Coherent X-ray Science Growing Tall PoppiesStudents took part in the Growing Tall Poppies program over a week and investigated the topic of Mathematics and Physics meeting Popular Culture. This included the study of Lasers, X-rays, Diffraction Patterns and even puzzles like sudokus and Latin Square problems. The students were required to work together as a team and produce a booklet and then prepare for a presentation that was to be held on the final day of their experience.

Index-Page 2 ::: What is Growing Tall Poppies?Page 2 ::: What is ARC and CXS?Page 2 ::: Weekly BriefPage 3 ::: Light Diffraction ImagingPage 4 ::: Amplitude and PhasePage 5 ::: IterationPage 6 ::: Use of Coherent X-ray SciencePage 8 ::: The Australian SynchrotronPage 12 ::: Acknowledgments

2010

Ivan Galic, Xavier de Bruyn, William Fleming, Adrian Marcato, Jordan Lo Presti, Xavier Butcher

St. Bernard's College11-15th October 2010

What is Growing Tall Poppies?

Growing Tall Poppies is a program that provides opportunities for students to participate in an authentic science experience. The aim of Growing Tall Poppies is to demonstrate the importance and relevance of the physical sciences to individuals, communities and societies. Growing Tall Poppies gives students insight to roles of scientists in today’s world, and inspires them to see that a career in the physical sciences contributes to solutions to the issues facing our world such as understanding diseases, producing designer drugs, modelling climate change and even finding alternative energy sources. The projects are designed specifically to involve students with the work of the ARC Centre of Excellence, and guide students in the science fields of biology, chemistry and physics, and follow both the VELS and IB standards. Groups of six students participate in a Growing Tall Poppies program at a university, and each program is accepted as work experience and is five days long. Programs are preceded with a project/team management workshop. The project briefs are developed and supported by Eroia Barone-Nugent from Santa Maria in collaboration with CXS. Growing Tall Poppies is implemented with the help of Akorn Educational Services.

What are ARC and CXS?

The Australian Research Council [ARC] Centre of Excellence for Coherent X-Ray Science [CXS] is a multi-institutional organization consists of leading Australian Researchers specialised in the field of X-ray physics. The organisation was first established in 2006, and is dedicated to the research and exploration of scientific problems. The technology used consists of an extremely fine and narrow X-ray targeted at a crystallised protein or other sample. The diffracted X-ray light is recorded and mathematical methods are used to reconstruct its molecular structure. The brightness and intensity of these sources gives the chance to have a description of biomolecules, and essentially solve many problems in today’s society. CXS is also looking into the primary issues in the use of these light sources, including interaction between intense and coherent X-rays and electronics. They strive to extend boundaries in biotechnology, which includes the non-crystallographic structural determination of membrane proteins. These proteins cannot form crystals that are suitable for analysis and therefore have unknown structures. There are less than 20 known membrane structures, and even the slightest discovery in this area of science would revolutionize rational drug design through the insight gained into the function of membrane proteins and be a very large advantage towards the pharmaceutical industry and cures for diseases. If the exact molecular structure is known, the function of the molecule can be identified and other molecules can be used to block or prevent further infection of cells. CXS also explores the application of short wavelength high-harmonic generation sources and free electron X-ray lasers that are under-developed worldwide.

Weekly brief

Following an early start to the day, Ivan Galic, Adrian Marcato, Xavier de Bruyn, Jordan Lo Presti, William Fleming and Xavier Butcher from St. Bernard's College each made their own way via trains, trams and other forms of public transport to the University of Melbourne for a 9am start. The group was introduced to each member of the CXS representatives and also their weekly mentors. They were then taken to two labs in which demonstrations of laser diffraction patterns were shown. On Tuesday, sudokus and Latin Square problems were focused on. Students solved several sudokus, and considered the iteration method. An excursion to the Australian Synchrotron took place later on in the day. The excursion consisted of a tour guide showing the students models of particular parts of the synchrotron and their functions. The two days before the presentation were used to write up and complete their booklet, presentation and cue cards. Friday, the last day of the program, was used to revise over cue cards and prepare for the final presentation planned for a 3pm start.

2

Light Diffraction Imaging

To understand the process of light diffraction imaging it is important to first understand what diffraction is. Diffraction occurs when a light wave passes through an aperture which has a diameter smaller than the light source, in this case the laser beam, but larger than the amplitude of the light waves. When the light passes through these apertures’ sharp edges, a new wave is then formed from the section that passes through the gap. It is easier to comprehend this process if light is though of as a ray. When the light is diffracted off the points where it hits the objects edges, a new set of waves is emitted spreading out in all directions from the aperture. This brings up the point of interference.

Diffraction is not limited to producing just one new set of waves once it has passed through an aperture. If the distance between the edges of the aperture is large enough, the light waves will diffract from both edges and not just one which means that there are two separate waves being emitted in all directions. Interference is anywhere where two or more of the waves overlap. This can cause one of two possible outcomes being either Constructive or Destructive interference. Destructive interference occurs when the overlapping waves collide out of phase and therefore cancel each others amplitude. This creates dark areas where the light is being diffracted because the amplitude is brought to zero hence no light is produced. Constructive interference occurs when the overlapping waves are in phase or coherent with each

other meaning the amplitudes combine to form a larger wave - It is important to note that this does not alter the wavelength. The result of constructive interference are the bright bands of light.

For a light to be fully coherent, we must know the relationship between the light’s constructive intensity and the phase everywhere in the beam. But this can be difficult to assess because although intensity is relatively easy to determine, the phase can be very difficult to measure, especially for X-Rays. An important thing to realise when trying to extract information from the phase is that it will change as it diffracts through an object. This means that the end data can be traced back in order to determine the object it has passed through and information can then be retrieved. The problem with this in terms of Coherent X-Ray Imaging is that X-Rays waves change only a miniscule amount when passing through an object. This is because X-Rays refractive index is extremely close to one, meaning it can pass through the small spaces in between atoms. This means that there is hardly any change in the phase through most objects besides very dense substances such as lead and steel which gives very little information to backtrace in order to determine which object the waves have passed through.

However, there is an important reason as to why they must be used. In order to view an extremely small object such as a single molecule, an extremely small wave length is required, which is where X-Rays are important. Because they have a wave length of approximately 0.5nm, they are able to diffract through the sample although not as normal light waves would. X-Rays do not diffract through an aperture like other larger light sources but rather bounce off the electrons inside the molecule sample which means that information can then be traced based on where the X-Rays were redirected to after colliding with the electrons contained in the molecule. But this still leaves the problem of not gaining any information on the phase. This can be overcome however by using parallel examples. By using the amplitude information that is gathered from the electron behaviour, a basic idea of the image can be determined, but because of the little to no information located on the wider parts of the image, detail cannot be found from the X-Ray diffraction alone. Phase

3

information from very similar objects found previously must be used. By using the phase information from these objects, details can be filled in on the image hence adding definition to the image and producing a relatively accurate phase through past knowledge.

This same process continues for objects that are smaller again in the way that smaller waves are required to view smaller objects such as Gamma Rays are used to view single atoms. The issue with using waves any smaller than X-Rays is that less and less information is able to be retrieved from the phase and less and less previous knowledge is known in order to create a similar phase. The smaller waves also add an element of danger with each use because there even lower refractive index, they have the ability to pass through extremely small gaps, such as the ones between nuclei - These can be cancerous as these waves can pass through cell walls and cause mutation to the cell itself.

Amplitude and Phase

The Amplitude of a light wave can also be described or identified as the height or size of the wave. We categorise light waves by their amplitude, which can also be interpreted as the intensity or the brightness of the wave. The intensity of a light wave is found by the equation I = A². The wavelength of a wave determines the type of light wave. The amplitude of light waves is directly influenced by the phase of the lights waves, as whether they are in-phase or not, determines the existence of constructive or destructive interference, which then changes the amplitude.

The type of light wave can vary depending on the wavelength or size of the light wave. Radio waves are the longest type of light wave, followed by microwaves and infrared light waves, all of which are visible to humans. Ultraviolet light is not visible to the human eye, measuring between 10nm to 400nm. Smaller than these waves are X-ray waves which can get as small as 0.01 nm to 10nm, and the smallest light waves are the gamma rays, which can be as minuscule as 10 picometres. The smaller the light waves get, the more dangerous and harmful they tend to become. Certain radioactive materials can omit gamma rays, and scientists using these types of rays for research need to be very careful. The length of the wave can also determine the colour of the light source, for example, a wave of length 600nm will be of a red colour, whereas a wave of length 300nm may be of a blue colour.

The phase of a light wave refers to the coherent nature of the wave, and whether the light waves are in sync with each other. The phase can also be interpreted as how far along the wave is in its cycle, and we can figure out if the light waves are coherent. This information can be gathered through coherent diffraction imaging. When light travels through a narrow space, also known as a slit, it bends through and diffracts where it can be captured on a detector. As the light travels through the slit, where can determine their coherence if we know their phase, or how far along the waves are in their cycle. If the light waves are in-phase, then they can be labelled as coherent, which can lead to constructive interference. This is when the wave lengths form a combined wavelength, and become more intense, forming light spots on a detector. The equation for the resulting amplitude can be written as A¹ + A² = A.

When the light waves are not in-phase, they are considered to be incoherent, and can therefore result in some destructive interference. This is when the light waves are not coherent, so they

4

cancel each other out, resulting in dark spots on the detector. The he waves are incoherent, so the combined amplitude is nullified, or results in zero. The equation for the amplitude of incoherent light waves can be written as A¹ - A² = A. The phase of light waves can change when they diffract through an object, making phase harder to ascertain.

The Fourier transform is another important part of Coherent diffraction imaging that helps to determine the phase of light waves. The Fourier transform consists of many complex equations and algorithms which by using the combined amplitude and diffraction pattern to determine the separate amplitudes of light waves and hence find if they are in-phase or not. The Fourier transform focuses on sine waves and cosine waves and through these complex equations it calculates the phase, known as phase retrieval, and with all of this extra information, it becomes easier to determine the image that the light rays passed through.

During the week we went to the Coherent Diffraction Imaging Laboratory to get a first hand look at coherent diffraction imaging. To achieve a perfect image the light source must be infinitely far away from the sample, which of course is impossible, which is why we can only make it as far away as desirable. The process involves a light wave, which in our case was slightly smaller than infra-red, being bounced off a series of mirrors. This process relates to the idea of having the CCD camera infinitely far away. This doesn't really make sense, but essentially means that the further away the CCD camera is from the light source, the wider the diffraction image is on the CCD camera. This allows us to retrieve more information from the phase, thus giving us a clearer and sharper final image of the sample. After being bounced off the mirrors, the light travels through a slit and then through the sample, before being photographed by the camera.

These photographs are then used by a computer and put through the iteration process. This process involves taking the raw image and iterates the rows and columns of pixels, alternates between reducing errors from both the rows and columns until it has the refined 2D image. This process can vary in length depending on the size of the sample and the amount of pixels it has.

Iteration

Iteration is the act of repeating a process, most commonly with the aim of approaching a desired goal, target or result. Each repetition of the process is also called an 'iteration'. The results from one iteration are used as the starting point for the next iteration.

The process first starts in the BeamLine Simulation Lab where a light is diffracted through an image. The diffracted light is reflected off many mirrors and ends at the CCD camera where we can capture the diffracted light. The pictures are then saved to the computer so that they can be put through the program and be iterated. The pictures are taken in 2048 x 2048 pixels so that when the picture is iterated it is much more clearer and you can read the image much better.

The pictures of the diffracted light taken by the CCD camera can be iterated to retrieve the image that the light diffracted through. In order to iterate a problem you need two sets. Set A and Set B. Set A is the pictures of the diffracted light that are taken by the CCD camera. Set B is the support that the image needs to fit into; you can set the outside of the object to zero so that you know that the object is less than that size. The computer then iterates the two sets. The computer fixes the minimum amount of errors in Set A and then it switches to Set B and

5

fixes the minimum amount of errors there. The results from Set A are used as the starting point for the iteration of Set B. It then switches back to Set A. This process repeats; each time reducing the error until eventually there is a clear picture of the image that the light was diffracted through.

The program solves the errors by matching numbers to colours. This means that each shade is matched to its own number so as the numbers are solved the shades match each other to make the image that the light was diffracted through.

The process sometimes works backwards to get the result; this is because when you are fixing the errors in a Set you may create other errors.

Within the process there are constraints. Constraints are fixed into position. If you reach a stagnation point then you keep switching between two different errors. If this happen you need to fix more than just the minimum amount of errors to get the solution. The constraints are there so that you only get one solution

This same process can also be applied to the popular puzzle Sudoku. The constraints are the numbers that you are given and these cannot be changed. Using these you can split the 9 x 9 grid into 9 separate 3 x 3 grids. From here you know each number can only appear once in each 3 x 3 grid and the same number can't appear twice in the same row or column, now you can solve each 3 x 3 grid by solving the rows and then the columns and you keep switching between the two, each time making the minimum amount of fixes required. If you reach a stagnation point you need to make more than the minimum amount of fixes so that you aren’t switching between two errors. When you put it back into a 9 x 9 grid, using the process of iteration, you can fix the columns, switch to the rows and fix the errors in them, by repeating this you eventually find the solution.

Biological uses of Coherent X-ray Science

History

The developments and use of Coherent X-ray Science is being applied to biology by scientists all around the world in an effort to r e v o l u t i o n i z e m e d i c i n e a n d o u r understanding of the functioning of molecules and viruses. The first major breakthrough using coherent x-ray science was in 1989 by scientists at the Victorian college of Pharmacy, Monash university. The group of researchers discovered a molecule that targets a protein called neuraminidase that is located on the surface of all viruses. The molecule which is called Zanamivir inhibits the transmission of this protein into another host, therefore blocking it's ability to reproduce and cause damage. This molecules specifically works for the influenza A and B viruses. The influenza virus or more commonly known as the flu, targets the body's respiratory cells which leads to damage due to swelling and inflammation. Symptoms of infection by this strain of influenza include sneezing, coughing and congestion and can lead to further complications such as pneumonia and in the worst of cases; death.

This discovery was the first of it's kind and can greatly assist the immune system in fighting off the influenza virus. The only problem with the medicine is that it has to be injected or exhaled into the body. As well as this, virus strains constantly mutate, which could deem this medicine ineffective in the future. This could create serious problems and complications for people that have been infected with the virus and other drugs would have to be created.

6

The Zanamivir molecule is also known as Relenza. The way that the Relenza works can be compared to the same process as an enzyme and its substrate. The molecule covers the transmission point for the virus which blocks the transferral of nutrients and substances in and out of the host. This eventually kills the host and eventually with it the virus. Whilst experimenting, the group of researchers from Monash university, used the diffraction pattern of the virus, to determine how large the the exchange point is and where it is located on the virus. After many attempts, they eventually found the molecule which stopped the protein exchange from occurring.

New developments and endless opportunity

Coherent X-ray Science can lead to many discoveries and developments which can change the way that we study and think about physical biology and science. Using the diffraction patterns that are obtained from observing viruses and other molecules under the laser light, we learn more about the structure of these things, which in turn can give us more knowledge about how they function.

This knowledge can create opportunities for scientists to discover cures for other diseases or v iruses, which can advance the world of immunisation and health. This could save many lives and may also open a larger field of science for people to study in and for research. With this process we will gain more knowledge about the functioning of organisms and about our surroundings.

Protein Crystallization

In order for proteins to be viewed and observed in detail, they must first be crystallised. This process can be very complicated, time consuming and has a very low rate of success. It requires so much time because the protein must first be separated from the cell and then purified, which can take several months to be completed. The time it takes to complete is influenced by the pH of the water where it is being purified, and the amount of dissolved substances within the water. After the purification process, the protein is ready to be crystallised. A crystallised protein will create a 3D shape and can be observed using x-ray crystallography. After a diffraction pattern is obtained, the information is iterated to produce a high resolution, clear 3D image of the protein.

Learning in detail / Structural biology

Biological imaging is a very important and vital part of biology. This is because biology is a science that is based on the observation of physical and chemical interactions within an organism.

The imaging helps to show specific details within molecules and cells. Biologists can then use this information to discover how substances cross the phospholipid bi-layer to exit or enter the cell. This will change what we know about the membrane and the exchange of nutrients, and the importance of this function to the cell or organism. However,

7

using the light to see the membrane proteins is time consuming and has only been successfully completed for very few of the total number of known membranes.

Biological imaging can provide great detail about the shape of the cell and the make-up of the membrane. Scientists can use this information to determine what nutrients and substances can cross these membranes and to identify what form the disease or virus is in, in order to discover what molecule can be used as a counterattack to the virus. This will stop the virus from spreading or infecting other cells. This will make medicine more effective and can lead to rapid developments in the technologies used to fight viruses. However, finding a molecule that is perfect shape can be a long and arduous task but it is a small sacrifice for finding a prevention or cure for diseases which can be life-threatening.

The information obtained from the scattered light image can also be used to identify substances which can cross over the cell membrane of bacteria or viruses, to kill the infected cells or to place a disease fighting substance directly into the bloodstream, and therefore diffusing into the cell's cytoplasm.

Current applications and studies using Coherent X-ray Imaging

The use of Coherent X-ray imaging is currently being used in an effort to discover the structures of diseases. Biologists then observe the information that these images provide, in aim to find a cure or prevention for several dangerous and severe diseases and viruses. These include malaria, HIV (aids), avian flu, swine flu, the common cold (rhinovirus) and a cure for the types of cancers.

There has been little success in the studies. The drug called tamiflu was developed in 2009, as a vaccine for the swine flu, which was a quickly spreading global pandemic in that year. The drug tamiflu works very similarly to Relenza as it is a neuraminidase inhibitor that binds to the active site of the virus or host cell, but is taken orally by tablet.

The antiviral drug zanamivir (Relenza) is shown in green. The yellow shows the different regions when compared to the Spanish flu and the avian flu. After the first two weeks of the initial outbreak of the disease, the virus mutated. These regions are shown in red.

Synchrotron

The Australian Synchrotron can be found in Clayton, Victoria. In simple terms, a synchrotron is a very large, circular, mega voltage machine about the size of a football field. From outside, the Australian Synchrotron, for example, looks very much like a roofed football stadium. But on the inside, it’s very different. Instead of grass and seating, there is a vast, circular network of interconnecting tunnels and high-tech apparatus.

8

How does it work?

Firstly, electrons are produced at the electron gun by heating a cathode ray, which gives the electrons energy. This causes them to jump off the cathode and into an electronic field, which pushes them into the linear accelerator.

Secondly, the linear accelerator (or linac) accelerates the electron beam over a distance of about 10 metres. After the first metre of acceleration in the linac, the electrons are already travelling at more than 99.9987% of the speed of light. They accelerate it by passing them through a series of charged tubes. The first tube being positive, which means that the electrons are attracted to it. As soon as they are in there, they swap the charge of the tube to negative, forcing the electrons to move away from the tube and onto the next one, which is positively charged. They do this for a while until they have almost reached the speed of light.Thirdly, the electrons are moved into the booster, which is an electron synchrotron 130 metres in circumference that accelerates the electron even further. The booster ring contains 60 steering and focussing electromagnets to keep the electrons inside the stainless steel vacuum chamber. The beam is accelerated by a simultaneous ramping of magnet strength and cavity fields. An electron spends about half a second in the booster ring and completes over one million laps.

Next, the electrons move into the storage ring, and is the final destination for the accelerated electrons. The storage ring is 216 metres in circumference and consists of 14 nearly identical sectors. Each sector consists of a 4.4 metre straight section and an 11 metre arc. Every arc contains two dipole ‘bending’ magnets where synchrotron light will be produced. Electrons that are accelerated that go around corners give off light, which can be harnessed to be uses in coherent x-ray imaging. Individual beam lines are positioned to capture the synchrotron light given off by the electrons in the storage ring. These range from infra-red to hard x-ray.Lastly, experiments employing synchrotron light are conducted in customised facilities called end-stations. Most of the end-stations are housed inside radiation shielding enclosures called ‘hutches’ to protect staff and visitors from potentially harmful x-rays. Each beam line utilises the synchrotron light to gather data in the form of images, chemical spectra, and/or scattered light. Because research scientists cannot enter the hutches during data collection, much of the equipment is controlled remotely via motors and robotic devices.

9

Parts and functionsElectron Gun:Electrons are produced at the electron gun by thermionic emission from a heated tungsten matrix cathode. Applying a 500 MHz voltage signal to the gun as it fires means the electrons are generated in bunches two nanoseconds apart. The emitted electrons are then accelerated to an energy of 90 keV (kilo electron volts) by a 90 kilovolt potential difference applied across the gun, and move into the linear accelerator.Linear Accelerator:The linear accelerator (or linac) accelerates the electron beam to an energy of 100 MeV (mega electron volts) over a distance of about 10 metres. This involves a series of RF (radio frequency) cavities operating at a frequency of 3 GHz. Due to the nature of the acceleration, the beam must be separated into discrete packets, or ‘bunches’, with a spacing consistent with the 3GHz acceleration frequency of the linac. This is done at the start of the linac, using several ‘bunching’ cavities. The linac can accelerate a beam once every second. After the first metre of acceleration in the linac, the electrons are already travelling at more than 99.99% of the speed of light.Booster:The booster is an electron synchrotron 130 metres in circumference that takes the 100 MeV beam from the linac and increases its energy to 3 GeV (giga electron volts). The booster ring contains 60 combined function (steering and focussing) electromagnets to keep the electrons inside the stainless steel vacuum chamber and a single 5-cell RF cavity (operating at 500 MHz) to supply energy for acceleration. The beam is accelerated by a simultaneous ramping of magnet strength and cavity fields. Each ramping cycle takes approximately one second for a complete ramp up and down. An electron spends about half a second in the booster ring and completes over one million laps.Storage Ring:The storage ring is the final destination for the accelerated electrons. It can hold 200 mA of stored current with a beam lifetime of over 20 hours. The storage ring is 216 metres in circumference and consists of 14 nearly identical sectors. Each sector consists of a 4.4 metre straight section and an 11 metre arc. Every arc contains two dipole ‘bending’ magnets where synchrotron light will be produced. Most of the straight sections have room for an ‘insertion device’. The storage ring also contains a large number of quadrupole and sextupole magnets used for beam focusing, chromaticity corrections and orbit corrections.Beamlines:Individual beam lines are positioned to capture the synchrotron light given off by the storage ring. The first section of every beam line is the photon delivery system (also called the ‘beam line optics’). It incorporates filters, monochromators, mirrors, attenuators and other devices to focus and select appropriate wavelengths for particular research techniques.

10

End-stations:Experiments employing synchrotron light are conducted in customised facilities called end-stations. Most of the end-stations are housed inside radiation shielding enclosures called ‘hutches’ to protect staff and visitors from potentially harmful x-rays. Each beam line utilises the synchrotron light to gather data in the form of images, chemical spectra, and/or scattered light. Because research scientists cannot enter the hutches during data collection, much of the equipment is controlled remotely via motors and robotic devices.

HistoryThe Australian Synchrotron is very unique. There are 3 other major ones like it in the world in the US, Switzerland and Germany. Construction of the Synchrotron started in 2003 after approval from state, local, and federal governments, and funding was organised. The Australian Synchrotron was opened in 2007. It cost around $270million dollars. Funding came from the federal government, state government, universities from around Australia and some individual groups. Both the State and Federal governments, and the Federal government of New Zealand are now contributing millions of dollars to help in operating costs. UsesSynchrotron light enables scientists to get pictures of molecules without damaging it (through staining/dissecting etc.), and gives a superior accuracy, clarity, specificity and timeliness to those older methods. This is particularly important if there are very few samples around. It is hoped that this technology will lead to vaccines for patients with viruses like Malaria, Hepatitis, HIV and Smallpox. The vaccine for the Influenza virus (Relenza) was found using this method of imaging. The general rule is that only bacterial infections can be treated at this stage in time.

11

Acknowledgements:

Growing Tall Poppies program

St. Bernard’s College

CXS

University of Melbourne

Australian Synchrotron

Akorn Education Services

Special Thanks to:

Keith Nugent

Eroia Barone-Nugent

David Rosel

Georgene Bridgeman

Bec Ryan

Evan Curwood

Tania Smith

Harry Quiney

Louie Pittas

Mark Reedy

Rita Krouskos

Thank you

On behalf of St. Bernard’s College, we would like to personally thank all of the above for offering us this wonderful experience, which has opened our eyes to a new expansion of career pathways and shown us the scientific world in a new, exciting light. We wish you the best of luck with your groundbreaking research and all the best in the future.

12