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

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 https://www.agarscientific.com/lm/slides-coverslips/microscope-slides), 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;

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

  • 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 ,

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;

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

 

The Neubauer/Improved Neubauer haemocytometer

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

A1

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;

A2

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;

A3

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;

A4

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.

A5

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

Therefore;

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;

https://www.agarscientific.com/the-england-finder

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;

https://www.agarscientific.com/eyepiece-graticules-e-f

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

Here Comes The Sun: The Science of the Summer Solstice

sun

Given the weather in the UK during most of May, you could be forgiven for thinking it was still spring (although, technically it was)! However, Sunday 21st June 2015 sees the summer solstice dawn upon us. So, what exactly is the summer solstice and have we really approached midsummer already?

The word ‘solstice’ derives from the Latin ‘sol’ meaning ‘sun’ and ‘sistere’ meaning ‘to stand still’. This standing still refers to the path of the sun and ‘declination’ which is a means of measuring the angle of a celestial body on the ‘celestial sphere’. This is an imaginary sphere around earth upon which all objects in the sky can be thought of as projected onto the underside of the sphere or dome. Similar to the latitude and longitude system used here on earth, it is a means by which astronomers can describe the position of stars and planets relative to a co-ordinate system. Declination specifies a position in the sky which is relative to the equator and the poles.

The earth doesn’t spin on an axis which is vertical, instead, the angle of tilt of the north/south axis is approximately 23.40 and this gives rises to the seasonal variations we experience on earth relative to the amount of sunlight we receive. At the Northern Hemisphere during the summer solstice, the North Pole is tilted towards the sun.

Factors affecting the exact date

The way in which a year is measured is not exactly an accurate science- it’s why we have leap-years. A year actually last for 365.24 days, so every four years we catch up with the extra day on February 29th. This means of measuring time also has an effect on the dates and exact times of each of the solstice and equinox dates. This year, it’s the 21st June, whereas in 2016 it will fall on the 20th June. The last time there was a summer solstice on the 22nd June was back in 1971.Other factors also influence the exact timing of the summer solstice. For example, the earth does not rotate at a constant speed around its elliptical orbit.

Axial precession

Another factor which occurs over a vast time scale is known as the ‘precession of the equinoxes’ (or ‘axial precession’). The earth is spheroid in shape and not a prefect sphere- indeed the equatorial diameter of the earth is 43 Km larger than the polar diameter. As described above, the earth it tilted in its orbit and this means that the ‘bulge’ around the equator is off-centre relative to the gravitational pull of the sun. This results in a small amount of torque as the gravitational force of the suns pulls harder on one side of the earth relative to the distant side. It’s not just the sun which exerts a gravitational pull on the earth- our own moon and other planets in the solar system also exert a pull on us. If the earth was a perfect sphere, precession would not occur.

If you extend an imaginary line through the centre of the earth (titled at 23.40), then this would trace a circle as the earth moves through the cycle of precession. One complete precession cycle takes 25,772 years. This cycle changes the exact times and dates of the solstices and equinoxes. Another interesting point is that the exact positions of stars in our sky also change through the cycle of precession. Polaris is our current ‘Pole Star’, however, 13,000 years from now, this will change and Vega (in the constellation of Lyra) will become our Pole Star. Polaris will get another turn at being the Pole Star in approximately 26,000 years from now!

Midsummer? But we’ve only just started!

The exact date of the solstice and what we know as midsummer do not fall upon the same days. Although midsummer festivities and celebrations are pagan in origin and would have fallen on the equinox days, the Christian church took over this festival and designated the date as the 24th June and is associated with the nativity of John the Baptist.

However, midsummer doesn’t mean the middle of summer. In meteorological terms, summer starts with the equinox and lasts for the months of June, July and August. In astronomical terms, the solstice marks the beginning of summer and it ends with the autumnal equinox on September 23rd. The average highest temperatures in the UK are usually in the months of July and August. This is partly due to the way that earth retains heat from the sun and the fact that we are an island. The oceans surrounding us act as a huge ‘heat sink’, absorbing and re-radiating the heat from the sun. Although the earth absorbs the most intense of the sun’s rays around the date of the solstice, it takes several weeks for the heat to be released, hence the fact that our hottest days are usually in July or August. If there were no oceans on earth, our hottest days would fall around the date of the solstice.

Depending on the location on earth, the summer solstice is generally regarded by those of us living in the Northern Hemisphere as the ‘longest day’. At the South Pole there will be 24 hours of darkness, whereas the North Pole will receive 24 hours of daylight. At the equator, there is approximately 12 hours of daylight. Here in the UK, the day length varies with London seeing around 16 hours and 38 minutes of daylight at the solstice, whereas in Edinburgh, the day length is approximately 17 hours and 36 minutes. Up in Shetland, they will see a day as long as 18 hours and 55 minutes.

Megalithic monuments and the solstice

Perhaps the most famous of the megalithic sites in the UK which is associated with the solstice is Stonehenge, although there are many other ancient sites in this country and around the world which are aligned to astronomical events.

Archaeologists have found evidence of very early wooden structures at the Stonehenge site which date back around 10,000 years ago. Even then, the post holes were aligned east/west which may have signified a link to the movements of the sun.

The earliest monumental building phase at Stonehenge was around 3,100 BC and consisted of a 110 metre circular bank and ditch with a gap facing the north east. The latter stages of monument building at the site maintained this NE/SW alignment. It was the English antiquarian and archaeologist William Stukeley (1687-1765) who first noted and recorded the rising of the summer solstice sun above the Heel Stone in the summer of 1720. The sun doesn’t rise exactly above the Heel Stone when viewed from the centre of the circle, but this is unsurprising when taking into account the vast time period and factors such as precession. Evidence has also shown that the Heel Stone was only one of a pair of such stones and the solstice sunrise would have been framed by these stones at the time of the building.

After much controversy and legal battles, English Heritage now grant access to Stonehenge at the times of the solstices and equinoxes (the stones are roped off to visitors at other times of the year);

http://www.english-heritage.org.uk/visit/places/stonehenge/plan-your-visit/summer-solstice/#

Author: Martin Wilson

Posted in General

Love in the Lab: Why Sexism is Still an Issue

women in science

Sexism in science reared its ugly and unwelcome head again recently when Sir Tim Hunt stood up on the 9th of June to address the World Conference of Science Journalists in Seoul, South Korea. In front of an audience of science journalists from around the world, he reportedly said the following comments;

“Let me tell you about my trouble with girls. Three things happen when they are in the lab: you fall in love with them, they fall in love with you, and when you criticise them they cry.”

He later gave an interview for the ‘Today’ programme on BBC Radio 4 stating that he did “mean the part about having trouble with girls”, but went on to try to apologise for his comments adding that he was “really sorry that I said what I said”, and that it was “a very stupid thing to do in the presence of all those journalists”. He had hoped his remarks were “intended as a light-hearted, ironic comment” but instead they were “interpreted deadly seriously by my audience”. Is the joke still funny when no one else is laughing?

Let me tell you about my trouble with these comments. Firstly, I have never worked in a laboratory which has employed ‘girls’ (or boys for that matter). Although laboratory visits from schoolchildren should be encouraged, they shouldn’t be made to don lab coats and crack on with an RT-PCR, well, not at least until they have left school! Many women view the word ‘girl’ as derogatory and its use in this context can imply emotional and intellectual immaturity.

Secondly, as humans, we all feel a huge range of emotions, from love to hate and everything in between. However, to let our emotions towards fellow scientists somehow hinder or cloud the science we are employed to do seem to hint at a lack of professionalism. Because of our emotions and judgements, there will undoubtedly be colleagues we like and those we dislike. But respect is the key and regardless of our feelings towards other scientists, if they are good at their job, then surely that is the fundamental issue.

Despite Tim Hunt’s comments, should we have rushed to judge him and publically head-hunt him through social media and the press? I’m not defending his personal viewpoints, but how would it have been perceived if a senior female scientist had made such comments about ‘boys’ in the lab? Would there have been such a media outcry? Unfortunately, there is still a huge gender gap between senior scientists; only 16 % of full-time professors in the STEM subjects (science, technology, engineering and maths) are women.

Tim Hunt is 72 and he attended a single-sex school in the 1960’s. His own views about ‘girls’ in the lab may be part of the conditioning from that era and from his educational environment. In his statement he also said “my trouble with girls”, implying this was a personal issue and problem for him. Indeed, in an exclusive interview with Tim Hunt and his wife, Professor Mary Collins in The Observer newspaper 1, Collins stated that “really it was just part of his upbringing.” Although she also admitted that “it was an unbelievably stupid thing to say”.

Shortly after the conference, a rather vicious flurry of social media comments ensued and he was described on Twitter as a “clueless, sexist jerk”. Yes, his personal viewpoints are sexist, but a clueless jerk? Tim Hunt was a Fellow of the Royal Society, but they immediately distanced themselves from his comments and in their statement said that “Too many talented individuals do not fulfil their scientific potential because of issues such as gender”. On the 11th June, the Royal Society issued a statement to say that Tim Hunt had resigned 2. They stated that although he had “made exceptional contributions to science in terms of his own research on the cell cycle and its implications for our understanding of cancer”, his “recent comments relating to women in science have no place in science.” The final comment in their statement is the most telling; “It is the great respect that he has earned for his work that has made his recent comments so disappointing”.

The Royal Society works to promote diversity in science and has many projects, awards and campaigns targeted not only at gender inequality in science, but also ethnicity, disability and those who may need flexibility in their work due to roles as carers and parents 3. Tim Hunt should have been aware of the work of the Royal Society in his professional role before making seemingly personal comments which contradict the vision of diversity promoted by the Royal Society.

Furthermore, Tim Hunt was an Honorary Professor with University College London (UCL) Faculty of Life Sciences- that is until 10th June 4. In the interview in The Observer, his wife was informed by UCL that he had to resign or face the sack (Tim Hunt was still flying back from South Korea at this point). With regards to his resignation, the university stated that “UCL was the first university in England to admit women students on equal terms to men, and the university believes that this outcome is compatible with our commitment to gender equality.”

What have these thirty seven words from Tim Hunt done to highlight gender inequality in science? They have shown that even at the highest level of academia, intelligence and (supposed) professionalism that such views still exist. It may partly be a generational issue, but inequality exists in our laboratories, institutes and universities. In real terms, this manifests as the fact that there are still far fewer women working in the STEM subject areas and these women are paid less than their male counterparts. More worryingly, one study examining trainees in scientific fieldwork has reported that women are 3.5-times more likely to experience sexual harassment than men 5. Whilst Tim Hunt’s statement was disappointing, it highlights  the very real issues of gender inequality in science and rather than head-hunting an individual, we should focus our attention on addressing the root causes with a view to achieving gender neutrality in science.

1 http://www.theguardian.com/science/2015/jun/13/tim-hunt-hung-out-to-dry-interview-mary-collins

2 https://royalsociety.org/news/2015/06/sir-tim-hunt-resigns-from-royal-society-awards-committee/

3 https://royalsociety.org/about-us/diversity/

4 http://www.ucl.ac.uk/news/news-articles/0615/100615-tim-hunt

5 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102172

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 Washing

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 Light

UV 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

The Origins and Development of the Confocal Scanning Microscope

It all starts in New York City in the summer of 1927 with the birth of Marvin Lee Minsky. Following his high school education (and a brief stint in the US Navy during WW II), he went on to gain a BA in mathematics from Harvard, and a PhD in the subject from Princeton before joining MIT in 1958. He founded the MIT Computer Science and Artificial Intelligence Laboratory is Professor of Electrical Engineering and Computer Science. In Isaac Asimov’s autobiography, he admitted that there were only two people he considered to be more intelligent than he was, one was Carl Sagan, and the other was Minsky. In addition, Minsky was also an advisor on the movie ‘2001: A Space Odyssey’.

The need to see neurons

In Minsky’s memoir on inventing the confocal scanning microscope, he explained that his home was always full of prisms, optics and lenses- his father was an ophthalmologist. At a young age, he started taking optical systems apart and rebuilding them. At both Harvard and Princeton, he studied biology, neurophysiology and neuroanatomy in addition to his mathematical studies. At the time, not much was known about how brains function and how nerve cells were connected. Minsky admits to being frustrated by this lack of understanding and although the shapes of many nerve cells were visible, what was needed was a three dimensional ‘wiring diagram’. However, with the imaging systems at the time (and given the sheer density of the nerve cells of the brain), it was impossible to distinguish such neural networks due to the scattering of light. Minsky knew that what was needed was an instrument capable of optical sectioning to eliminate the out of focus light.

Zirconium arcs and a military surplus radar

Whilst studying at Harvard, he was fortunate enough to be given a room in the physics laboratory with permission to use and buy whatever equipment he needed. Minsky designed his symmetrical microscope with an objective lens and a pinhole at either side of the specimen to eliminate the scattered and out-of-focus light. When he made his invention, there were no lasers suitable to illuminate the specimen with the intensity of light needed, so he used zirconium arcs instead which was a time-consuming process- each point scan taking up to 10 seconds. The final image was produced on a military surplus radar scope which he had acquired.

The next design problem he faced was whether to move the specimen or move the optics for the scanning procedure. Minsky admitted that the prospect of moving two tiny pinholes simultaneously daunted him, so he decided to keep the optics in a fixed position and move the stage instead. He machined all of the parts for the prototype himself, spending months in the machine shop at Harvard- the skills he learnt during this time also helped him to design and build a robotic hand and arm 10 years later.

1955: The first patent for a scanning microscope

Were it not for the fact that his brother-in-law was a patent attorney, Minsky’s invention may never have been documented at all. Minsky admitted that he didn’t keep notes or write down what he was working on! On November 18th 1955, he sent a patent letter entitled ‘Double focussing stage scanning microscope’ and the confocal was born.

1966-1967: A Nipkow disc confocal

In 1966, a patent was filed by two Czechoslovakian scientists, Mojmir Petran and Milan Hadravsky for a tandem-scanning microscope. This confocal used a Nipkow Disc. Briefly, this is a mechanical spinning disc which has a spiral pattern of thousands of individual pinholes drilled in it. The effect of the Nipkow disc is that thousands of points on the specimen are illuminated simultaneously. This was actually the first commercially available confocal scanning microscope being sold by a small company in Czechoslovakia and by Tracor-Northern in the US. The first scientific paper describing the instrument and its uses was published in the journal Science in 1967. However, the instrument only worked well for the brightest of specimens.

1969: The first laser scanning confocal

The first true laser scanning confocal microscope was designed and built by M. David Egger and Paul Davidovits from Yale University who published a paper on their findings in Nature in 1969. This used a 633 nm He-Ne laser and opposed to the earlier confocal systems (and the next one in the time-line) the sample remained stationary and was illuminated by the movement of an objective lens.

1977-1979: Naming the confocal

It wasn’t until 1977 that the term ‘confocal’ was used to describe such microscopes in a publication on theoretical analysis in ‘Optica Acta: International Journal of Optics’ by two scientists from Oxford, Colin J. R. Sheppard and A. Choudhury. The term ‘confocal’ means ‘having the same or common focus’. There is another claimant to using the term ‘confocal microscope’ for the first time- a Dutch physicist called G. Fred Brakenhoff developed a laser scanning microscope in 1979. He published his findings in the Journal of Microscopy and it seems to be the first time the term Confocal Scanning Light Microscope (or ‘CSLM’) was used.

Most of these early confocals proved to be too inconvenient for biological applications being both too slow and too sensitive to vibrations.

The 1980’s: Time to buy!

In 1982, a company called ‘Oxford Optoelectronics’ (since acquired by Bio-Rad) offered the first commercially available stage scanning CSLM which was connected to a computer (the ‘SOM-25’). This was commercialised from a design by a team based at Oxford University.

In 1986, at the Medical Research Council Laboratory of Molecular Biology in Cambridge, a team of scientists were busy working inside a tent made from World-War II blackout material. Inside the tent, a new prototype CSLM capable of scan speeds in excess of 4000 lines per second was being developed. This instrument used a galvanometer-driven mirror for frame-scanning and a rotating polygonal mirror for line scanning. The line scanning mirror was proving to be problematic and producing chromatic aberrations when green and red fluorophores were used. The polygon was subsequently replaced with two oscillating galvanometer mirrors. Further developments were made to allow adjustable scanning as well as dealing with the chromatic aberration and the patent for the design has since been used by Bio-Rad for all of their confocal point-scanning systems. The team had approached other leading microscope manufacturers including Zeiss and Leica, but this proved to be unfruitful. However, a commercial agreement was soon concluded with Bio-Rad and the ‘MRC 500’ was launched to an excited audience in 1987.

It wasn’t long until the other main microscope manufacturers were developing and building commercially available instruments, either based upon the MRC 500, or in parallel to this microscope. The development of the confocal microscope continues and in the years since the MRC 500 was launched we have seen major advancements including multi-photon techniques and the ability to image 90 separate colours in a single sample. It is an exciting and rapidly expanding field- watch this space!

AUTHOR: Martin Wilson

Posted in General