How the Scanning Electron Microscope works

Preparing samples for scanning electron microscope imaging

Blog post by Clodagh Dooley

Scanning electron microscopes (SEM) are amazing tools that allow imaging far beyond the resolution capabilities of light microscopes. SEMs work by creating a focused beam of electrons that scans over a sample; interacting with and exciting its atoms and generating signals that can be used to derive an image of the samples topography.

SEM blog

Fig. 1.Sage leaf surface hairs. Images by Dr. Clodagh Dooley, AML, CRANN, TCD.

SEM blog2

Fig. 2. The scanning electron microscope used to image the Trinity Tree samples. The tool is based in The Advanced Microscopy Lab, CRANN, TCD. The microscope is a Zeiss Ultra FESEM with Gemini column and Quorum Cryo preparation chamber.

To avoid scattering of the electron beam, the electron source and the sample chamber are under vacuum. This causes no issue when working with a dry sample, such as a metal or ceramic, but can cause big problems when working with a sample with a high water content. Samples with a high water content, such as plant material, use the water as support for its structure, if this water is removed rapidly, as will happen when placed in a vacuum, then the sample will dehydrate and collapse.

SEM blog3

Fig. 3. The vacuum chamber of the SEM showing the loading stage.

SEM blog4

Fig. 4. The vacuum chamber of the SEM showing the loading stage.

The first step in preparing a biological sample for SEM is to preserve it using glutaraldehyde, a chemical fixative that prevents any breakdown and degradation of the structure. The next step is to deal with the water. This is usually done in one of two ways; removing the water in a slow, controlled manner with help of solvents or solidifying the water as ice and imaging a frozen sample.

In the first method the water in the sample is slowly replaced with alcohol by immersing it in deionised water/alcohol solutions with increasing alcohol concentration until it is in a 100% alcohol solution. The alcohol immersed sample is placed in the critical point dryer (CPD) chamber and the alcohol solution is very slowly replaced with liquid CO2. Once the alcohol within the chamber has been completely replaced with liquid CO2 the system is brought to what is termed as the ‘critical point’ for CO2. This is where the pressure and temperature within the CPD chamber causes the liquid CO2 in the sample to turn from a liquid to a gas. The gas can then be vented off without any distortion of the sample due to surface tension.

SEM blog5

Fig. 5. The Quorum Critical Point Dryer

The second method involves the use of liquid Nitrogen. The sample is plunged into liquid Nitrogen slush (-190°C) to flash freeze. It is then transferred, under vacuum, to a cold stage within the SEM imaging chamber where it is held at this temperatures throughout imaging. With this method of preparation, chemical fixation can be avoided as the flash freezing preserves the sample structure and prevents degradation.

SEM blog6

Fig. 6. Cryo preparation chamber on the side of SEM chamber.

The final step before we can image a biological/hydrated sample is to coat it with an electron conductive coating; commonly used coating materials include carbon, gold, palladium and platinum. Conductive coating prevents charging of the specimen, which is caused by accumulation of static electric fields and makes imaging difficult.

SEM blog7

Fig. 7. Cressington 208 Turbo sample coater.

SEM blog8

Fig. 8. Plant material coated in a 10nm layer of Gold Palladium.

Tree Spotlight no.6 – the Yew

pacific yew overview image

Taxus baccata is a conifer native to western, central and southern Europe, northwest Africa, northern Iran and southwest Asia. It is the tree originally known as yew, though with other related trees becoming known, it may now be known as English yew, or European yew.

yew bark image

Description: It is a small to medium-sized evergreen tree, growing 10–20 metres (33–66 ft) (exceptionally up to 28 metres (92 ft)) tall, with a trunk up to 2 metres (6 ft 7 in) (exceptionally 4 metres (13ft)) diameter. The bark is thin, scaly brown, coming off in small flakes aligned with the stem. The leaves are flat, dark green, 1–4 centimetres (0.39–1.57 in) long and 2–3 millimetres (0.079–0.118 in) broad, arranged spirally on the stem, but with the leaf bases twisted to align the leaves in two flat rows either side of the stem, except on erect leading shoots where the spiral arrangement is more obvious. The leaves are poisonous.

The seed cones are modified, each cone containing a single seed, which is 4–7 millimetres (0.16–0.28 in) long, and partly surrounded by a fleshy scale which develops into a soft, bright red berry-like structure called an aril. The aril is 8–15 millimetres (0.31–0.59 in) long and wide and open at the end. The arils mature 6 to 9 months after pollination, and with the seed contained, are eaten by thrushes, waxwings and other birds, which disperse the hard seeds undamaged in their droppings. Maturation of the arils is spread over 2 to 3 months, increasing the chances of successful seed dispersal.

Yew trees do not need rich soil but they do need a well drained site, preferably not too exposed to wind or frost. Many yews are single sex, but most Irish yews are female and so bear fruit. Even if the flesh is removed, these may be slow to germinate. The best seeds are those that have been eaten by birds and have passed through them; such bare seeds may be collected from under yew trees. The Yew tree is in fact a good tree for wildlife as birds roost and nest in it.

Longeivity: Taxus baccata can reach 400 to 600 years of age. Some specimens live longer but the age of yews is often overestimated. Ten yews in Britain are believed to predate the 10th century. The potential age of yews is impossible to determine accurately and is subject to much dispute. There is rarely any wood as old as the entire tree, while the boughs themselves often become hollow with age, making ring counts impossible. Evidence based on growth rates and archaeological work of surrounding structures suggests the oldest yews, such as the  may be in the range of 2,000 years, placing them among the oldest plants in Europe. One characteristic contributing to yew’s longevity is that it is able to split under the weight of advanced growth without succumbing to disease in the fracture, as do most other trees. Another is its ability to give rise to new epicormic and basal shoots from cut surfaces and low on its trunk, even at an old age.

fortingall-2000 year old yew

The Fortingall Yew in Perthshire, Scotland, has the largest recorded trunk girth in Britain and experts estimate it to be 2,000 to 3,000 years old, although it may be a remnant of a post-Roman Christian site and around 1,500 years old.

yew arch lo res

The Yew arch in the Golden bridge Cemetery, Dublin


In Ireland the yew is native and may be found in old woods although it is often seen in the artificial surroundings of estates or churchyards. An evergreen conifer (although an unusual one), yew is a dramatic tree with its dark foliage and red berries encasing a single seed. Reenadina wood on the Muckross Peninsula, Co. Kerry is Ireland’s only native yew wood.

yew red berries

Toxicity: All parts of a yew plant are toxic to humans with the exception of the yew berries (however, their seeds are toxic); additionally, male and monoecious yews in this genus release cytotoxic pollen, which can cause headaches, lethargy, aching joints, itching, and skin rashes; it is also a trigger for asthma. These pollen grains are only 15 microns in size, and can easily pass through most window screens.

The foliage itself remains toxic even when wilted, and toxicity increases in potency when dried. The major toxin within the yew is the alkaloid taxine. Horses have a relatively low tolerance to taxine, with a lethal dose of 200–400 mg/kg body weight; cattle, pigs, and other livestock are only slightly less vulnerable. Several studies have found taxine LD50 values under 20 mg/kg in mice and rats.

Symptoms of yew poisoning include an accelerated heart rate, muscle tremors, convulsions, collapse, difficulty breathing, circulation impairment and eventually cardiac arrest. However, there may be no symptoms, and if poisoning remains undetected death may occur within hours. Fatal poisoning in humans is very rare, usually occurring after consuming yew foliage. The leaves are more toxic than the seed.

Uses:

Wood from the yew is classified as a closed-pore softwood, similar to cedar and pine. Easy to work, yew is among the hardest of the softwoods; yet it possesses a remarkable elasticity, making it ideal for products that require springiness, such as bows. One of the world’s oldest surviving wooden artifacts is a Clactonian yew spear head, found in 1911 at Clacton-on-Sea, in Essex, UK. Known as the Clacton Spear, it is estimated to be over 400,000 years old.

Yew is also associated with Wales and England because of the longbow, an early weapon of war developed in northern Europe, and as the English longbow the basis for a medieval tactical system. The oldest surviving yew longbow was found at Rotten Bottom in Dumfries and Galloway, Scotland. It has been given a calibrated radiocarbon date of 4040 BC to 3640 BC and is on display in the National Museum of Scotland. Yew is the wood of choice for longbow making; the heartwood is always on the inside of the bow with the sapwood on the outside. This makes most efficient use of their properties as heartwood is best in compression whilst sapwood is superior in tension. However, much yew is knotty and twisted, and therefore unsuitable for bow making; most trunks do not give good staves and even in a good trunk much wood has to be discarded.

TAXUS BACCATA (YEW), TOPIARY, TREE CLOUDYew topiary tree cloud

Today European yew is widely used in landscaping and ornamental horticulture. Due to its dense, dark green, mature foliage, and its tolerance of even very severe pruning, it is used especially for formal hedges and topiary. Its relatively slow growth rate means that in such situations it needs to be clipped only once per year (in late summer).

Tree spotlight no.5 – the Hophornbeam

Hophornbeam

Hophornbeam or Ostrya is a genus of eight to ten small deciduous trees belonging to the birch family Betulaceae. Its common name is hophornbeam in American English and hop-hornbeam in British English. It may also be called ironwood, a name shared with a number of other plants.

The genus is native in southern Europe, southwest and eastern Asia, and North and Central America. They have a conical or irregular crown and a scaly, rough bark. They have alternate and double-toothed birch-like leaves 3–10 cm long. The flowers are produced in spring, with male catkins 5–10 cm long and female gaments 2–5 cm long. The fruit form in pendulous clusters 3–8 cm long with 6–20 seeds; each seed is a small nut 2–4mm long, fully enclosed in a bladder-like involucre.

hophornbeam details

The wood is very hard and heavy; the name Ostrya is derived from the Greek word ostrua, “bone-like”, referring to the very hard wood. Regarded as a weed tree by some foresters, this hard and stable wood was historically used to fashion plane soles. Ostrya species are used as food plants by the larvae of some Lepidoptera species, including winter moth, walnut sphinx, and Coleophora Ostryae. 

Colour/ Appearance: Wide sapwood is white to pale yellow.

Heartwood is a light brown, sometimes with a reddish hue.

Overall appearance can be very similar to birch.

Rot resistance: Heartwood is rated as non-durable to perishable regarding decay resistance, and is also susceptible to insect attack.

Workability: Overall, a difficult wood to work. Hophornbeam has high cutting resistance, (which also means that the finished wood product has good wear resistance). Reacts poorly to steam bending attempts. Turns, glues, and finishes well.

Odour :No characteristic odor.

Allergies/ Toxicity: Hophornbeam has been reported to cause skin irritation.

Pricing/ Availability: because of its small size, Hophornbeam is seldom harvested commercially. Likely to be limited in availability, even within its natural range. Expect prices to be high for a domestic hardwood.

Sustainability: This wood species is not listed in the CITES Appendices or on the IUCN Red List of Threatened Species.

Common Uses: furniture, canes, tool handles, and other turned objects.

Sierra Exif JPEG

plane tool

Celtic sacred trees – The crab apple

As the Crab apple tree was this week’s highlighted tree I thought I would include a blog about it’s mythological and sacred nature. At a later stage I will also look at the stories and symbolism associated with the Yew tree.

wild apple wood twigs

In general many types of trees found in the Celtic nations are considered to be sacred, whether as symbols, or due to medicinal properties, or because they are seen as the abode of particular nature spirits. Historically and in folklore, the respect given to trees varies in different parts of the Celtic world. On the Isle of Man, the phrase ‘fairy tree’ often refers to the elder tree. The medieval Welsh poem Cad Goddeu (The Battle of the Trees) is believed to contain Celtic tree lore, possibly relating to the crann ogham, the branch of the ogham alphabet where tree names are used as mnemonic devices.

The pome fruit and tree of the apple is celebrated in numerous functions in Celtic mythology, legend, and folklore; it is an emblem of fruitfulness and sometimes a means to immortality. Wands of druids were made from wood either of the yew or of the apple.

Hand carved druid wand from apple wood

Crab apples have long been associated with love and marriage. It was said that if you throw the pips into the fire while saying the name of your love, the love is true if the pips explode. Apple wood was burned by the Celts during fertility rites and festivals, and Shakespeare makes reference to crab apples in A Midsummer Night’s Dream and Love’s Labour Lost.

To follow are brief synopsis of some legends that talk include references to the apple tree, which I found in Wikipedia.

The soul of Cú Roí was confined in an apple that lay in the stomach of a salmon which appeared once every seven years. Cúchulainn once gained his escape by following the path of a rolled apple. An apple-tree grew from the grave of the tragic lover Ailinn. In the Irish tale Echtrae Conli (The Adventure of Connla), Conle the son of Conn is fed an apple by a fairy lover, which sustains him with food and drink for a month without diminishing; but it also makes him long for the woman and the beautiful country of women to which his lover is enticing him. In the Irish story from the Mythological Cycle, Oidheadh Chlainne Tuireann, the first task given the Children of Tuireann is to retrieve the Apples of the Hesperides (or Hisbernia). Afallennau (Welsh, ‘apple trees’) is a 12th-century Welsh narrative poem dealing with Myrddin Wyllt. The Breton pseudosaint Konorin was reborn by means of an apple.

Tree spotlight no. 4 – The Crab Apple

 

Malus-sylvestris-Native-Crab-Apple-Ireland

Crann fia-úll (Malus sylvestris)

Like the wild cherry, crab apple has been deliberately grown around old farmsteads (and the fruit used for crab apple jelly) but is also a truly native species found in old woodland. Crab apple is found in hedgerows throughout the Irish countryside. Unlike modern hybrid apples, crab apples grow true from the apple pips.


It is a small tree, very suitable for gardens. It bears attractive pink/white apple blossom in the spring, while the apples provide an autumn feature in the garden, as well as a useful crop.

crab apple tree in the middle of a large green field

What does crab apple look like?

Overview: one of the ancestors of the cultivated apple (of which there are more than 6,000 varieties), it can live to up to 100 years. Mature trees grow to around 10m in height. They have an irregular, rounded shape and a wide, spreading canopy. With greyish brown, flecked bark, trees can become quite gnarled and twisted, especially when exposed, and the twigs often develop spines. This ‘crabbed’ appearance may have influenced its common name, ‘crab apple’.

The crab apple is one of the few host trees to the parasitic mistletoe, Viscum album, and trees are often covered in lichens.

Leaves: the brown and pointed leaf buds form on short stalks, and have downy hair on their tips, followed by glossy, oval leaves, which grow to a length of 6cm and have rounded triangular teeth.

Flowers: in spring, the sweetly scented blossom is pollinated by bees and other insects, which develops into small, yellow-green apple-like fruits, around 2-3cm across.

Fruits: sometimes the fruits are flushed with red or white spots when ripe. Birds and mammals eat the fruit and disperse the seeds.

Look out for: it has a ‘crabbed’ or spiny appearance because of gnarled and twisted twigs.

Where to find crab apple

Crab apple thrives best in heavy, moist, well-drained soil and areas of scrub. They grow throughout Europe.

Value to wildlife

The leaves are food for the caterpillars of many moths, including the eyed hawk-moth, green pug, Chinese character and pale tussock. The flowers provide an important source of early pollen and nectar for insects, particularly bees, and the fruit is eaten by birds, including blackbirds, thrushes and crows. Mammals, including mice, voles, foxes and badgers also eat crab apple fruit.

CrabApples_large

How we use apple

The trees are often planted in commercial orchards as their long flowering period makes them excellent pollination partners for cultivated apples. The fruit can be roasted and served with meat or added to ales or punches. More commonly it is used to make crab apple jelly, and also as a natural source of pectin, for setting jams.

The pinkish wood has an even texture and makes good quality timber, and lends itself particularly well to carving and turning. It also makes a sweetly scented firewood. In Ireland a yellow dye was extracted from the bark to colour wool.

crab-apple-bark-2-lo-res

Threats

The crab apple is susceptible to a variety of fungal infections, including apple scab, honey fungus and apple canker.  As you can see above the Crab Apple tree in the Rose Garden in Trinity College Dublin suffers from apple canker, the white furry residue on the bark. 

Tree spotlight no.3 – Snakebark Maple

snakebark maple leaves and stripey bark

Snakebark maples are maples belonging to the taxonomic section Acer sect.Macrantha. The section includes 18–21 species, and is restricted to eastern Asia (the eastern Himalaya east to Japan) with the exception of one species in eastern North America.

The various species of snakebark maples are most easily distinguished from other maples by their distinctive bark, smooth (at least on young trees), and usually patterned with vertical dark green to greenish-brown stripes alternating with stripes of light green, pinkish or white, sometimes with a bluish tone.

Other characters include stalked buds with just one pair of scales, and flowers on arching to pendulous racemes. The samaras are small, and often numerous.

They are small deciduous trees, typically 5–15 m tall, rarely to 20 m tall, fast-growing when young but soon slowing down with age, and often short-lived; they typically occur as under-storey trees in mountain forests, often along stream-sides.

acer_red_flamingo_snakebark

Some have good fall colour with tones of of reds and orange, while others tend toward a pale yellow which is less impressive.  All are relatively hardy compared to many other species of maples, and many are widely cultivated as ornamental trees for their bark. 

Rag trees in the Rose Garden

 

When you visit the Rose Garden in Trinity College you will see that a few of the trees have many ribbons and scraps of fabric tied to the branches. See images above.   I haven’t been able to find out who initiated this tradition or the reasons behind them. I can only presume though that they follow the traditions of the Rag Trees that are scattered around the country on the roadsides.

A ‘ragtree’ or ‘raggedy bush’ can be completely covered in rags or scraps of clothing. Usually, though not always, the trees are close to Holy Wells, and they are almost invariably Hawthorn trees.

The custom of hanging rags on trees is particularly strong among Ireland’s Traveller community, an indigenous minority of nomadic people whose culture is very old and who still maintain many ancient customs which have largely died out among the rest of the population.

Usually the rags are placed there by people who believe that if a piece of clothing from someone who is ill, or has a problem of any kind, is hung from the tree the problem or illness will disappear as the rag rots away. Sometimes the rag represents a wish or aspiration which will come to pass as the rag rots.

St-Gob-RagTree-Mont

When I encounter a tree where someone has left a token behind for whatever reason it always makes me wonder about the person or the intention tied to the token. I think these Rag Trees are an important conduit, allowing for a private intention to be expressed in a public sphere.