Introducing… CorrStub™

A new SEM specimen stub for correlative microscopy

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

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

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

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

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

CorrStub includes the following unique and useful features:

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

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

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

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

Find out more about CorrStub here:

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

 

 

Posted in General

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.

 

 

References

(1)   http://www.nature.com/nchem/journal/v5/n2/full/nchem.1546.html

(2)   http://www.microscopy-analysis.com/editorials/editorial-listings/dna-probes-without-damage

(3)  http://www.nature.com/ncomms/2015/151001/ncomms9497/full/ncomms9497.html

(4)   http://spirochrome.com/

AUTHOR: MARTIN WILSON

Posted in General

Santa-ology: The Science of Christmas Present Delivery

santa

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’.

 

References

  1. http://cartoons.osu.edu/digital_albums/thomasnast/santa_camp.htm
  2. http://www.pewforum.org/2013/12/18/celebrating-christmas-and-the-holidays-then-and-now/#religious-observance-of-christmas
  3. http://nationalzoo.si.edu/SCBI/AnimalCare/News/25daysreindeer.cfm
  4. http://www.noradsanta.org/
  5. http://web.archive.org/web/20091224050527/
  6. http://www.norad.mil/about/Santa.html

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.
References

  1. http://www.sciencemag.org/site/feature/data/genomes/254-5029-184.pdf
  2. https://www.celera.com/
  3. http://www.the-scientist.com/?articles.view/articleNo/12765/title/Clinton–Blair-Stoke-Debate-on-Gene-Data/
  4. https://www.sciencemag.org/content/291/5507/1304.full
  5. http://www.bio-itworld.com/archive/111202/horizons_venter.html
  6. http://www.jcvi.org/cms/research/projects/gos/past-voyages/#c1302
  7. https://edge.org/conversation/what-is-life
  8. http://www.sciencemag.org/content/317/5838/632.abstract
  9. http://www.sciencemag.org/content/329/5987/52.full
  10. http://www.jcvi.org/cms/research/projects/first-self-replicating-synthetic-bacterial-cell/overview/
  11. http://www.syntheticgenomics.com/150513.html
  12. http://www.wired.co.uk/magazine/archive/2013/11/features/j-craig-venter-interview

JAY

J. Craig Venter

 

AUTHOR: MARTIN WILSON

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;

http://www.agarscientific.net/koehler-illumination-a-history-and-practical-set-up/

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;

http://www.agarscientific.net/looking-down-and-looking-through-the-optics-of-a-microscope-2-the-objectives/

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!

 

References;

  1. https://ceb.nlm.nih.gov/proj/ttp/flash/hooke/hooke.html
  2. Bradbury, S 1967 The Evolution of the Microscope Pergamon Press, London.

AUTHOR: MARTIN WILSON

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.

 

References

AUTHOR: Martin Wilson

Posted in General

Do I have to share a microscope with the likes of you?!

Sharing microscopes with fellow scientists is part of working in science and research, however, this inevitably means having to suffer the laziness and dirty habits of other users.

If you are already working in a shared facility, you will no doubt recognize the following scenarios. If you are new to the wonderful world of microscopy, here are some of the joys you can look forward to.

 

Immersion oil- it’s for the whole microscope, not just for objectives

I often wonder if the people I share microscopes with come from an engineering background and somehow get confused between a delicate optical instrument and a steam engine. It seems that some users think that microscopes need liberal oiling before use. In reality, you only need one drop of immersion oil when using an immersion objective, but this is obviously lost on some people. No, one drop of oil would never be enough to get this machine running smoothly- we need oil on all of the other objectives, on the stage, on the focus wheels and we might as well oil the desk whilst we are at it.

It’s a wonder that some people don’t just slide of the microscope bench.

 

My work is so good, I’ve left it for you to see

There is a subtle difference between a glass recycling point and a microscope facility. However, some poor souls get confused between the two. Why put a slide in a glass bin when you can leave it on the stage for others to see? You too can marvel at how terrible their staining techniques are. Better still, why not leave piles of slides delicately balanced on the bench- microscope slide Jenga anyone?

 

Too busy and important to change out of surgical scrubs

Having worked in a research facility within a teaching hospital, real scientists had to share microscopes with medical students. Medics need to let everyone know how important they are by wearing their surgical scrubs absolutely everywhere. This uniform is great for the operating theatre, but surely they could take two minutes to change before coming into the microscope suite? I really don’t want to share space with someone wearing the same outfit they had on when they were rummaging in some body cavity or orifice. What’s even more worrying is that the medics presumably go back into theatre in the same scrubs after gracing us with their presence. Well, at least with all of that immersion oil splattered everywhere it should make orifice examinations run a bit more smoothly!

 

Free make-over whilst you work!

Eye make-up is wonderful for enhancing the eyes of some women, male and female goths/emos and so on, but it doesn’t quite have the desired effect on aging male professors. The rubber eye-cups around the microscope eye pieces are great for collecting eye liner, mascara and eye shadow and then inadvertently re-applying on the next user.

You too can have longer lashes after looking at your slides.

On a more serious note- the eye cups can also harbour eye infections such as conjunctivitis. If you are unfortunate enough to have such an infection, it’s best to refrain from microscopy until it’s cleared up lest all the other users of the microscope end up with red and weeping eyes.

Here’s an idea- after using a microscope, simply clean the eye cups ready for the next user.

 

The microscope facility refectory

You don’t usually see microscopes in the refectories and cafes within an institute and there’s a reason for that. However, it would seem that eating and drinking in a microscope suite is perfectly fine for some. It’s still a laboratory though with plenty of potential for ingesting nasty chemicals along with your coffee and muffins.

Before you know it, the microtome will be getting used to slice ham and cheese for sandwiches.

 

Scared of the dark

If you are afraid of the dark, the microscope facility can be a scary place. This must be the reason that microscope lamps are left on. All of the time. On full power. This might be news for some people, but the dial which controls the light intensity can also be turned back down. Try it the next time you have finished an imaging session (but perhaps take a torch with you if the fear gets too much).

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 http://www.agarscientific.com/lm/slides-coverslips/microscope-slides.html ), 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 WashingAcid 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;

http://www.agarscientific.com/lm/slides-coverslips/polysine-microscope-slides.html

  • 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.

AUTHOR: MARTIN WILSON

 

 

Posted in General Tagged ,