Essentially, for a while, the SCQ has been interested in presenting a phylogeny related section, which would use some type of creative form as its driving force. And why not haikus (or is it Haiki?). So this is essentially a call for some Haiku’s on various organisms, preferably ones that work in the phylogenetic context.

Can you imagine it? A phylogenetic database derived solely from 5,7,5 syllabus prose. Should be quite interesting, and hopefully, we’ll be able to set something up so that Haiku submissions can be dynamic (i.e. real time).

Anyway, to get your creative juices rolling, here is a sampling that have come in so far:

DROSOPHILA MELANOGASTER DISASTER
fruit fly punnett squares
tic-tac-toeing genotypes
gives birth to Mothras

~Scheherazade

THE HAIKU CHEAT
Tyrannosaurus
Is a bitch for a haiku
Too many sybbles

~Paul Clarkson

UNTITLED
Cynics declare that
The vestigial organ
Is the human brain.

~Jonathan Cohen

OSTRICH
Plumply arching in
the dry Aussie African savanna,
head stuck in the ground

~Robert Isenberg

E.COLI IN MY BUTT
I learnt this today
there’s e.coli in my butt
also in my gutt

~Henry James

On page 1420 of the old Second Edition Webster’s Unabridged my father bought over 20 years ago for my brothers and me, it states that in music, a prelude is an introductory section or movement of a suite or fugue, and that since the 19th century it has become any short romantic composition.

I was not quite four when my family moved to Vancouver from Manila. I remember it was the nearing the end of the rainy season, and it was overcast and grey. I was wearing my best dress – crinoline and lace that made a wedding cake of my middle. Tito Jimmy and Tita Boubot, my mother’s siblings, each held one of my arms, and I was swung over the gigantic puddles that the rains had carved into the dirt roads. My shoes got wet, despite their best efforts.

The collective of moving parts in a piano that is responsible for striking the string is called the action. When a key is depressed, a domino-like cascade of events ultimately results in vibrations from the hammer’s contact with the string. These vibrations are carried along the length of the string and pass over a short straight bridge for the bass notes, or a long curved one for the treble, and are kept in place by steel pins. These pins clearly delineate the terminal node of each string and aids in sound transmission to the soundboard.

When I was nine, my parents rented our first piano, and I started lessons with Nancy, a quiet awkward Chinese girl who had just finished grade 12 of the Royal Conservatory of Music Piano Studies. She was just barely nineteen, and lived in a new Vancouver Special on the east side, a white stucco two storey house with red brick paneling the height of the faux oak double doors. It took my father two months to warm up to the idea of allowing my lessons, and another two to rent the piano. It was an old plywood upright that dominated our small living room, but soon I was well on my way to conforming to the vogue that many immigrant families could boast of; a child prodigy.

A typical piano has 226 strings. From the extreme bass, 10 notes each have a single string. To produce the low pitch of these notes, these strings have a steel core which are wrapped in copper or iron wire to reduce the speed of vibration. To avoid being overpowered by the thicker and louder bass notes, the next 18 notes have two strings each, and the 60 notes that lead to the top treble each have 3 strings. The bass notes are strung in a diagonal across the treble to centre them on the soundboard, and to conserve space.

On Tuesday nights, I was allowed to accompany my brothers to their scout meetings at the old church on 41st Avenue. While they learned to tie knots and mend buttons, I would sneak into the dark chapel, turn on all the lights and play Studies on the baby grand to an empty hall. I’d look up at the vaulted ceiling and play without looking at my fingers, liking the reverberations the chapel provided me but refusing to acknowledge its size. I used to hate it when sounds of my struggles attracted curious parents to leave their sons to their ropes and seek out my solitude, and I would always stop and leave, saying I didn’t really know any songs – I was just learning.

A piano frame is called a harp, and is usually made of iron cast in a single incredibly strong structure that can withstand the tension wrought by the strings. The average upright has a combined pull of 50 000 pounds of pressure. Attached to the harp is a hardwood pinblock which houses the steel tuning pins to which each string is coiled. To maintain the proper tension in the strings, the pinblock must be able to hold each pin by friction alone.

My first piano was a monstrosity of an upright. It was stood five feet high, its long thin frame bearing tiny scars where the finish had chipped on the right front corner and in a cloud above the pedals to reveal cream coloured striations. It had a rather ornate music stand that would unfold impossibly from the case, and housed yellowed ivory keys, chipped at B and high D. An octave would barely be enclosed by my five fingers; my little right pinky was perpetually being caught by B’s sharp teeth. And sitting on the edge of a bench that was a just a bit high, my bare feet still recall the places where the brass had worn away on the pedals, and where the geography of these seas pressed against my soles.

The two bridges transmit the insubstantial sounds made by vibrating strings to a thin wooden diaphragm called the soundboard. This is slightly crowned towards the strings to maintain compression and vibrancy, and to keep it from buckling under the tension of the strings. Through a balance of rigidity and flexibility, the soundboard radiates the vibrations into the air.

On page 720, a fugue is described as a polymorphic composition constructed on one or more short subjects or themes, which are harmonized according to the laws of counterpoint, and introduced by the various instruments or voices in succession with various contrapunctal devices.

My father never understood why I couldn’t play sonatas right away. I found myself playing quieter and quieter so as not to disturb him, cutting short my practice time to cut short the time he would berate me for not progressing fast enough. I took to playing just until he was due home from work, and learned to have the case closed and the piano books away well before he came in the door. As the colours of my Piano Studies books slowly deepened from yellow to wine red, I started coming home from school later and later so that I would have to spend only a short while in front of the monster.

In music, pure tones are rarely heard. There is no such thing as an ideal string – one composed of the perfect alloys to allow it to vibrate without any stiffness, and at any frequency. Instead, we hear notes made of a matrix of pure and over tones determined by the vibrational capacity of the materials from which the sound originates, and these in turn, allow us to distinguish the sound of one instrument from another.

By the time I turned a gangly, bucktoothed thirteen, my father had been promoted to master controller at Pacific Coach Lines, and he invested in a new piano. It was smaller; a sleek apartment sized Royale with only two pedals. It stood three foot six, and only just over four across, and was stained a dark mahogany. Looking at it, I felt as if it was missing something that had so appealed to me in the beginning with the first piano. Looking at it, I’m sure he felt it a great achievement, something so much greater than polishing shoes for 50 centavos on the streets of Manila.

An instrument’s timbre is the fusion of all the separate tones the vibrating system produces. Warmth is attributed to the number and relative loudness of the partial tones that accompany the fundamental. Woodwind and most stringed instruments produce harmonic overtones that are simply ordinals of the fundamental. However, there is a force that governs the vibrations of any string that seeks to restore it to its original position after being displaced, and is influenced by the stiffness of the string itself. In a piano, this stiffness generates partial tones that depart from simple harmony as the notes climb to the upper registry.

The less I played, the more clumsy my fingers became on the piano, and I started to forget how to move my hands above the keys. As a teenager, on the very few occasions that I was left the house to myself, I would sit in front of the piano and play snippets of music that had somehow gotten caught in the confused composite that was my memory; old studies, fragments of my imagination. It was at these times that I wished that I had never started piano lessons, and felt shame that I couldn’t be what my father wanted me to be. And I wouldn’t close my eyes, for at any moment, the door could open and I would be caught and reprimanded. I became more quiet and introverted, hidden by baggy clothes and braces.

The resonance of two or more frequencies produce beats equal to the difference in cycles per second between each tone that is sounded. Small differences in fundamentals are amplified in a piano’s inharmonic partial tones. The beats sounded by a chord, or by the multiple strings in 78 of a piano’s keys sound a remarkable simultaneous aural complexity.

A suite is a set or series of related things; an early form of instrumental composition consisting in a series of dances in the same or related key, a modern composition in a number of movements, page 1823.

I left home early. Got involved with a boy who slowly but steadily devoured my confidence. I lost value in my father’s eyes. I remember what it felt like to have the birthday gift I offered as a truce to the silence between us, refused. How afterward I went home, and tore the handmade card into a thousand, thousand pieces. It would be four years before he acknowledged me again.

Sound is a vibration of matter. A vibrating source transmits its movement to adjacent air molecules that in turn agitate their neighbours. In this manner, compression waves travel from the source in spherical ripples through the air.

In my first year of university, I hired movers to transfer the piano from my parent’s home to mine. I sat without words in my living room the first night, my hands resting on the smooth cool surface of its case, making up excuses as to why I couldn’t, shouldn’t play. For years afterward, it sat as a beautiful shelf, holding incidentals – plants, CD’s, the occasional drink. It was rare to see the stark dichotomy of the black and white keys, and even rarer hear anything, even discordant clusters of notes, from the soundboard.

Sound waves are collected by the pinna. The fine membrane of the eardrum is stimulated by the force of each compression wave and vibrates. In a well timed reversal, this drum begins a cascade of vibrations that begin with the hammer to the coils of the cochlea, then carried and transformed along the strings of our neurons from raw data to music in the cerebral cortex of the brain.

Now and then I tell myself I’m saving it for my future children, this misplaced, good intention of my father’s. It took me a long time to get over my anger, and gain a small understanding of what it must’ve been like to leave a life, a country behind and begin by working the graveyard shift at the local corner store to support a family of six. I hope that my children will never know what it is like to be ashamed of your accent when you had mastered English back home, nor have an ocean to separate you from your roots. And we never speak, my father and I, of old disappointment.

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(REPRINTED FROM ISSUE TWO, SEPTEMBER 6th, 2005)

Want to expand a genome? Previous thinking suggests you only need some transposable elements, often nicknamed “jumping genes”, to repeatedly, and irreversibly, insert into the genome. Time will take care of the rest.

However, new research is now challenging this view by revealing that transposable elements can also be deleted during evolution.

The new findings were recently reported by Louie van de Lagemaat, Dixie Mager and their colleagues from the Terry Fox Laboratory in Vancouver, BC. They compared the human, chimpanzee and Rhesus monkey genomes and found 37 instances where transposable elements were present in the primitive Rhesus monkey, but lost in the human and chimpanzee genome.

In finding that transposable elements can be deleted during evolution, van de Lagemaat and colleagues also discovered that such deletions occur between short flanking repeats of DNA — a finding that points to a mechanism for how genome size my be attenuated during evolution.

“Our work strongly suggests an important role for short, non-adjacent, identical segments of DNA in genomic deletions,” said Dr. Mager in a press statement, “and it lends insight into deletion mechanisms that help to counterbalance genome expansion in primates.”

Transposable elements are a well known cause of genomic expansion, and in mammals, have been reported to comprise upwards of 50% of the genome. The prevailing theory is that such expansions are unidirectional, with transposable elements becoming irreversibly maintained in a population over time.

However, this theory wasn’t what van de Lagemaat and colleagues were originally interested in. Instead, they were simply looking for new transposable elements in the human genome.

“We were looking at supposedly new transposable elements but they instead appeared to be of a very old variety,” said van de Lagemaat, who is the lead author of the current study. “So we started to wonder if some of them were deleted,” he said, thereby making them look new when they weren’t.

van de Lagemaat and colleagues therefore went about aligning the entire human, chimpanzee and Rhesus monkey genomes, to see how many transposable elements had deletions. They estimate that 0.5 to 1.0% of transposable elements that look like insertions are instead deletions. The results of the study appear in the September issue of Genome Research.

“The baseline belief is that anything can happen but some things are more frequent than others,” said van de Lagemaat about what he first thought when he saw the data. “This is one of those ‘any-things’ that isn’t very frequent,” he said.

Although the frequency of precise transposable element deletion is rare, van de Lagemaat said the mechanism that causes their deletion is not.

The mechanism involves flanking identical repeats of at least 10 base pairs of DNA, which play a role in recombining and removing DNA sequence between them. van de Lagemaat and colleagues suggest that 19% of genomic deletions between 200-500 base pairs since humans diverged from chimpanzees are due to these repeats.

“We are looking at a generalized deletion mechanism,” said van de Lagemaat, “and such deletions are important in a lot of circumstances like disease, giving us the potential to more deeply understand them.”

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(REPRINTED FROM ISSUE TWO, SEPTEMBER 6th, 2005)

Picture a group of scientists exploring tropical forests to collect plants, fungi or microorganism samples. They are bioprospecting. In other words, they are looking for new compounds that may help remediate so-far incurable diseases. Picture them settling in villages and discussing with the shaman to learn their traditional way of using plants to heal their people. These local medicine men are often rich of a secular oral tradition about nature’s secrets. Now, picture the scientists coming back to their wealthy country with their suitcase full of unexplored drug candidates. Ten years later, after intense research work and numerous trial and errors, one of the plants used by the shaman is found to be a revolutionary cure for cancer. At first glance, this seems like blockbuster story! But is it, really? Alas, the reality is more complex, and the happy end is not always within reach for all the story’s characters.

Bioprospecting refers to the search of biological products with characteristics interesting for humankind. In the past, bioprospecting has focused on the quest for new chemical compounds with medicinal or anti-microbial properties, and has many times been successful. In 1958, for example, a research group was able to isolate two new therapeutic agents from a Madagascar plant called the rosy periwinkle The plant was found using cues from local shamans and spiritual herbalists [1]. If bioprospecting leads to a successful commercial product, it is likely that the company financing the initial “hunting” effort will want to protect their intellectual property rights on the product. But how are the country or the indigenous communities that traditionally use the beneficial natural resource going to be acknowledged or rewarded for their contribution? Moreover, how are they going to be affected by the sudden commercial value of the species producing the active compound? For instance, a curing plant that was a free commodity for the local communities of a tropical country may become a marketed one, now unaffordable for the people who discovered its virtues. Even worst, maybe the patent filed by the pharmaceutical company that developed the drug in its commercial form will prohibits the use or sell of the plant by indigenous groups.

These questions and issues are now getting even more complex, as advances in biochemistry and molecular biology make the own genetic material of living organisms very easy to extract and exploit for commercial purposes. In effect, with the recent sequencing and analysis of a growing number of plant and microbial genomes, biologists have gained a great knowledge about the genes allowing for the synthesis of interesting chemical compounds. These genes, which encode for enzymes capable of converting one molecule into another, are the best tools you can possibly dream of to push even farther the limits of chemical diversity that may be generated in a laboratory, and thus increase the production of new molecules to be tested as drug candidates. As a result, scientists believe that the future of bioprospecting lies in the search for genes rather than for chemical compounds [2]. Once an interesting gene has been found in a given species, bioengineers are able to isolate the DNA stretch corresponding to it and even to modify its sequence in order to alter the structure of the protein resulting from the translation gene. They can then insert the modified gene into another organism and control when and where it is going to be expressed. In this case, after such a number of modifications and transfers, should which organism originally contained the gene and the country it comes from still be acknowledged? At this point it seems difficult to bridge the gap between the people who discovered a product through trial and errors of the course of their culture history and a the biotech company which can, by highly technical and costly efforts, transform and improve that product to make it efficient and available to the greatest number of people [3].

When it comes to the expropriation of land for mining purposes, several indigenous communities were previously able to obtain a financial compensation. Therefore, it would be logical that, in a similar way, they get a share of the benefits resulting from the use or exploration of their genetic resources, especially if they provide cues to scientists as where to look. Unfortunately, the current patent laws clearly favors “western science” and innovative economies rather than traditional medicine and oral wisdom. As a result, many civil society groups and activists describe bioprospecting as a form of piracy. They argue that bioprospecting leads to a loss of power of indigenous people over their own resources, which is particularly threatening to their lifestyle since most of these people heavily depend on the local biodiversity for their survival [4]. Some countries are now developing strict measures in order to restrict access to their resources by foreign companies. For example, India has recently established a national gene bank and created a new body of legislation in order for all exportations of plant genetic resources to be highly regulated by the government. The new legislation forbids open access to the gene bank by all American agri-businesses, revoking an agreement previously made with the US government. These measures where taken in reaction to several cases of controversial exploitation of resources by first world parties. One of these cases involves a plant called neem, which has been used for millennia in India for its anti-microbial properties, but on which a corporation recently tried to put patent rights [5].

Unfortunately, many developing countries do not have the economical power or the political commitment necessary to put in place similar national protective measures. They also face great pressure by countries lending them money to open their frontiers to the latter’s companies. In addition, the World Trade Organization (WTO) recently brought about an international agreement on intellectual property rights that supports the idea that government control of resources is in itself an obstacle to economical growth and should therefore be avoided. Having individuals empowered to negotiate the price for the share of their biological resources is considered an obstacle to the creation of a free global market [3]. The treaty imposes no requirements for the bioprospecting parties obtain approval from local communities or their government before proceeding to natural resource hunting. In addition, the benefits obtained for the commercialization of a product obtained by bioprospecting do not have to be shared with indigenous communities. Despite these controversial aspects, all countries that are members of the WTO are under great pressure to sign this agreement because failure to do so can lead to trade sanctions.

At first glance, it really seems like the indigenous people’s claims on bioprospecting are hopeless, a typical David and Goliath battle. However, the story is not as dark as it sounds. Indeed, there are still considerable efforts made to integrate bioprospecting with the needs and rights of the developing countries. For example, The Convention on Biological Diversity, brought about in 1992 by the United Nations (UN), stipulates that bioprospecting shall not be done without the consent of the host country. According to that convention, the exploitation of local resources for drugs and medicine purposes shall be approved and actively involve local traditional communities, and the benefits made form such resources be shared in a fair and equitable way [6]. Another promising initiative is the International Cooperative Biodiversity Group (ICBG), a network of bioprospecting projects funded by the US government. The main goal of ICBG is to find plants bearing chemical compounds that could cure key diseases in the United States. However, countries that are hosting the searches can expect fair rewards and benefits. Typically the projects are set up so that the first extraction steps and analysis of candidate compounds are carried out in local laboratories, therefore creating new jobs and the development of a certain expertise. If a compound shows desired properties, further research and clinical trials are transferred to laboratories in the United States. If the compound leads to commercialized drug, 50% of the royalties are invested in a community development fund run by indigenous people and another 30% goes to research on tropical diseases.[7]

Finally, bioprospecting is also beginning to foster a renewal of awareness toward the conservation of biodiversity [1]. In fact, it is appears ironic that the countries where biodiversity is the richest and the most extensive are often some of the poorest in the world. Bioprospecting actors such as European and North American research institutes and pharmaceutical companies are of course interested in the preservation of such biodiversity, as they see it as bounties of potential new drugs. However, for developing countries, preserving biodiversity is luxury that they cannot afford, especially when these natural resources can provide a quick profit. The spread of initiatives such as the ICBG that propose a fair share of the benefits made out of bioprospecting activity is likely to influence governments of biodiversity-rich countries regarding the management of their natural resources. On these bases, bioprospecting has the potential to bring not only hope to human health but also to social justice and environment conservation. Now that is a great blockbuster story!

References

1. Onaga, L., Cashing in on nature’s pharmacy: Bioprospecting and protection of biodiversity could go hand in hand. EMBO Rep, 2001. 2(4): p. 263-5.

2. Firn, R.D. and C.G. Jones, Avenues of discovery in bioprospecting. Nature, 1998. 393(6686): p. 617.

3. The complex realities of sharing genetic assets. Nature, 1998. 392(6676): p. 525.

4. ETC-Group, From global enclosure to self enclosure, in ETC group Communique. 2004. p. 1-14.

5. Jaymaran, K.S., India seeks tighter controls on germplast. Nature, 1998. 392: p. 536.

6. Masood, E., Social equity versus private property: striking the right balance. Nature, 1998. 392(6676): p. 537.

7. Masood, E., A formula for indigenous involvement. Nature, 1998. 392(6676): p. 539.

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(REPRINTED FROM ISSUE TWO, SEPTEMBER 6th, 2005)

REFERENCE:
Potential Effects of the Next 100 Billion Hamburgers Sold by McDonald’s. (2005) American Journal of Preventive Medicine 28(4) :379-381

ABSTRACT:
Background: McDonald’s has sold more than 100 billion beef-based hamburgers worldwide with a potentially considerable health impact. This paper explores whether there would be any advantages if the next 100 billion burgers were instead plant-based burgers.
Methods: Nutrient composition of the beef hamburger patty and the McVeggie burger patty were obtained from the McDonald’s website; sales data were obtained from the McDonald’s customer service.
Results: Consuming 100 billion McDonald’s beef burgers versus the same company’s McVeggie burgers would provide, approximately, on average, an additional 550 million pounds of saturated fat and 1.2 billion total pounds of fat, as well as 1 billion fewer pounds of fiber, 660 million fewer pounds of protein, and no difference in calories.
Conclusions: These data suggest that the McDonald’s new McVeggie burger represents a less harmful fast-food choice than the beef burger.

The Participants

Charles Shaw: A brand of wine, widely known as “two buck chucks” for its affordability (although in Ohio, one can’t seem to find it for no less than $3.33). An acquaintance of both participants in this dialog.

Herman Kahn—Arguably the most celebrated and controversial nuclear strategist of his time. Often said what was on everyone’s mind, was Director of the Hudson “think tank” and wrote “On Thermonuclear War.” Also a rather large man.

Me– Pacifist. Has the last name Kahn, but is probably unrelated to Herman Kahn and related to Justin Kahn only in his own imagination. Definitely smaller than Herman Kahn though.

The Dialog

Charles Shaw: Let’s party. For tomorrow we die.

Herman Kahn: That fact that nuclear war is a terrible prospect does not mean that we can justify avoiding thinking about the possibility. In order for a threat of retaliation to be credible, we must be willing to make provisions for survivors in the event of thermonuclear war.

Me: Tolstoy believed that drinking and war were both ubiquitous human experiences, that could be universally abstained from if people were at all happy.

Charles Shaw: You both need to relax.

Herman Kahn: I joke about a nuclear war. But that’s because I want people to recognize that this is a serious reality which deserves to be treated as an eventuality, not as some impossibility. I believe that if we are fully prepared to face a nuclear war, we can avoid it. At least until Mr. Kahn learns how to dance.

Me: I’m sorry my mind was elsewhere. I don’t want to die. What’s going on?

Charles Shaw: I serve Dionysus. Think wine, think freedom. I could teach Mr. Kahn to dance.

Herman Kahn: I serve the city, the organization and protection of people. I already know how to dance. Oh, right. The other Kahn.

Me: I’m sorry what’s the difference?

Charles Shaw: The divine grape hurts no one.

Herman Kahn: Anyone who refuses to think about the nature of modern warfare is hurting. I refuse to allow the emotions or other components of the individual overtake the concerns of the future. Our children deserve better.

Me: You two deserve each other.

Charles Shaw (to Herman Kahn): Will you be mine?

Herman Kahn: I have been so concerned with applying systems analysis to artificial scenarios, that I forgot how wonderful it is to be human, to feel the warmth of a cup of coffee. O.K. I stole that coffee bit from the Wim Wender’s film.

Me: I love you both. I hate myself for it.

Charles Shaw: Love is a strong word. But not strong enough.

Herman Kahn: Hate is a strong word. But not strong enough.

Me: What I was thinking would be ideal would be to have Herman set up a war game, so I could get a sense. I mean try everything at least once. Charles, I was thinking one drink, maybe two, just to take the edge off of things.

Charles Shaw: You don’t understand me, because you overestimate thought.

Herman Kahn: You don’t understand me, because you overestimate sobriety.

Me: So what am supposed to do?

Charles Shaw & Herman Kahn: We will leave you here to think about that.

Me: Alone?

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(REPRINTED FROM ISSUE TWO, SEPTEMBER 6th, 2005)

(August, 2004)

Seeing a cell is an essential aspect of cell biology. To the small world of the cell, confocal microscopy is a major advance upon normal light microscopy since it allows one to visualize not only deep into cells and tissues, but to also create images in three dimensions [1]. There are many aspects to a confocal microscope that make it a much more versatile instrument than a conventional fluorescence microscope. Although the confocal microscope is often thought of as an instrument that can create 3D images of live cells, the great versatility of the machines means that many creative ways of examining not just the structural details, but also the dynamics of cellular processes, are being developed [1].

An Introduction to Confocal Microscopy

The term confocal refers to the condition where two lenses are arranged to focus on the same point1. The major optical difference between a conventional microscope and a confocal microscope is the presence of the confocal pinholes, which allow only light from the plane of focus to reach the detector (see Figure 1). This forms the principle of a confocal microscope where “out of focus” light is removed from the image by the use of a suitably positioned pinhole. This produces images of exceptional resolution and clarity, and also allows the user to collect optical slices of the object for use in creating a 3D representation of the sample. How does this work? It involves changing the level or plane at which the sample is observed. If the plane of focus is changed, or the object moved, a series of images at different positions can be produced through the thickness of the object, i.e. a series of X-Y images at different Z positions. Such a series of images (a stack) is a three dimensional representation of the object being looked at, produced by optical (as opposed to physical) sectioning (see Figure 2). This is the main feature and a major advantage of confocal microscopy over conventional microscopy [1].

Figure 1: An Example of a Confocal Pinhole

In practice, the confocal principle is combined with a scanning system utilizing a laser light source [1-2]. This builds up an image by scanning a point of laser light across the sample in X and Y directions (see Figure 2). In the case of a laser scanning system, the detector of reflected light from the sample is generally a photomultiplier tube. The signal from the photomultiplier tube is converted to a digital form that contains information on the position of the laser in the image and the amount of light coming from the sample. A computer is used to store the intensity value of each point from the detector, and presents these in the correct order on a high-resolution video monitor to display the image. The image is displayed on the computer screen as a shaded gray image with 256 levels of gray that can be suitably coloured later for presentation or publication. To collect a series of images, the computer then shifts the focus by a fixed amount and the object is scanned again to produce the next image at the different Z position. This image is stored and the process repeated to build the 3D data set.

Confocal microscopes possess several advantages over conventional microscopy. First, confocal microscopy produces images of improved resolution, up to 1.4 times greater than standard microscopy, by eliminating out-of-focus light. Confocal microscopes also have a higher level of sensitivity compared to conventional microscopes, due to highly sensitive light detectors and the ability to accumulate images captured over time. Another key advantage of confocal microscopy is the ability to produce 3-dimensional reconstructions of specimens as mentioned above. Computer software is then used to digitally reconstruct 3D representations of the sample. Confocal microscopy is also a less invasive form of imaging. This is due to the use of high-power laser illumination and the reduction in light-scattering artifacts, allowing the non-invasive imaging of thick sections of semi-transparent tissues [2].

Figure 2: Confocal Scanning of a Sample

Setting up To View a Sample on a Confocal Microscope

Unlike samples for conventional microscopy, tissue and cell preparations for confocal microscopy do not need to be thin [3]. There is little or no advantage in viewing a thin section with a confocal microscope. It is possible to optically section several millimeters into a transparent tissue at low magnification (20X objective) while the practical limit with a normally opaque tissue with a 63X objective lens may be 20 micrometers or less.

In confocal microscopy, as in conventional microscopy, it is necessary for the tissue specimen of interest to demonstrate some form of optical contrast between different areas of the sample for visualization [1-3]. This requires staining of the tissue specimen using a label that either absorbs or reflects light or is fluorescent. As a general guide, fluorescent labels are most versatile and represent a good starting point. Most staining protocols that work well for conventional microscopy can be easily adapted for confocal microscopy, although for thick tissue sections, the diffusion of labels into tissue is often a major limiting factor. It may be useful to increase the degree of staining for some tissue sections in confocal microscopy since thin optical sections of the samples are used. In other cases, it may be necessary to considerably increase incubation periods and to use detergents (such as 1% Triton-X100 in PBS for 4 hours) or other treatments to allow the label to penetrate [4]. Vibratome sections of solid tissues (50-100 _m thick) are a useful way to prepare thick tissue for confocal microscopy [4].

Different Types of Confocal Microscopes

With a prepared sample, it is next necessary to choose what type of confocal microscope to use? Two types of microscope are most common: (1) a laser scanning confocal microscope (LCSM) and (2) a two-photon confocal microscope. Both of these microscopes are used to give optical sectioning of a sample, but do so in a different manner.

In a typical set-up for a laser scanning confocal microscope, a pinhole is placed in front of the light source to produce a distinct and spatially constrained illumination point (See Figure 3). The light passing through this pinhole is focused on the sample, while a second pinhole is placed in front of the light detector. If the optical distance from the detector pinhole to the focal point (the point in which the light is focused) is exactly the same as that between the focal point and the illumination pinhole, only the light generated at the focal point will reach the detector since the pinhole will block out the out-of-focus light. The signal from the detector is then digitized and passed to a computer. The image of the sample is digitally built up by scanning the sample in the X and Y directions and then special software is used to reconstruct a digital image [1-3].

Figure 3. Laser Scanning Confocal Microscope setup versus Two-Photon Confocal Microscope setup. In a confocal microscope, the detector must be the same distance as the light source from the illuminated portion of the sample to effectively block out-of-focus light. The position of a detector in a two-photon system is variable

Two-photon, also referred to as multi-photon, microscopy setups are virtually the same as laser scanning confocal microscopy, except that the need for pinholes is eliminated. Optical sectioning of the sample is instead achieved through the use of a mode-locked Ti:sapphire laser which operates in the near-infrared (see Figure 3). The laser produces a high photon density (tens of kilowatts of peak power in a series of low-energy pulses that are approximately 10nJ per pulse) that is tuned to a wavelength about twice that of the intended absorption wavelength of the sample. What this means is that two or more photons are required at a single point to produce an optical signal (i.e. excitation) that can be detected. The probability of such a two-photon event occurring is limited to the focal plane where there is an extremely high photon density. As a result, in 2-photon imaging, excitation occurs only at the plane of focus [2].

There are several major advantages in the use of the rather expensive and technically sophisticated two-photon confocal microscopy versus laser scanning confocal microscopy. First, out of focus bleaching (the loss of optical signal production due to sample damage from the laser) is reduced [2]. Two-photon microscopy also increases sample penetration because of the reduced absorption of near-infrared radiation. This allows thick, live tissues to be imaged with little damage to the sample environment [5]. In addition, two-photon microscopy is believed to increase sensitivity since the elimination of the pinhole allows the entire signal to reach the detector [2].

Applications of Confocal Microscopy

Confocal microscopy is now being used in a large variety of scientific fields [5-6]. Some of the applications include the use of confocal microscopy for live cell imaging. This is often done by making use of a green fluorescent protein (GFP) (Figure 4) or other fluorochromes attached to cellular components (proteins, genes, cell structures) and imagining their position or movements. Confocal microscopy is also used to analyze subcellular functions, such as pH gradients and membrane potentials, using specific fluorescent dyes and to measure intracellular changes in ion concentrations of molecules such as calcium, sodium, magnesium, zinc and potassium.

Figure 4. This image shows the distribution of GFP in Arabidopsis cells when no localization tag is present. [7]

Confocal microscopy, whether laser scanning or the novel two-photon system, has generated a tremendous amount of excitement in the research community. This technique has rapidly become the technique of choice for researchers, particularly in the biosciences. With the recent availability of more affordable confocal and two-photon microscopes, this technology holds a great deal of potential for allowing the visualization of the cellular world.

Additional Reading

1. Nikon Microscopy U. An Introduction to Confocal Microscopy
2. Matsumoto B. 2002. Cell Biological Applications of Confocal Microscopy. San Diego: Academic Press. 507 p.
3. Diaspro A. 2002. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances. New York: Wiley-Liss. 567 p.
4. Wright SJ, Wright DJ. 2002. Introduction to Confocal Microscopy. Methods Cell Biol 70: 1-85.
5. Piston DW. 1999. Imaging Living Cells and Tissues by Two-Photon Excitation Microscopy. Trends Cell Biol 9(2): 66-9.
6. McWilliams A, et al. 2002. Innovative Molecular and Imaging Approaches for the Detection of Lung Cancer and its Precursor Lesions. Oncogene 21(45): 6949-59.

References

1. Hibbs A. 2000. Confocal Microscopy for Biologists: An Intensive Introductory Course. BIOCON ed. pp. 2-9.
2. Rawlings S, Byatt J. 2002. How Microscopy Produces a Sharper Image. In “Scanning Microscopy.” Biophotonics International: Laurin Publishing Co. Inc. pp. 1-4.
3. Bacallo R, et al. 1990. Guiding principles of specimen preservation for confocal fluorescence microscopy. In “Handbook of Biological Confocal Microscopy” New York: Plenum. Pp. 197-205.
4. Humphrey E, Norman K. 2001. A Practical Introduction to Confocal Microscopy: for Users of the Bio-Rad Radiance 2000 Confocal Microscope. University of British Columbia, pp. 1-15.
5. Michalet X, et al. 2003. The Power and Prospects of Fluorescence Microscopies and Spectroscopies. Annu Rev Biophys Biomol Struct 32: 161-182.
6. Stevens JK, et al, eds. Three- Dimensional Confocal Microscopy: Volume Investigation of Biological Systems. London: Academic Press.
7. Carnargie Institude of Washington, Department of Plant Biology, Cell Imaging.

(Art by Jane Wang – note that high res versions of image files available here)

(August, 2003)

Flow cytometry is a technology that allows a single cell to be measured for a variety of characteristics, determined by looking at how they flow in liquid [1]. Instruments used for this can gather information about cells by measuring visible and fluorescent light emissions, allowing cell sorting based on physical, biochemical and antigenic traits.

Flow Cytometers

A flow cytometer, sometimes called a Fluorescence Activated Cell Sorter (FACS), has several key components [2] (see Figure 1):

1. A light or excitation source, typically a laser that emits light at a particular wavelength; 2. A liquid flow that moves the suspended cells through the instrument and past the laser; and 3. A detector, in this case a photomultiplier tube, which is able to measure the brief flashes of light emitted as cells flow past the laser beam.

Figure 1. A rendition of a flow cytometer.

In a flow cytometer, single cells move past the excitation source and the light hitting the cells is either scattered or absorbed and then re-emitted (fluorescence). This scattered or re-emitted light is collected by the detector (see Figure 2).

Light Scatter

Scattered light is a consequence of a light beam making contact with a cell, resulting in either reflected or refracted light reaching the detector. The pattern of light scattering is dependent on cell size and shape [1,2], giving relative measures of these cellular characteristics as cells flow through the beam. This can be quite useful, as cells can be sorted on the basis of size or shape to different collection tubes using a technique called electrostatic deflection, which employs charged plates to change the path of the cell [2] (see Figure 2).

Figure 2. A schematic of fluorescence detection by a flow cytometer and a schematic of cell sorting by a flow cytometer.

Fluorescence

Fluorescence-based detection depends on the absorption of light by the cell and the subsequent re-emission of this light at a different frequency. Flow cytometers make use of this technology by employing filters to block the original light source from reaching the detector, while the fluorescence emission is allowed through for detection, which allows only a very low background of stray light to reach the detector [2].

In flow cytometry experiments, fluorescence is often achieved by the deliberate labeling of a cellular component using a fluorescent marker, usually a type of dye [3]. These dyes fluoresce only when light of the appropriate wavelength (specified by the frequency of the laser) hits them, causing the emission of secondary light at a different wavelength. Detection of the second wavelength is used as a measure of the presence of the dye on the cell and thus the component it is labeling.

Various fluorescent dyes are commercially available [4] and their emitted colors are reminiscent of the reds and greens seen in fireworks. The most common fluorescent dyes are Texas-Red, fluorescein isothiocyanate (FITC) and phycoerythrin (PE). Table 1 shows some of the different fluorochromes, their excitation and emission wavelengths, and their emission color.

Table 1. Common flourescent dyes.

Display and Interpretation of Flow Cytometry Data

The amount a cell scatters or fluoresces light is measured by the detector and subsequently need to be displayed for interpretation [1,2,5]. These profiles of cells are normally displayed as dot plots or histograms [5].

Dot plot displays use two parameters to graph the data generated by flow analysis, with each dot representing the passage of one cell through the detector (see Figure 3). The X- and Y-axes measure the different emissions, displaying a dot for each of the cells that show that particular emission. In Figure 3, the dot blot shows an example where two populations of cells have been analyzed by flow cytometry. A cell of a particular population type will show up as a dot in the quadrant of the dot plot designated for that population.

Figure 3. A Dot plot of flow cytometry data.

Histograms can also be used to display data from flow cytometry experiments. In these plots the X-axis shows the intensity of the detected signal and the Y-axis measures the number of events (cells) counted. Histograms often display the output of two (or more) samples using a single fluorochrome. In an experiment determining the presence or absence of a particular cell marker or a relative increase or decrease of a marker after experimental treatment, a histogram shows the shift in the fluorescence intensity of the sampled cells (see Figure 4).

Figure 4. A histogram of flow cytometry data.

Applications of Flow Cytometry

Flow cytometry is used in a variety of different fields including immunology, pathology and medicine, all the way to plant breeding [5-7]. A few of the most common applications are listed below:

DNA Content

Fluorescence staining of DNA followed by flow analysis has been used to determine a cell’s DNA content [8]. Stained cells with one copy of their genetic material (a haploid cell) will be half as bright as cells with two copies (a diploid cell). A cell varies between these states during the cell cycle and flow cytometry can be used to determine its position in the cell cycle based on its DNA content.

Evaluation of Cell-Surface Markers

Immunologists frequently use flow cytometry to determine the types of markers and receptors on the surface of a cell. For these experiments, a fluorescent dye is attached to antibodies or receptor ligands [3]. These cells can then be subjected to flow cytometry and the amount of the receptor on their surface detected as a level of fluorescence.

These experiments can be designed to incorporate more than one fluorescent marker at a time, giving the ability to detect multiple cell-surface markers simultaneously. Building upon the example of staining for particular markers or receptors, staining with more than one fluorescence dye allows researchers to determine whether there are populations of cells that contain multiple receptors.

A specific example is the analysis of the markers on T-cells. T-cells are a type of immune cell, which have cell surface marker proteins known as CD4 and CD8. In mammals there are T-cells that are CD4 positive, cells that are CD8 positive and cells that are positive for both markers. To determine the relative abundance of cells carrying the different markers, FITC-attached CD4 antibodies (normally termed FITC-conjugated CD4 antibodies) and Texas Red-conjugated CD8 antibodies could be incubated with T-cells. In flow cytometry analysis cells that were CD4 positive fluoresce green, while cells that were CD8 positive fluoresce red and cells that were positive for both markers give off green and red light (see Figure 5). Detection of the levels of each fluorescent color would give a measure of how many of each type of T-cell was present in the original mixture.

Figure 5. CD4 and CD8 T cells analyzed by flow cytometry.

Cell Sorting

Flow cytometry can be used to select and purify a specific subset of cells within a population [6,7] (see Figure 2). This is a popular application with researchers, since it allows the selection of cells expressing a particular receptor, in a phase of the cell cycle, or perhaps expressing a particular transgenic protein, followed by the culture of these cells as a pure population. Amazingly, a FACS can sort cells as fast as 15,000 cells/sec with very high purity (over 98%).

Texts Consulted and Additional Reading

1. Nunez R. 2001. Flow Cytometry for Research Scientists: Principles and Applications. Wymondham, Norfolk, UK: Horizon Press. 110p.
2. Tsieh S. 2002. Flow Cytometric Analysis of Hematologic Neoplasms: A Color Atlas & Text. Philadelphia: Lippincott Williama & Wilkins. 276p.
3. Stewart CC, Nicholson JKA, eds. 2000. Immunophenotyping. New York: Wiley-Liss. 442p.
4. A list of flow Cytometry links on the web

References

1. Givan A. 2001. Flow Cytometry: First Principles. New York: Wiley-Liss. 273p.
2. Owens MA, Loken MR. 1995. Flow Cytometry: Principles for Clinical Laboratory Practice. New York: Wiley-Liss. 288p.
3. Roederer M. 1997. Conjugation of monoclonal antibodies.
4. Molecular Probes Handbook.
5. Recktenwald DJ. 1993. Introduction to Flow Cytometry: Principles, Fluorochromes, Instrument Set-Up, Calibration. J Hematotherapy 2(3): 387-94.
6. Marti GE, Stetler-Stevenson M, Bleesing JJ, Fleisher TA. 2001. Introduction to Flow Cytometry. Sem Hematology 38(2): 93-9.
7. Weaver JL. 2000. Introduction to Flow Cytometry. Methods 21(3): 199-201.
8. Ross JS. 1996. DNA Ploidy and Cell Cycle Analysis in Pathology. New York: Igaku-Shoin.

(Art by Jane Wang – note that high res versions of image files available here)

– Click here to launch virtual lab –

(Screenshot #1)

This is the virtual version of the UBC Advanced Molecular Biology Laboratory’s experimental kit #2 (see Restriction Digest of Lambda DNA and Gel Electrophoresis for details) which features a common and important molecular technique used in laboratories to analyze DNA. This molecular technique can be used to isolate a gene of interest and to identify DNA sequences that are different. This molecular technique is particularly useful in forensics where it is used to analyze DNA from crime scenes! In this virtual experiment, analysis is performed on lambda DNA and will consist of two main steps. The first step is to use restriction enzymes to cut lambda DNA into fragments of different length. The second step is to perform gel electrophoresis where the DNA fragments of different length are separated by size and dyed for visualization forming a band pattern.

(Screenshot #2)

This picture is a good example of how the basic squirrel fishing model works. It’s best to find a fairly open space with enough room to walk around a bit. We stayed away from a group of ne’er do-well hippie hooligans who were playing games, as they were likely to inadvertently interfere with our fishing agenda. Keep in mind that squirrels often live in public places, so it might take time to find a secluded area. Trust me, though: it’s worth it in the long run. It doesn’t matter if people are somewhat close (see the man in the picture). Once they see what you’re doing, they tend to keep their distance.

There are two ways to go about casting a line. Evan opted to use his line sans rod, using a key to weigh the end down. This method was what I initially tried, but on my first cast, I wasn’t holding on to the string tightly enough. A squirrel grabbed the bait and began to run away. I chased the squirrel in an effort to regain my equipment, but he was too quick for me. I wound up falling on my arse and slipping in the grass. Chattering, the squirrel ran up a tree and defiantly nibbled on the nut. After a considerable amount of work, I was able to reclaim my fishing gear, but I decided to create a makeshift fishing rod.

Just be sure to select a sturdy twig and tie the line tightly around the end. I wouldn’t suggest using an actual fishing rod and reel, as that could cause problems with ornery park rangers who do not appreciate the fine art of squirrel fishing. Plus, a shorter pole allows greater contact with your friends the squirrels, and isn’t that what we’re all looking for?

Form and Technique:
Here, Evan demonstrates how to effectively lure the squirrel. Notice his slight crouch and bent knees. This position says to the squirrel, “Hello, squirrelie! I am your friend! I’m not a big scary human – I’m a nice human who wants to meet you!” The squirrel, though hesitant, will approach slowly. It’s important to refrain from sudden or jerky movements; this will frighten the squirrel, who usually scampers up a tree.

I’ve found that the human voice is music to a squirrel’s ears. Squirrels seem to be entranced by a soft coo or a gentle greeting. Evan and I took different approaches to the vocal lure.

Me: “Oh, hello, lovey! Hello, squirrelie! Oh, come HERE, I have a lovely treat for you, sweetie! Come on, lovey! That’s nice!”

Evan: “C’mere. C’mere, bub.”

You can guess who the squirrels came to see first.

Zen and the Art of Squirrel Fishing Maintenance:
Assuming that the aforementioned steps were maintained, a happy little squirrel should be within reach. But the rodent does not yet trust the human; the squirrel is by nature a skeptical creature, and he requires careful surveillance.

Gauge the squirrel’s temperment. Research has shown that squirrels may appear to be relaxed, but if they turn their backs to you or fluff their tail, they are not completely prepared to relax. You can help to de-stress your squirrel by being patient and tempting it with the bait. Eventually, the squirrel will become so intoxicated with the nut that he will overcome his fears.

Watch the squirrel and get to know his style. Some squirrels are skittish and jumpy; these tend to be the thinner, smaller ones. On the other end of the spectrum lie the chubby squirrels, who tend to be less inhibited when it comes to approaching humans. Go for the roly-poly ones. They’re friendlier, and fat for a reason.

As this picture illustrates, it’s fairly simple to bring the animal near you. Nuts entice the squirrel, rendering him under your spell. The larger the nut, the closer the squirrel.

Capturing the Creature:
Jackpot! We’ve caught a squirrel!

If the bait is tied securely to the string, then you should be able to play a bit with your catch. Try playing the classic tug-of-war game. Or pull the bait up with your pole, watching as the squirrel toddles about on his hind legs. These variations are always amusing and adorable.

Keep in mind, though, that teasing the squirrel too much will result in frustration for the little tree rat. After a bit, it’s important to give the squirrel what he wants, and that’s the nut. Sometimes they are clever enough to bite the nut off the string, in which case you will just tie a new nut to your equipment. However, this does not always happen, and on occasion you should just toss out a nut to the squirrel. Yes, it’s giving away nuts for free, but it wouldn’t be fair to just taunt the squirrel. In addition, giving the squirrel a nut will help create a special bond of trust between human and animal.

Once you’ve caught the squirrel, you should appreciate the fine features of his appearance. Take time to notice his cute little nose and his plump, furry belly. As he looks at you, his paws curling around the nut, know that this is your reward for your work in squirrel fishing.

The Joy of Squirrel Fishing:

Look, it’s a happy and satisfied squirrel! And it’s all because of squirrel fishing, the sport of the future.

For more information on squirrel fishing, please visit Yasuhiro Endo, who inspired our adventures.

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(REPRINTED FROM ISSUE TWO, SEPTEMBER 6th, 2005)