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.

Posted in General

Reading for Pleasure, Writing for Science

man and books

Essentially, when you author a paper or make a poster you are telling a story (albeit non-fiction!). You want to create a narrative and impart something unique to your audience.

Tell us a story

Your story should have a beginning (Introduction), middle (Methods and Results) and end (Results and Conclusions). Okay, so it’s not exactly ‘and they all lived happily ever after’! More like, ‘this work warrants further investigation’ and the characters wander off into the sunset in search of the elusive grant.

I’ve read more papers than I care to remember. I say ‘read’ in the loosest possible sense of the word. Some of the papers which I needed for my thesis and further research were dealt with in a vulture-like manner, stripping nuggets of information from the bones and body of the work (obviously in a manner which does not plagiarize). You know the sort of thing- you’ll read a review article and in amongst the thousands of words there’ll be that single sentence which confirms results you’ve seen.

A written word niche

Each and every one of us will develop our own writing style and this will be influenced by our work environment (and to a greater or lesser extent your mentor/professor/supervisor), but also by our environment of the written word in which each of us inhabits our own unique niche. No two people will ever read the same combination of blog/newspaper/journal/textbook/novel/comic. And, unfortunately, some of us may not even read for pleasure.

Reading for pleasure vs reading for work

When I was doing my PhD, it really turned me off reading for pleasure. Similarly, when I moved into different areas of research, the steep learning curves meant that I had to read another ton of articles. Reading for work really put me off reading for pleasure.

During my early career, I was an avid book worm. I would consume at least one novel or non-fiction book a week. I was living in the time of a new Scottish renaissance of writing with authors such as Iain Banks, Alasdair Gray, Irvine Welsh and AL Kennedy. At the same time, popular science books were really taking off- we’d had the early works of Richard Dawkins and the like, but now publishers were releasing books on topics ranging from viruses to quantum physics.

At this stage, I wasn’t required to read many papers or write a huge amount. I was running assays, gaining a grounding in histology and microscopy, trying (and failing!) with in-situ hybridisation. I had my name on posters, papers and abstracts, but merely as the person who had carried out the work. As my career progressed, it came with the expectation that I would start to contribute sections to publications. I started with the easy stuff- writing methods. As I was the one doing the work, this was no great hassle. However, it meant that I started reading more journal articles to examine and explore different styles and to try to replicate the way in which information is expected to be imparted to the readership of each of the journals. As I spent more time reading articles, it naturally meant less and less time for the books which I cherished.

To visualise and imagine

In my opinion, one of the most important aspects to being a good scientist is your ability to visualise and have a good imagination. Anyone with a reasonable memory can learn and regurgitate pathways and processes. But having a good memory doesn’t preclude that you have an understanding of the workings and mechanics of cells, biology and organisms.

I like to think that I have a reasonably visual imagination. I have absolutely no proof of this, but it may be partly due to the fact that I grew up reading comics and have continued to do so to this day. Comics are still regarded by some as ‘childish’- but, there are a large percentage of comics which are written especially for adults and mature readers. Some studies have shown that when we think like children, we are actually more creative. So, perhaps my years of reading comics has created a more visual and imaginative network in my neurons.

It was when I embarked on my part-time PhD that both my time for reading, and the material that I read was substantially narrowed. I had moved into a new field of research and facing the near vertical wall of learning a new area, I read very little else apart from journal articles.

Read far and wide and see the big picture

As you become more and more embedded in your own field of research, it can be difficult to see over the edges and to see the bigger picture. It’s an easy trap to fall into. If possible, try to continue reading for pleasure and reading around the edges of your own specific field. It all helps to see how your relatively tiny area of work fits in and can make a difference to the world- and the way you write.

Posted in General

Arise SiR Hoechst! A New Far-Red DNA Stain

Some of the most common cell-permeable DNA stains for imaging are the so-called ‘Hoechst Stains’ which are named after a German chemical company of the same name. These stains, which are also known as bisbenzimide stains, are part of a family of blue fluorescent stains which bind to the adenine-thymine regions of DNA. The number which follows each Hoechst stain refers to the sequential production of compounds by the company, so Hoechst 33342 is actually the 33,342nd compound which was synthesised by the Hoechst AG Company (which is now part of the Sanofi Group).

The problems with Hoechst

Although the Hoechst stains are generally easy to use, and non-toxic at low concentrations, their excitation wavelengths fall into the ultraviolet spectrum from around 350 to 390 nm. When imaging live cells, such blue light can be phototoxic. In addition, due to their DNA binding nature, the Hoechst stains can interfere with normal replication in long term live cell imaging experiments. Furthermore, such DNA stains are incompatible with super resolution microscopy techniques such as stimulated emission depletion (STED) microscopy.

A combined answer

Researchers at the Ecole Polytechnique Federale de Lausanne (EPFL) in Switzerland have been working on the aforementioned problems. Previous work from the group has focussed on the fluorophore known as silicon rhodamine (SiR) which is a photostable probe with excitation and emission wavelengths in the near-infrared part of the spectrum (ex 650 nm/em 670 nm) 1. This probe binds to a part of the DNA double-helix known as the ‘minor groove’ and due to its specific binding properties, does not interfere with normal cell function and replication 2. In addition, SiR probes exist in an equilibrium between fluorescent and non-fluorescent states and only become fluorescent upon binding to cellular targets.

The team at EPFL have synthesised an SiR conjugate known as ‘SiR Hoechst’ with excitation/emission wavelengths of 652 nm/672 nm 3. In their Nature Communications paper, the research group reported that SiR Hoechst possessed the highest specificity for nuclear staining when compared to three other commercially available far-red DNA probes 3. Although the probe SYTO 61had a higher fluorescence intensity than SiR Hoechst in the experiments, this comes at the cost of a very high background signal.

For the live cell confocal imaging experiments, HeLa cells were chosen and examined for up to 24 hours. The three commercial probes which were compared to SiR Hoechst substantially sensitised cells to phototoxicity over this time period as well as inhibiting cell proliferation even in non-imaging control experiments 3.

From cells to whole organisms

In addition to the HeLa cell line, primary human fibroblasts were also stained with SiR Hoechst and imaged using the super resolution technique known as STED. In these live-cell experiments, chromatin structures were revealed at a resolution of less than 100 nm. In contrast, two commercially available probes were incompatible with the 775 nm laser used for STED. The SYTO 61 probe was compatible with the STED system in these experiments, however, this was found to be inferior when compared to SiR Hoechst due to a decrease in staining specificity and an increase in toxicity 3.

Whole organism imaging was demonstrated using the pupal stage of Drosophila with a spinning disc confocal microscope. In the epithelial cells of the pupae, the team were able to visualise chromosomes during cellular division with SiR Hoechst for several hours without the detrimental effects of phototoxicity 3.

On sale now!

Through the EPFL, the team behind the research established a start-up company called ‘Spirochrome’ 4. The new SiR Hoechst probe, along with a number of other SiR based fluorophores, is now commercially available to the research community.







Posted in General

Santa-ology: The Science of Christmas Present Delivery


It’s that most wonderful time of the year when the big bearded man in the red suit drops down our chimneys and leaves a stack of presents for all the good boys and girls of the world. But, where did he come from and how does he manage his deliveries in a single night?

A very brief history of Santa
Saint Nicholas was a 4th Century Greek bishop who was known for giving gifts to the poor and, up until the 16th Century, children were given gifts in his honour on the evening of the 6th December. After the Reformation, the date for giving gifts was moved to the 25th December. However, the origins of Father Christmas date back further than Christianity, especially in the Germanic countries of Europe. One of the figures attributed to Father Christmas is the Norse god Odin who is associated with the festival of Yule. Clad in his blue hooded cloak, long white beard flowing, Odin would ride his eight-legged horse Sleipnir through the midwinter sky delivering gifts to his people. Even Santa’s laugh of ‘Ho-Ho-Ho’ is thought to have been the hunting cry of Odin. Although the original bishop robes were red and white, it was in 1862 that the American illustrator Thomas Nast who drew the Santa we know today- a portly fellow dressed in a red suit with white fur trim and large belt 1.

Lots of kids
So how many children does Santa visit these days?

  • According to UNICEF, there are an estimated 2.2 billion children in the world.
  • Although not everyone in the world recognises Christmas, a recent research poll suggested that around 90% of Americans celebrate Christmas, regardless of their religion 1.
  • Tentatively extrapolating this data would mean that Santa Claus visits1.98 billion children.
  • Assuming there are 2.5 children per household, this would mean Mr Claus would have to squeeze down 792 million chimneys.

In order to carry out such a feat of mass delivery, Santa flies with the sun, from east to west, in order to maximise the hours of darkness. This gives the Christmas courier 32 hours to do his work, but means he only has 145 microseconds to visit each house. This obviously goes some way to explaining why we don’t always see Father Christmas, or the fact that he doesn’t always have time to consume the mince pies and sherry which we leave out for him.

Dasher, Dancer, Prancer and Vixen
Let’s wildly assume that everyone wants a Thunderbirds Interactive Tracy Island for Christmas (and who doesn’t?!), each one weighing three kilograms. The total payload for his sleigh will weigh in at around six billion metric tons.

Of course, the power to pull such a weight falls to Santa’s reindeer. The average reindeer can pull twice its weight and males can weigh up to 120 Kg 2. So, Santa would need 25 million reindeer to pull all of those Tracy Islands.

Well, that’s your normal Cervidae species, but traditionally (according to the 1823 poem ‘The Night Before Christmas’) there are eight flying reindeer. If the sleigh squad hasn’t recruited extra numbers, each of these colossal beasts would need to weigh in at 375 million tons each. Those are big deer.

Santa on the radar
With such large beasts pulling a huge sleigh through the skies at speeds of up to 1800 miles per second, it’s no wonder that this object can be tracked using radar. From the 1950’s until 1996, the North American Aerospace Defense Command (NORAD) Santa Tracker has used its radar equipment to track the yearly voyage of Father Christmas, broadcasting his progress via radio, television and running a phone line.

Since 1997, the NORDA Santa Tracker has been available online 3. Using the tracker, people can view Santa’s progress across the globe with videos posted from famous landmarks around the world.

The NORAD Santa Tracker actually came about by accident. In 1955, a department store in Colorado Springs posted an advertisement in a local newspaper inviting children to phone Santa Claus on his direct line. Except the phone number was misprinted and was actually for the Colorado Springs’ Continental Air Defence Command Centre!

Colonel Harry Shoup was on duty on Christmas Eve, 1955. Instead of hanging up the phone on all the children who were calling, he instructed each of the air defence staff to find and report the location of Santa and his sleigh to the callers. Harry later became known as the ‘Santa Colonel’.



AUTHOR: Martin Wilson

Posted in General

All Synthetic Life is Here: The Work of J. Craig Venter

Reading about some of the achievements and goals of J. Craig Venter, you can’t help but feeling that this maverick and innovative scientist is thinking further out of the box than his contemporaries when it comes to understanding the genome and what we can do with it.

Sheer lunacy
Admittedly, his work draws controversies and criticism. Back in 1990, whilst working for the National Institutes of Health (NIH) on a project to sequence short cDNA fragments in the human brain, Venter announced to Congress that they were planning to file patents for these sequences at a rate of 1000 sequences per month 1. This went down like a lead balloon. James Watson described the plan as “sheer lunacy” and was “horrified” at the prospect 1. A public outcry ensued and the NIH withdrew the plans for patent applications.

Faster and cheaper
In 1998, Venter (along with the Applera Corporation) founded ‘Celera’- a company with a goal to sequence the human genome within three years 2. To do this, Celera pioneered the use of ‘shotgun sequencing’- a method rejected as being too inaccurate by the publically funded Human Genome Project (HGP). Celera was determined to sequence the entire genome faster and cheaper than the HGP. With the aim of patenting still in mind, Celera initially announced they would seek patents on a few hundred genes, but eventually filed preliminary patents for up to 6,500 partial and whole genes. However, in March 2000, President Bill Clinton and Prime Minister Tony Blair made a brief announcement that there should be free access to the information from the human genome 3. Celera welcomed the statement, albeit defending their business at the same time. The Clinton/Blair statement led to a drop in biotech stocks, including a 19% in Celera shares 3. Despite this, Celera went on to sequence the human genome at a fraction of the cost of the HGP ($300 million versus $3 billion) and in February 2001, the HGP published their results in Nature, followed a day later by Celera who published their results in Science 4. It turned out that Venters’ DNA sample was one of the five used for sequencing by Celera. Venter was fired from Celera in 2002, although he had already made up his mind to leave the company 5.

Sampling the oceans
Later in 2002, Venter set up The Center for the Advancement of Genomics which became one of the divisions of the J. Craig Venter Institute (JCVI). In 2003, Venter turned his attention to the worlds’ oceans and following a pilot project, Venter and his team set off in 2004 in his own yacht (the Sorcerer II) to circumnavigate the globe for the Global Ocean Sampling (GOS) Expedition 6. The aim was to better understand the diversity of marine microbiological organisms, ecosystem functioning and to discover genes which play an evolutionary role. Covering around 32,000 nautical miles, this expedition led to the discovery of six million new genes and many thousands of new protein families 6. Further expeditions were launched in 2007/2008.

Changing species and synthesising life
In 2012, Venter gave the 70th Anniversary Schroedinger Lecture at Trinity College, Dublin. In it he stated “Life is based on DNA software. We’re a DNA software system, you change the DNA software, and you change the species.” 7. A team at JCVI had done just that in 2007. They had successfully transplanted the whole genome of one bacterial Mycoplasma species into another. On examining the recipient cells they found that none of the original proteins existed- the recipient hardware was running the donor software 8.

Not content with species swapping, scientists at the JCVI set out to create synthetic life. In 2010, Venter and colleagues published the results of a synthetic Mycoplasma species containing computer designed DNA 9.  Named ‘JCVI-syn1.0’, these cells are capable of functioning and reproducing in the same way as naturally occurring bacteria. This proof of concept synthetic cell has societal and ethical implications- something which the JCVI welcomes discussion about. However, the applications of this technology are wide-ranging, from creating new medicines to advanced biofuels 10.

Setting his sights on space
In 2005, Venter founded Synthetic Genomics Inc. with an initial focus on biofuels. The work of this company has rapidly expanded into other areas including synthetic vaccine production 11. In addition, they have created a DNA synthesiser dubbed a ‘digital biological converter’ (DBC) or ‘biological teleporter’. This DBC can take digital DNA code and re-synthesise the sequence in the lab. In 2013, Venter gave an interview to ‘Wired’ magazine about his latest book, ‘Life at the Speed of Light: From the Double Helix to the Dawn of Digital Life’ 12. In it, he proposed that if the Mars Curiosity Rover were able to sample Martian DNA, this sequence could be transmitted back to a laboratory on earth where a DBC could re-build Martian life in a maximum containment facility. Alternatively, the astronauts we send to Mars could take a DBC with them to enable mission control to send genetic information enabling them to ‘print off’ medicines.

Although this ambitious work is still at early stages, you can be sure that Venter and his colleagues will be behind this and future innovations, bringing with it much ethical debate and conversation.



J. Craig Venter



Posted in General Tagged , ,

Microscope Condensers -The Overlooked Optics

The two main optical components of the microscope which are adjusted and changed whilst in use are the objectives and the eyepieces. However, the other optical component which is sometimes overlooked, especially by beginners unaware of Koehler Illumination, is the microscope condenser.

To learn more about Koehler Illumination, please see this article from the Agar Scientific Blog;

On an upright microscope, the condenser is found beneath the stage (often referred to as a ‘sub-stage condenser’) and on an inverted configuration, it is found above the stage and the specimen. The condenser serves to focus the light waves from the light source to provide an even illumination and intensity across a specimen view. An integral part of the condenser is the aperture diaphragm and adjustments to the diaphragm, as well as varying the vertical position of the condenser, serve to change the contrast, depth-of-field and resolution of the image.

The history of the condenser

Robert Hooke (1635-1703) was an English scientist and natural philosopher. His interests and knowledge spanned many disciplines, from microscopy and palaeontology to the theory of gravity. In 1665, he published his book entitled ‘Micrographia’ which was to be a significant work in the field of microscopy and biology 1. Hooke was credited as being the first person to use the word ‘cell’ within the book. The microscope which Hooke used for his observations had a simple external condenser which consisted of a large globe filled with salt water and a plano-convex lens to focus the light on his specimens.

The modern achromatic objective lenses were developed in the 18th Century and such lenses corrected for chromatic aberration. When white light passes through a convex lens, it is split into its component wavelengths. The corrected (achromatic) lenses bring the red and blue wavelengths to approximately the same focal point as the green wavelength light. With such developments, it became apparent that there was a consequent need for an equal improvement in terms of condensers.

The first achromatic condenser was developed in 1837 by the French biologist Felix Dujardin (1801-1860) 2. However, improvements were subsequently made in the 1840’s by three prominent microscope makers; Andrew Ross, Hugh Powell and James Smith 2. In 1841, the Microscopical Society commissioned Hugh Powell to make a microscope for them and this particular one was fitted with an achromatic condenser which is thought to be the first ever to be produced in England 2. In contrast, the use of the condenser was not widespread in other European countries until 1870.

The Abbe condenser

Ernst Karl Abbe (1840-1905) was a German mathematician and physicist who not only co-founded Schott Glassworks in 1884, but also co-founded the Zeiss Optical Works with Carl Zeiss in 1866. Abbe was also the first person to define and describe numerical aperture (NA).

In 1870, Abbe invented the ‘Abbe Condenser’ which is still the most widely used design in light microscopy. This type of condenser consists of two or three lenses along with an associated diaphragm to control the amount of light with which the specimen is illuminated. The uppermost lens (on a three lens substage Abbe condenser) can be flipped into the light path which ensures that the light completely fills the field of view when using higher power objectives.

Condenser numerical aperture

Not only does the iris diaphragm control the light intensity, but adjusting this also determines the NA and size of the light cone from the condenser. Condensers have a mechanism similar to the focus wheels of a microscope which controls the vertical height of the condenser body. To realise the full optical potential and NA of the condenser and objectives, the condenser must be adjusted to the correct height for each objective used (see the above link to the article on Koehler Illumination). Whilst the Abbe condenser is adequate for most brightfield applications, its limitations come when using high magnification objectives as the maximum NA of a low-end Abbe condenser is around 0.6. It should be noted that to achieve an NA of greater than 0.95 requires the use of immersion oil between the top lens of the condenser and the underside of the specimen slide (conversely, ‘dry’ condensers have an NA of less than 0.95). Ideally, the condenser should have an NA which is equal to that of the highest power objective on the microscope.

Corrected condensers

As with the objectives, condensers are classified depending on their optical correction. Briefly, there are two types of aberrations for which condensers are corrected; spherical and chromatic (see above). Spherical aberrations occurs when light is focussed through a curved lens and the resulting light rays are spread along the optical axis instead of coming together at one focal point. For a more detailed description of aberrations, please see the article on objectives from the Agar Scientific Blog;

A simple two lens Abbe condenser has no optical corrections. An ‘aplanatic’ condenser is corrected for spherical aberration and may typically have a potential NA of up to 1.4. Achromatic condensers are corrected for chromatic aberration which brings the blue and red wavelength light to approximately the same focal point as the green wavelength. The highest level of correction is found in the aplanatic-achromatic condensers which are corrected for both spherical and chromatic aberrations.

In conclusion, the condenser plays a critical role in achieving the optimum resolution and highest potential NA from a microscope, therefore it is good practice to learn the correct set up for Koehler Illumination and not to overlook the importance of these optics!


  2. Bradbury, S 1967 The Evolution of the Microscope Pergamon Press, London.


Posted in General Tagged , ,

Home for Winter: How Birds Utilise Quantum Physics in Migration

It’s around this time of the year that we watch some species of birds leave our country and our over-wintering visitors returning. Swallows make the six week journey back to Africa between September and October, covering around 200 miles each day. Old folklore and superstitious beliefs held that these birds hibernated in village ponds over winter- a belief which persisted until around the 19th Century. As we lose the swallows of summer, we welcome our winter visitors such as barnacle and pink-footed geese which arrive from the Arctic and Iceland.

But how do birds navigate the thousands of miles between continents?

One of the proposed methods for navigation is the utilisation of cryptochrome proteins which are found in the avian retina with which birds can actually ‘see’ the magnetic field of the earth. Cryptochromes are a class of flavoproteins and are found in plants and animals. In plants, they are partly responsible for phototropism (directional growth) and also play a role in circadian rhythms in plants and animals.

Can we see it too?

The human retina expresses the cryptochrome CRY2 which means that we too have the capability to sense geomagnetic fields. However there is much debate and controversy surrounding the research as to whether or not humans are capable of sensitivity to magnetic fields. A study which generated a transgenic Drosophila line with human CRY2 has shown that this protein can function as a light-sensitive magnetoreceptor 1. But it remains unclear whether CRY2 function can be translated downstream within the human eye.

How cryptochromes (possibly) work

Cryptochromes are activated when exposed to blue light and via an electron transfer chain involving FAD (Flavin adenine dinucleotide) 2. It has been hypothesised that when the reaction returns to its resting state, a radical pairing occurs between flavin and the superoxide radical and this radical pair reaction responds to geomagnetism 2. Herein could lie the answer as to why humans cannot actively perceive magnetic fields in the way our avian counterparts can. Superoxide is a highly reactive free-radical and causes cellular damage. Humans have developed an efficient anti-oxidant system which prevents free-radicals from damaging our cellular systems. Although our highly effective superoxide dismutase enzyme prevents cellular disruption and cell death, it could also prevent superoxide from building up to levels where we could utilise the cryptochromes in our eyes 3. Our evolution has chosen preservation over visibility of geomagnetism.

The quantum physics part

In physics, particles can be described by their quantum state which depends on energy, electron spin and so on. Two separate particles can interact with one another in what is described as ‘quantum entanglement’. What happens to one particle will have an effect on another, despite being separated. As with many quantum systems, we don’t know until we look and the process of examining a quantum system will change it.

The current thinking with the superoxide/cryptochromes model is that when this molecular interaction is activated by light, it produces electron entanglements 4. These molecules would each have unpaired and entangled electrons. If the downstream molecular products of this interaction are dependent on the spin of the electrons, then an external magnetic field could change the orientation of the spins and therefore affect any chemical reactions in the retina of migratory birds 5.

Bird’s eye view

Despite years of research, the exact workings of the avian compass remain to be elucidated. What’s even more speculative is how birds actually ‘see’ the magnetic field of the earth. The molecular reactions involving cryptochromes may vary according to the angle of the magnetic field lines, therefore birds flying close to the equator may sense horizontal lines, whilst nearer the poles, these lines could become more angular. The actual bird’s eye view might be lighter or darker regions, or spots. We’ve come a long way from our folklore beliefs surrounding hibernating swallows, but we’ve still got some way to go to understanding how they actually navigate their 10,000 mile journey.


AUTHOR: Martin Wilson

Posted in General

Helping Cells and Sections to Stick: Cleaning, Sterilising and Coating Slides and Coverslips

Although we very briefly touched on slide coating in a previous article, I wanted to describe in more detail the many ways in which slides and coverslips are prepared for different purposes including growing cells on them and helping sections to stick to them through the process of immunohistochemistry (IHC).

In this article, we will look at cleaning and rinsing of slides/coverslips, sterilising slides/coverslips for growing cells and different methods of coating slides/coverslips. There is no single correct way to prepare slides or coverslips and it will depend on factors such as the cell-line being used.

Cleaning slides and coverslips

Although many slides are supplied as pre-washed (such as these ones, many labs and scientists still prefer to wash their own slides/coverslips and it is obviously more important to do so if using items from a packet which is already open. A freshly opened box may look clean, but there may be a thin film of grease from the manufacturing process which will prevent optimal adherence of cells.

  • Washing with PBS/water

Although many protocols advocate washing in water or PBS before use, such washing may not get rid of any oil/grease based films from the surface of the glass and the addition of PBS may in fact leave salt crystals on the surface when it dries.

  • Acid washing of coverslips is recommended particularly if you are planning to grow adherent cells on the surface and it helps polypeptides to adhere to the glass. It is a relatively long protocol though, so it is best to plan in advance and make up a batch of acid-washed coverslips and store them in a clean container.
  1. Separate the coverslips and incubate them in 1M HCl at 500C to 600C for between four and 16 hours.
  2. Allow the HCl to cool to room temperature and then rinse two times in double-distilled or ultrapure water.
  3. Add double-distilled or ultrapure water to the coverslips and sonicate for 30 minutes. Repeat this twice using fresh water each time.
  4. Sonicate the coverslips for 30 minutes in a solution of 50% ethanol (make up all the ethanol solutions with double-distilled or ultrapure water).
  5. Sonicate the coverslips for 30 minutes in a solution of 70% ethanol.
  6. Sonicate the coverslips for 30 minutes in a solution of 95% ethanol.
  7. Remove the final ethanol wash and store the coverslips in 95% ethanol until use.

A slightly shortened version of this protocol involves taking the coverslips from step (3) above, rinsing them in 100% ethanol, drying and storing them in a clean airtight container until use. Other similar methods involve the use of commercial glass cleaners in place of the hydrochloric acid.

Sterilising coverslips

  • Ethanol

Most protocols for sterilising coverslips advocate the use of ethanol in concentrations ranging from 70% to 100%, either with or without further sterilisation steps.

If using ethanol alone, the coverslips can be placed in the bottom of a sterile 6-, 12- or 24- well plate and covered with 70% ethanol, left for five minutes and repeated three times. Follow this with a final wash in 100% ethanol.

A more thorough method (and more time consuming) is to dip a coverslip in 70% ethanol and flame with the blue flame of a Bunsen burner. There’s an obvious hazard here though- keep the ethanol stock well away from the flame! Flaming will eliminate most micro-organisms and spores from the surface of the glass.

  • UV LightUV light is often used in conjunction with 70% ethanol. Dip the coverslips in 70% ethanol and then expose to the UV light in a tissue culture hood for between 20 and 30 minutes. Alternatively, UV exposure can be used on its own.
  • Autoclaving

This is one of the easiest methods to sterilise coverslips. Simply place the coverslips in a glass petri dish and send through the dry cycle of an autoclave. This is typically a 20 minute step. Some protocols advocate a 70% ethanol wash beforehand, but autoclaving alone will ensure that the glass is tissue culture sterile. Just remember to open the petri dish in a tissue culture hood and use sterile forceps to remove each one.

Coating slides and coverslips

As with cleaning and sterilising, there are many methods for the coating (or ‘subbing’) of slides and coverslips. The coatings used depend largely on the cells which you wish to grow on the glass. The most common coating is poly-lysine, but we will also take a look at collagen, laminin and fibronection.

  • Poly-Lysine

Poly-Lysine enhances the electrostatic interaction between the positively charged ions on the surface of the glass and the negatively charged attachment ions of the cell membrane. Poly-Lysine not only increases the electrostatic bond between cells and the glass surface, but also helps to ensure that frozen and paraffin embedded sections remain stuck to slides during the many IHC steps.

Two types of poly-lysine are available and the choice will depend on the cells type you are using. Poly-L-lysine is the most commonly used and is also suitable for tissue sections. However, some cell lines can excrete proteases which break down Poly-L-lysine, so it is recommended to use Poly-D-lysine instead.

Poly-L-lysine has been shown to be a suitable coating for cells such as primary neurons, neuronal cell lines, PC12 cells and HEK293 cells. As with many of these cleaning and coating methods, there are a variety of options, but below is a standard method for poly-lysine coating;

  1. Clean the slides/coverslips before coating. If you are coating coverslips to grow cells on, then it is recommended to acid-wash these beforehand.
  2. Make a 1 mg/ml poly-lysine solution in sterile water. If coating coverslips, place these in a sterile plastic petri dish and cover with poly-lysine solution. If coating slides, place in a clean slide rack in a dish and again, cover with poly-lysine solution. The optimum ratio of poly-lysine solution to glass surface is 1 ml: 25 cm2 (this works out around 900 slides/litre of solution).
  3. Place slides or coverslips on a rocker and rock gently for 5 minutes at room temperature.
  4. For coverslips, remove solution by aspiration and wash with sterile water. Allow to dry for at least 2 hours before use. For slides, pour off poly-lysine and dry in a 600C slide oven for an hour.
  5. Coated glass can be stored at 40C in a sterile container for up to three months.

Pre-coated poly-lysine slides are also commercially available, such as these ones from Agar Scientific;

  • Collagen

Collagen is an extracellular matrix protein which can be used on coverslips to promote adherence of cells such as epithelial and endothelial cells as well as cells lines such as CHO and HEK293 cells. The most commonly used collagen is type I (isolated from rat tails). Other types used for adherence include type II and type IV, but also use the recommended type depending on the cells you wish to grow on the coverslips.

  • Fibronectin

Fibronectin is also an extracellular matrix protein which contains an RGD sequence (Arg-Gly-Asp) which mediates cell attachment. For this matrix protein, the species source is unimportant as all contain the RGD sequence. Many cell types will attach to fibronectin coated glass including endothelial cells, fibroblasts, neuronal cells, and CHO and HEK293 cell lines.

  • Laminin

Laminin is an extracellular matrix protein and is a major component of one of the layers of the basement membrane. Laminins bind to cell membranes through integrin receptors and other plasma membrane molecules. Laminins are a suitable coating and substrate for a wide variety of cell types ranging from fibroblasts to neuronal cells. Some protocols advocate pre-coating of slides/coverslips with poly-lysine before laminin coating for extra adherence.


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Haemocytometers: You Can Count On Us!

Apart from the incubators and the Class II hoods, perhaps one of the most overlooked but important pieces of equipment in the cell culture lab is the haemocytometer. For accurate cell-seeding experiments and splitting cells, this thick glass slide is an essential tool for everyone working with live cells.

There are a number of different haemocytometers on the market and each one has a different grid pattern as well as different recommended uses.

Louis-Charles Malassez (1842-1909) was a French histologist and anatomist and is the person credited for inventing the haemocytometer. As the name suggests, this device was originally intended for the quantitative counting of blood cells.

The most frequently used haemocytometer is the Neubauer (or ‘Improved Neubauer’) chamber. Other haemocytometers include the Burker, Thoma and Fuchs-Rosenthal. Most of the haemocytometers are manufactured from crystal glass and generally measure 30 x 70 mm with a thickness of 4 mm. Two vertical lines are ground from the glass to define the counting area and the double cell counting chambers have a ground out ‘H’ shape. A selection of haemocytometers is available from Agar Scientific;


The Neubauer/Improved Neubauer haemocytometer

The cell counting area of the Neubauer measures 3 mm2 (with each of the main squares measuring 1 mm);


The grid is subdivided and the 16 squares at each of the four corners measure 500 mm2. In a Neubauer chamber, the central square consists of 16 smaller squares each one with the same width/height the corner squares. Each of the 16 central squares is further subdivided into 16 smaller squares. The glass covers for haemocytometers are specifically designed with regards to thickness and size (generally 22 mm2). Do not be tempted to use a coverslip as this may result in an inaccurate cell count. When the coverglass is placed over the counting area, this leaves a specific area for which to introduce the cells/liquid to be counted. The Neubauer chamber is designed to leave a gap of 100 mm between the top surface of the counting area and the bottom surface of the coverglass.

The Improved Neubauer has a slightly different grid pattern compared to the ‘old’ Neubauer chamber. The overall size of the central counting area is still the same, but the square in the central cross area are divided into 25 smaller squares, each one measuring 400 mm2;


Each of the 25 squares in the centre of the cross grid are further subdivided into 16 smaller squares which measure 2.5 mm2 (or 0.0025 mm2 as etched onto the surface of the Improved Neubauer slide). In other words, the central cross square contains 400 small squares.

The Neubauer chambers are designed with these grid patterns for the counting of blood cells (although, these chambers are suited for the counting of most mammalian cell lines/primary cells etc.). The larger squares at the four corners are designed for the counting of white blood cells whereas the central smaller squares are for counting red blood cells and platelets.


The Burker Haemocytometer

The Burker chamber has the same dimension of grid (and same depth) as the Neubauer slide;


The main difference is that the central square is not sub-divided into the 16 or 25 smaller squares. The grid pattern runs across the whole counting area and the central square contains the same number of smaller squares (16) as the corner and side squares.

The Thoma Haemocytometer

The Thoma chamber has the same dimensions and depth as the Neubauer chamber, however, this haemocytometer does not have the four larger corner squares;


The Thoma haemocytometer seems to be the favoured counting chamber for microbiologists.

The Fuchs-Rosenthal Haemocytometer

This counting chamber is specifically designed for the counting of cells from cerebro-spinal fluid. The main difference (apart from the counting grid pattern) is that the distance between the coverslip and the chamber surface is 200 mm. This gives a larger volume of cells/liquid which can be counted and is also used for counting blood cells.


The grid pattern of the Fuchs Rosenthal is similar to the Burker chamber, except that the Fuchs Rosenthal haemocytometer is designed to accommodate twice as much volume compared to the other chambers mentioned above.

Counting cells using a haemocytometer

For this example, we will consider the counting of a sample of cells using an Improved Neubauer haemocytometer (although this can be applied to any of the haemocytometers above excluding the Thoma chamber).

Firstly, you’ll need to clean and prepare the chamber- the easiest way is with 70% ethanol. Dry the chamber (preferably with a lens cleaning tissue). You will then need to moisten the edges of the chamber and the most common way is with the exhaled breath with mouth fully open. Slide on the coverglass using gentle pressure and you should see something called ‘Newton’s Rings’ where the coverglass is in contact with the chamber (rings caused by an interference pattern).

  • Prepare the cell suspension

The cells and fluid (such as culture medium, PBS etc.) should be well mixed to provide a homogenous sample. You can do this by gently pipetting the cells up and down a pipette tip or by gently agitating the flask/container. Don’t be tempted to use excessive pipetting or a vortex mixer as this may shear cells.

Quickly remove a volume of cells and transfer to an Eppendorf tube. Add an equal volume of trypan blue and gently mix by pipetting or by tapping the tube (note- this is an example using a 1:1 dilution, but you may need to adjust depending on the cell density). Using trypan blue will help to distinguish between dead (blue) cells and living (clear) cells.

  • Loading and counting cells
  1. With a pipette, carefully draw up around 20 ml of the cell mixture. Place the pipette tip against the edge of the coverglass and slowly expel the liquid until the counting chamber is full. Capillary action will help to ensure that the counting chamber is full, but care should be taken not to overfill the chamber. A volume of 10 ml is sufficient to fill one counting chamber.
  2. Place the haemocytometer on the microscope stage. Using the 10X objective, focus on the grid lines. The general rule for counting of cells which fall on the edges of the squares is only to count those cells which are on the top and the left hand side grid lines. This rule ensures that you do not count the same cells twice.
  3. It is advisable to use a hand tally to keep count of cells. Count the number of cells in one of the corner squares (which are further separated into 16 squares). Count all of the clear cells within the squares and those touching the top and left hand grid lines. Another tip is to count in a ‘turret’ pattern, i.e., count along the top row of squares from left to right, down to the next square and count from right to left and so on until all the cells in the 16 small squares have been counted.
  4. Move to the next corner square and repeat until you have counted the four larger corner squares.
  5. The volume of liquid between the coverglass and the counting chamber is such that the number of cells which are counted in one set of 16 small squares is equivalent to the number of cells X 104/ml.


  • Calculating the total number of cells

Add the four counts together and divide by four to give an average over the whole counting chamber.

Multiply by 2 to take into account the 1:1 dilution made when adding the trypan blue to the cell mixture.

The total count from 4 sets of 16 corner squares = (cells/mL x 104) x 4 squares from one hemocytometer grid.

In other words;

Total cells (X104)/ml = Total cells counted x (dilution factor/number of squares counted)

For example;

-Total cell count was 180

-Number of squares counted was 4

-100 ml of trypan blue was added to 100 ml of cell suspension


Total cells (X104)/ml = 180 x (2/4)

Which gives 90 X 104 cells/ml.

If you find that there are too many cells in your suspension, then simply increase the dilution factor of cells to trypan blue (but remember to take this into account in the final calculation of cell density).

For an accurate determination of the total number of cells, the number of cells in one of the large squares should be between 15 and 50.


AUTHOR: Martin Wilson

Posted in General

Finder Slides: Finding and Re-Locating Areas of Interest on Microscope Slides

How often have you been scanning a new batch of slides and found an area of interest which you wish to show to a colleague or come back to at a later date? Marking the slide or the coverslip with a marker or an engraver is impractical as this could easily crack the coverslip, or obscure the area of interest under a blob of ink.

This is where the ‘Finder Slides’ are invaluable accessories. These slides have a grid pattern of co-ordinates usually over the entire surface of the slide. They are not overlaid on top of the specimen slide, instead, the area of interest is found on a slide which is then carefully removed without disturbing the XY stage controls and replaced with a finder slide. Looking back down through the eyepieces, you will then see co-ordinates which relate to the area of interest.

Maltwood’s Finder (1858)

One of the earliest examples of a finder slide was the ‘Maltwood’s Finder’. This finder slide was invented by Thomas Maltwood (1827-1921), a fellow of the Royal Microscopical Society. These early finder slides were manufactured by Smith, Beck & Beck of London. James Smith started making microscopes in 1839 and went into partnership with Richard Beck on 1847. Four years later, Richards’s brother Joseph joined the firm. Smith, Beck & Beck started making the Maltwood’s Finder slides in 1858.

Maltwood initially drew the grid on a sheet of paper measuring 10 inches square which was photographed to give a negative measuring one inch square. A positive microphotographic plate was printed from this original negative and hand-mounted on a slide measuring 3” x 1 ¼”. On this plate the lines were only 1/50th of an inch apart with a total of 2,500 co-ordinate squares. These glass slides were hand engraved with the manufacturer names and each one was numbered.

Webb’s Finder (1880)

William Webb (1815-1888) is well known amongst antique microscopy enthusiasts for his microscopic writings etched onto glass slides. Webb had constructed a micro-engraving machine which reduced his hand movements to incredibly small scales, but the details of the machine are lost and it is thought that he destroyed it shortly before his death. Amongst the many curious slides which he made were epigrams measuring only 1/5000th of an inch and the entire Lord’s Prayer etched into glass and measuring 1/500th of a square inch. He proposed that these slides were “the best, the most simple, and unerring tests for objectives”. In 1880 he put his micro-engraving machine to more practical use and he produced finder slides and he boasted that these were of greater accuracy that the Maltwood’s Finder slides in that the Webb’s Finder contained 16 squares in the space of one square of a Maltwood’s Finder.

Gage Finder/Marker (1895)

Simon Henry Gage (1851-1944) was an American microscopist and Professor of anatomy, embryology and histology at Cornell University. In 1895, he published a paper in the Proceedings of the American Microscopical Society and he describes the problems of finding exact points on a specimen slide; “in one’s private study or for exhibition to friends, the special point that is on a slide is often the last to be found, much to one’s discomfiture.” This device differed from the classic finder slides as it actually marked the coverslip surface with a delicate brush filled with “colored shellac or other varnish”. This apparatus replaced one of the objectives and when an area of interest was found, the apparatus was swung into position and the marker turned to make a circle around the area of interest.

The England Finder

Today, one of the most common finder slides is ‘The England Finder’ which is available from Agar Scientific;

The England Finder is not named after the country, but after its inventor, Charles Norman England. He devised the original layout of the finder over 50 years ago and working with a company which was called ‘Graticules Ltd.’, they created a photomask layout as a freehand plot. Graticules Ltd. later became ‘Psyer-SGI Ltd.’ but the mastering of each of these slides is still derived from the original freehand plot which not only makes each Finder slide identical, but also a very unique product which cannot be reproduced by modern methods.

In a letter dated 1959 to the New Scientist from the Quekett Microscopical Club, Graticules Ltd. were described (in relation to the Maltwood’s Finder) as having “performed a public service in reintroducing an accessory of considerable usefulness in microscopy.”

The England Finder has a vacuum-deposited chromium grid which covers the entire slide surface and is particularly suited for lower-power magnification work. Finder slides mean that the microscopist can record the co-ordinates of any area of interest on a microscope slide which can then easily be found at a later date or by a colleague using the same (or a similar microscope). The England Finder can be used on any microscope with an X-Y stage movement of 75 mm by 25 mm. The grids line are situated at 1 mm intervals- along the X axis, the grids are labelled 1 to 75, whilst the Y axis grid lines are labelled A to Z (excluding ‘I’ to avoid confusion with the number ‘1’);

This gives a total of 1875 positions across the whole slide. In addition, each co-ordinate is divided into five (central circle and four quartiles) for even greater positioning accuracy (there are 9045 locations on each slide).

When using a finder slide, it is also useful to have a cross-hair graticule placed in one of the eyepieces of the microscope for even greater accuracy. Selections of such graticules are available from Agar Scientific;

To use the England Finder;

  • Firstly mark the slide orientation (X/Y) on the label or frosted section of the specimen slide. Make sure that the microscope stage is referenced to its maximum X and Y stop positions.
  • Locate the centre of the area of interest under the cross-hairs of the graticule.
  • Being careful not to move the position of the microscope stage, carefully remove the specimen slide and replace with the England Finder making sure the X/Y orientation is the same as the specimen slide.
  • Bring the England Finder grid into focus and make a note of the numbers and letters which are under the cross-hairs. Record the letter and numbers of the main circle followed by the number of the quartile (if the exact spot falls into one of these sections). For example, ‘U39/1’. If the point of interest lies in the centre of one of the circles, then this should be recorded as ‘0’.
  • To find an area of interest from a co-ordinate, ensure the stage is correctly referenced to X and Y, place the England Finder on the stage and locate the co-ordinate, then carefully replace the finder slide with the specimen slide. The area of interest should now be in the centre of the field of view.

AUTHOR: Martin Wilson

Posted in General