Company. Little did I know that the company had recently gone into liquidation and my suggestion of founding a new company called The London Resin Company, only raised painful memories in the minds of the trustees. However, they said that if I wanted to use the name that was fine by them. JL and Wally Goss had retired by then so I invited their successors, Jocelyn Germain and Roy Gillette, to join me in the venture.
I was to make the resin in my garage in Basingstoke, what could be simpler. Well for a start, commercial resins and monomers are sold in 200kg barrels, not handy Winchesters, also the barrels were delivered by juggernauts from Rotterdam at 6:00 in the morning to my house in a twee new development with narrow roads. I heard the juggernaut reversing down the road and groaned as I saw it missing neighbours’ cars by inches. The friendly Dutch driver asked me where the factory and my forklift truck were, after he stopped laughing, we had to come up with a cunning plan. We took the mattresses of the beds and placed them next to the lorry in a pile, he rolled the 200kg barrel off the lorry and onto the mattresses and I stopped it from flattening my neighbours’ roses. As he drove of down a sea of twitching curtains, it dawned on me that three more barrels were on their way. By the time they arrived I had made a ramp and winch mechanism. However, I was at The London when the next barrels arrived so my wife Chris got the other barrels off the lorries.
At this time a search of the liner the QE ll at Southampton docks had revealed bomb making equipment and the local TV that night were appealing for the public to report any strange deliveries of bulk chemicals. No one snitched!
Forty years on and two factories later, Agar Scientific has taken over the manufacture of the LR White and LR Gold resins in their new resin manufacturing facility at Stansted. I quite miss my old factory in Berkshire, but I miss the chance to answer customer queries more.
The introduction of uncatalysed LR White arose from just such an exchange. Soon after the launch of LR White I got a letter from the Clove Diseases Research Centre in Madagascar asking if we shipped to the tropics, the only way to find out was to send them a free bottle, which duly arrived pre-polymerised, not a great start. So, I made up a special batch with no catalyst, sent it off with the catalyst in a separate bottle and the diseased cloves were embedded without further ado. Uncatalysed LR White is still in production today.
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.
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!
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.”
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.
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.”
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
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
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
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
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.
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
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.
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.
if both hydrophilic and hydrophobic treatments are required?
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.
Glow Discharge Methods are supported?
EasiGlow supports both hydrophilic and
hydrophobic treatments with positive and negative charges, as shown in the
Carbon-coated TEM grids
Air (with subsequent magnesium acetate treatment)
Nucleic acid adhesion to carbon films
Proteins, antibodies and nucleic acids
Positively charged protein molecules, (e.g. ferritin, cytochrome c)
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.
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.