As we are all experiencing the impact of COVID-19, we would like to take this opportunity to inform you of the actions we are taking as a company to protect our employees and ensure the business runs smoothly for our customers and distributors.
We have set up systems so our office team can work effectively from home to prevent interruption to any communication, response times and order confirmations. Inevitably, as with any new systems, there will be some bedding in while we all adapt to new ways of working but we hope any disruptions will be minimal.
For our warehouse and lab teams, who cannot work from home, we are implementing other measures including split shifts, reconfiguring production sections and rearranged welfare areas, all to help protect the health of our team.
We are actively liaising with suppliers to monitor any effects on our supply chains and any changes will be reflected in the lead times on our website. Presently we have good stock and have not seen any major disruptions – but be assured we are keeping a close watch.
We are also keeping a close eye on government and WHO advise and will navigate accordingly if and when things change.
Please do not hesitate to get in contact with any of our team members if you have any questions or concerns.
From all the Agar Scientific Team – we hope you and your colleagues and families are keeping safe and well in this difficult time.
Last year, Agar Scientific launched a new direct replacement for Uranyl Acetate – UA-Zero. In this blog post we describe the protocol for using UA-Zero for tobacco Mosaic Virus (TMV) negative staining.
UA-Zero EM Stain is a patented solution developed as a direct replacement for Uranyl Acetate. UA-Zero does not contain any radioactive material and is non-toxic. UA-Zero can be used as a direct substitute for Uranyl Acetate with no changes to standard user protocols.
UA-Zero is supplied in an opaque dropper bottle, which should be stored long-term in the lab fridge at around 4°C. For daily use, the sealed bottle can be stored in normal laboratory conditions away from direct sunlight.
It has been shown that UA-Zero EM stain works well for various protocols that specify Uranyl Acetate as a staining solution for TEM sample preparation in life science. This blog post describes one example of a protocol for preparing samples and subsequent imaging using TEM. Uranyl Acetate and UA-Zero were directly compared and the images generated are shown later on in this post.
Staining the sample on a TEM grid, with a support film, requires just a few simple steps before drying.
Protocol used for TEM sample preparation
All steps performed at room temperature unless otherwise stated. Parts of the same sample have been stained using UA-Zero EM Stain and Uranyl Acetate, for direct comparison.
Suggested dilution for virus titre in solution between 1:20 and 1:100 for negative stain.
From TMV stock, make 1:20 dilution on Phosphate-Buffered Saline (PBS)
Incubate 20μl TMV solution on pioloform 300 mesh copper grids – 1 min
Blot and allow to dry – 5 mins
Staining: UA-Zero EM Stain in 20% ethanol OR 3% Uranyl Acetate in water – 1.5 mins
TEM images of sample prepared using UA-Zero EM Stain in 20% ethanol as staining solution
TEM images of sample prepared using 3% Uranyl Acetate in water as staining solution
Samples prepared and imaged courtesy of Prof. Dr. Paul Verde and Dr. Chris Neal, Wolfson Bioimaging Facility, School of Medical Sciences, University of Bristol.
When using LR White embedding resin for electron microscopy, very few changes need to be made to the regime used for epoxy resin embedding.
Every laboratory has its own individual embedding schedule, but we have laid out a “typical” schedule in this blog post for LR White as guidance for its use.
No change from normal fixation should be made if EM analysis is only required from the final blocks. However, if good ultrastructure and a wide range of LM staining is required then we have found that the use of aqueous paraformaldehyde in a phosphate buffer pH 7.2 with 2.5% w/v sucrose is the best compromise. Glutaraldehyde alone and Karnovsky’s glutaraldehyde-formaldehyde mixtures may need to patchy LM staining and some stains not working or giving “false positives” (e.g. PAS) whereas normal formalin fixation yields unacceptable EM ultrastructure.
For dual LM/EM applications, Osmium Tetroxide should be avoided due to its effect on many LM stains but 1% phospho tungsten acid (w/v) in the first absolute ethanol step of dehydration improves electron contrast without adversely affecting most LM stains. Osmium Tetroxide may be used if the blocks are required for electron microscopy only.
A graded ethanol series is the method of choice when embedding in LR White. Acetone acts as a radical scavenger in the resin system and therefore traces of acetone left in the tissue at curing can interfere with this polymerisation. For this reason, the use of graded acetone series and dimethoxypropane (which generates acetone) are best avoided. If the use of dimethoxypropane is considered vital we recommend either a protracted resin infiltration or washing the tissue with dry ethanol prior to infiltration in order to minimise the chance of acetone contamination of the final resin.
The extremely low viscosity of LR White may be exploited by allowing the use of short infiltration times or large specimens – but not both! A 1mm cube of animal tissue will be adequately infiltrated in about 3 hours if 4-6 changes of LR White at 60°C are employed during this period.
An overnight infiltration at room temperature, followed by two short changes of resin will often be more convenient, however, the long shelf life and low extraction rate of LR White allows specimens (e.g. reserve tissue) to be stored safely in resin for many weeks at 4°C if required. Larger blocks do require significantly longer infiltration times compared to small blocks.
Samples stained with Osmium Tetroxide should not be “cold-cured” with the accelerator. This process is strongly exothermic, and the dark colour of the tissue leads to a focal heat accumulation which can cause local problems in and around the tissue.
If the issue is not fixed with Osmium Tetroxide then curing with LR White accelerator may be employed. As with curing blocks for light microscopy we recommend cooling the moulds during polymerisation, but there is no need to exclude oxygen from the surface of the curing block.
Thermal curing should be used for osmicated specimens and may be used for all specimens. Here it is important to limit the contact of oxygen with the resin while polymerisation occurs. The most convenient way of achieving this with capsule-type embedding is to use gelatin capsules (available from our website), fill up to the brim and slide the other half of the capsule on.
If flat embedding is required for cutting orientation then the surface of the resin must be covered to prevent contact with oxygen.
One convenient method is to utilise the JB-4-type moulds and chucks, useful for light microscopy, and after polymerisation may be sawn off the stub and mould re-used.
Polymerisation time and temperature are fundamental to the physical characteristics of the final block to a much higher degree than with undercured epoxy systems.
We strongly recommend a temperature of 60°C +2 for a period of 20-24 hours. Some ovens are not capable of controlling polymerisation temperature so closely, and if faced with over brittle blocks, this is the first parameter to check.
LR White has extremely good powers of penetration and can penetrate and soften some low-density polyethylene capsules, which causes them to distort and collapse. Also, polyethylene is not impermeable to oxygen and may allow enough contact with atmospheric oxygen to give the blocks an inhibited “tacky” surface.
Both these problems may be overcome by the use of gelatin capsules (size 00 is similar to the popular polyethylene capsule size) and these are much cheaper and easier to seal during polymerisation.
Resin may be used straight from the refrigerator and has a very low toxicity both in monomeric and polymerised state, unlike epoxies. The cold cure accelerator does have some toxic risk and contact with the skin and eyes should be avoided.
For cold curing the accelerator should be used at one drop per 10ml of resin and this should cause polymerisation in between 10 and 20 minutes. If polymerisation occurs faster than this, we recommend either more careful metering of the one drop of accelerator or a higher volume of resin per drop of accelerator.
Trimming and cutting
Trimming the block may be done with a jewellers saw, razor blade or with a glass knife on the ultramicrutome as with epoxy resin blocks. Cutting mayo be performed in the same way as for epoxy resin with glass or diamond knives. A typical cutting speed of about 1mm per second is suitable.
All the common section stains give good results on tissue embedded in LR White resin. Stains made up in ethanol or methanol should be avoided as these solvents soften the resin and may remove sections from grids. As an alternative to Uranyl Acetate, 1% phosphotungstic acid has proved a good general-purpose stain, both as a block stain, as mentioned earlier, and as a section stain.
In the Electron Microscope
An initial reduction in electron density may accompany the initial exposure of the resin to the beam. This is thought to represent a loss of water, imbibed from knife-boat or staining solutions. Thinning as such does not occur and specimens have been kept stationary under a 120kV electron beam for 3 hours with no obvious signs of damage.
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.”