1 ACMM Newsletter AUSTRALIAN CENTRE FOR MICROSCOPY & MICROANALYSIS IN THIS ISSUE 01 LIVE-CELL IMAGING 03 LIGHT & OPTICAL: NEW ADDITIONS 04 THE XL30 SEM FINDS A NEW HOME 05 SCHOOLS OPEN DAYS 06 THE FRENCH CONNECTION 07 G’DAY FROM JAKARTA AUSTRALIAN CENTRE FOR MICROSCOPY & MICROANALYSIS FEATURED MICROGRAPHS LIVE-CELL IMAGING OF CELL-TO-CELL INTERACTIONS DURING CANCER PROGRESSION BY PAUL SOU & GUY LYONS Paul Sou is a PhD candidate under the supervision of Dr Guy Lyons from the Department of Dermatology in Faculty of Medicine here at the University of Sydney. Paul’s project is investigating cell-to-cell interactions during cancer progression in vitro and is using live-cell imaging here in the ACMM to determine whether or not different cells cooperate with each other to increase overall cell motility. ISSUE 31 JULY-AUGUST 2011 Electospun elastin scaffold with primary human dermal fibroblasts. Bacterial contamination evident as rod shaped cells. Sample by Jess Almine and Anna Waterhouse; image by Steven Moody. (ACMM). Technique: environmental scanning electron microscopy. The horizontal field width is 232µm. This image has been color-ehnanced. Underside of lavender leaf, by Ian J. Kaplin. You can see densely branched lavender leaf trichomes (hairs), and spherical glands which produce aromatic oil. The horizontal field width is 600µm. This image has been color enhanced. Cell motility, the way that cells move, is important in a variety of devel- opmental and biological processes, such as wound healing and embryo development. But cell motility has a dark side too, because the migration of cancerous cells within the body is how cancers spread, or ‘metastasise’, from the primary site to other areas of the body; often with fatal results. By using a cell-culture approach, we are investigating how intercel- lular cooperation of cancer cells affects cell motility. We have transfected HN13 cells, a cell line derived from the explant of a tongue squamous-cell carcinoma, with either Yellow Fluorescent Protein (YFP) as the control, with Snail2 (Cerulean), or Hras (mCherry). Snail2 is a zinc-finger transcriptional factor, specifically a transcriptional repressor of E-cadherin. E-cadherin is responsible for the cell-to-cell junction of epithelial cells, so that when this protein is repressed, normally non-mobile epithelial cells gain mesenchymal properties such as motility. This phenomenon is known as an epithelial–mesenchymal transition. Hras is a small GTPase that conveys extracellular growth signals to intracellular cascades. Activating mutations in Ras are found in 20% of all human cancers.
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ACMM Newsletter
AustrAliAn Centre for MiCrosCopy & MiCroAnAlysis
in tHis issue
01 live-Cell iMAging
03 ligHt & optiCAl: new Additions
04 tHe Xl30 seM finds A new HoMe
05 sCHools open dAys
06 tHe frenCH ConneCtion
07 g’dAy froM JAkArtA
AustrAliAn Centre for MiCrosCopy & MiCroAnAlysis
feAtured MiCrogrApHs
live-Cell iMAging of Cell-to-Cell interACtions during CAnCer progressionBy pAul sou & guy lyons
Paul Sou is a PhD candidate under the supervision of Dr Guy Lyons from the Department of Dermatology in Faculty of Medicine here at the University of Sydney. Paul’s project is investigating cell-to-cell interactions during cancer progression in vitro and is using live-cell imaging here in the ACMM to determine whether or not different cells cooperate with each other to increase overall cell motility.
issue 31 July-August 2011
Electospun elastin scaffold with primary human dermal fibroblasts. Bacterial contamination evident as rod shaped cells. Sample by Jess Almine and Anna Waterhouse; image by Steven Moody. (ACMM). Technique: environmental scanning electron microscopy. The horizontal field width is 232µm. This image has been color-ehnanced.
Underside of lavender leaf, by Ian J. Kaplin. You can see densely branched lavender leaf trichomes (hairs), and spherical glands which produce aromatic oil. The horizontal field width is 600µm. This image has been color enhanced.
Cell motility, the way that cells move, is important in a variety of devel-opmental and biological processes, such as wound healing and embryo development. But cell motility has a dark side too, because the migration of cancerous cells within the body is how cancers spread, or ‘metastasise’, from the primary site to other areas of the body; often with fatal results.
By using a cell-culture approach, we are investigating how intercel-lular cooperation of cancer cells affects cell motility. We have transfected HN13 cells, a cell line derived from the explant of a tongue squamous-cell carcinoma, with
either Yellow Fluorescent Protein (YFP) as the control, with Snail2 (Cerulean), or Hras (mCherry). Snail2 is a zinc-finger transcriptional factor, specifically a transcriptional repressor of E-cadherin. E-cadherin is responsible for the cell-to-cell junction of epithelial cells, so that when this protein is repressed, normally non-mobile epithelial cells gain mesenchymal properties such as motility. This phenomenon is known as an epithelial–mesenchymal transition. Hras is a small GTPase that conveys extracellular growth signals to intracellular cascades. Activating mutations in Ras are found in 20% of all human cancers.
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Figure 1. HN13 cells were transfected with YFP (Control), Snail2 (Cerulean) or Hras (mCherry). Cells were co-cultured: YFP (Ai, ii), Cerulean (Bi, ii), mCherry (Ci, ii), YFP/Cerulean (Aiii, iv), YFP/mCherry (Biii, iv) and Cerulean/mCherry (Ciii, iv) in 24-multi-well glass-bottom plates and cell migration was monitored over time on an Olympus CellR microscope for up to 24 hours. Time points shown are 5 hrs (i, iii) and 15 hrs (ii, iv) after removal of the silicon insert.
The plasmids were made as fusion proteins with the genes bound to different fluorescent tags. This allowed us to track the different population of cells by fluorescence microscopy. The transfected HN13 cells were subsequently co-cultured in two-well silicon inserts placed in 24-multi-well glass-bottom plates. Prior to imaging, the silicon inserts were removed so that the two populations of cells were able to migrate towards each other, and then images were collected for up to 24 hours with the Olympus CellR live-cell microscope. This microscope is equipped with a fully enclosed 37°C stage-top incubator and CO
2 controller,
which allowed us to image the samples over the course of a day. Thanks to the Marzhauzer motorized stage, which can be programmed to return to the same point in multi-well plates, we were able to do multi-point live-cell imaging.
Preliminary results reveal that the control cells had increased collective motility, but the motility of each individual cell was low (Figure 1. Ai, Aii). In contrast, Snail2 cells individually moved faster (Figure 1. Bi, Bii), but the individual and collective motility of Hras cells was low (Figure 1. Ci, Cii). Snail2 cells are known to undergo an epithelial-to-mesenchymal transition and, when cultured with non-Snail2 cells, this property is imparted to adjacent non-Snail2 cells (Figure 1. Aiii, Aiv, Biii, Biv). Thus, Snail2 cells have increased individual motility and are able to
impart this property to adjacent non-Snail2 cells, showing a cooperative effect that results in an increase in motility in non-Snail2 cells.
The advantage of using multi-point positioning on the Olympus CellR microscope was that experiments that would have previously taken months can now be done in one day. This particular experiment required the acquisition of 72 separate positions (X, Y, Z) and resulted in a 20 GB dataset. The largest challenge now is how to handle such large datasets, not to mention the amount of image analysis required.
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The ACMM has an Olympus CellR microscope, which is specially designed for live-cell imaging (see previous story). Current live-cell projects that are making use of this microscope include observing: cancer cell migration in collagen matrices, actin dynamics during treatment with anti-cancer drugs, and viral plaque progression. The key to ensuring successful live-cell imaging experiments is to maintain cell health and viability during transport and equili-bration of conditions prior to imaging on the microscope. The light and optical area has recently acquired two new CO
2 incubators to assist with users’ projects that require
live-cell imaging.
The Galaxy 14 S Micro CO2 incubator is a convenient
bench-top 14-litre incubator ideal for short-term storage of cell cultures that come to the ACMM from external facilities and that require equilibration prior to live-cell imaging. Ideally, cell cultures should be brought into the ACMM for equilibration overnight in this incubator before commencement of imaging the following day, so the Galaxy 14 S is perfect for this.
The second newcomer is an LEC portable incubator, which can maintain a temperature of 37°C for several hours, run-ning on battery alone. It can also be pre-injected with CO
2
to maintain the pH of culture media. The portable incubator is designed to allow users to transport cell cultures across campus to ensure cells remain healthy prior to live-cell imaging. This incubator can be booked via the ACMM booking system.
In addition, a new digital camera has been purchased that can be transferred from an inverted to an upright light microscope. The Leica DFC400 camera has a highly sensitive 1.4 Megapixel CCD sensor, ideal for rapid capture (up to 40 frames per second) of colour images. The Leica (inverted) and Nikon (upright) transmitted light/fluorescent microscopes are located in room 111 and can be booked online via the ACMM booking system.
ligHt & optiCAl: new AdditionsBy MinH HuynH
Live-cell imaging is a powerful technique for observing inter-cellular and intra-cellular interactions over time. Here is an introduction to some new equipment in the ACMM that will help users in this area.
A number of our users will have noticed the large space in lab 121, formerly occupied by one of our much-loved workhorse SEMs, the Philips XL30 CP.
In June, the XL30 made the short move across to the Redfern Technology Park on the other side of Redfern Sta-tion, and more specifically to the laboratories of Bandwidth Foundry Pty Ltd. Bandwidth Foundry is a company special-ising in the development of photonics technologies, working very closely with the Department of Physics and a number of other research groups in the University of Sydney, includ-ing the ACMM. In the future we aim to equip the XL30 with an experimental electron beam lithography (EBL) system, but most importantly the SEM is still owned and maintained by the ACMM, and is still available for use by any of our users, given the appropriate training, of course.
Naturally, many users might have questions concerning the procedure for accessing the XL30, so here we have tried to answer some common issues that might arise:
wHere is tHe Xl30 loCAted? Bandwidth Foundry is located on the ground floor of the Biomedical building on Central Avenue, in the Redfern Technology Park. This is approximately a 15–20 minute walk from the Madsen Build-ing. A detailed map can be found on the Bandwidth Foundry website (bwfoundry.com)
How CAn i Book tHe Xl30? The XL30 remains on our booking list, but is now called “XL30 at Redfern Technology Park”.
i wAnt to get trAined to use tHe Xl30 – sHould i ContACt BAndwidtH foundry direCtly? No; please contact the SEM staff at the ACMM and we shall organise your training as necessary.
i AM AlreAdy A CAtegory 2 user on tHe Xl30 And i would like to use tHe seM onCe AgAin. sHould i Just turn up At BAndwidtH foundry?
No – for the first time, it is best that you liaise with one of the SEM staff at the ACMM, and we can show you how to access the laboratory and introduce you to the relevant Bandwidth Foundry staff. On subsequent visits you can book and use the XL30 independently.
i AM A CAtegory 3 user on tHe Xl30 – CAn i use tHe seM in tHe evenings And At weekends? No – the laboratory is only accessible during business hours.
Are tHere Any sAMple prepArAtion fACilities witH tHe instruMent? Only limited facilities are available, such as carbon tape, tweezers etc. Full sample preparation and mounting should ideally be carried out beforehand at the ACMM or in your own laboratory.
Are tHere Any restriCtions ConCerning tHe use of tHe Xl30? Yes – the SEM is housed in a clean room. This means that you will have to wear appropriate protective clothing (overall, mask, shoes etc.) to access the instrument; these are all supplied at the lab. Details regard-ing the clean room rules will be explained to you at the first session, but this should not limit the samples that you may want to image or analyse.
We hope that many of you will continue to use the XL30 as before, and we especially encourage those of you that are located at or close to the Redfern Technology Park to view this as a real opportunity, with a great, all-round SEM on your doorstep.
Any questions concerning the XL30 should be sent to Pat Trimby ([email protected]).
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Schools Open Days are an annual event in the ACMM, hav-ing been occurring since 1985 in some form. Throughout their history, the open days have always sought to get school children excited about science by exposing them to the hidden microscopic world around them.
This year’s open days were no exception. The program followed the time-honoured format in which students, and their frequently gobsmacked teachers, spend two-hour blocks in the centre, getting hands-on with light microscopes and electron microscopes. The samples that the students examine in the microscopes have been chosen to reinforce the lessons that the students, mostly from Year 11 biology classes, are learning in their curriculum. Other activities during the visits give the teenagers an understanding of the differences between different types of microscopes, as well as a brief introduction to the history of microscopy and some of the major scientific advances that microscopes have enabled in the past. Some of the schools also take advantage of being on campus to bookend their time in the ACMM with visits to one or more of the University’s museums – apparently, the Pathology Museum is particularly popular (and ‘gross’).
This year’s 255 students came from 10 high schools across the Sydney region, including Strathfield Girls HS, Sydney Boys HS and Al Faisal College. Some of the schools are repeat customers each year, but several new schools took part for the first time courtesy of the University of Sydney’s Identified Schools Program of LSES schools. This program paid the modest fees for Schools Opens Days, allowing the participation of students from these lower socioeconomic areas, consistent with the University’s goals in social inclu-sion and in attracting and supporting promising students from diverse social and cultural backgrounds.
The regular positive feedback from teachers after the open days gives a sense of how beneficial these two days are for the students. For instance:
“We all had a great day and it was wonderful to see and use the electron microscopes etc. All staff were so approach-able and the two hours made the students think about future careers in science, which was great. Loved the cow bell at the end of each session. So, once again it was a fantastic day. Job well done!!”
“The boys had a great time last week. Thank you for the opportunity to attend.”
After the successful introduction of new schools this year, we are hoping to make other changes in Schools Open Days next year to further the University’s social-inclusion goals and to support a more research-centred approach to science for the students who take part.
sCHools open dAysBy Jenny wHiting
June 22 and 23 heralded one of the major outreach activities of the ACMM year: Schools Open Days. Here we enjoy the Cook’s tour of this year’s event.
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tHe frenCH ConneCtion
Besides having international users and research collaborators, the ACMM also plays host to researchers from around the world each year. Some of the regular visitors have come from universities in France, where international internships are a necessary part of the courses. Here two of our current French interns give their perspectives on working at the University of Sydney and the ACMM.
niColAs MArtin
“I’m a French student studying general engineering at the Ei.CESI Engineering School in Paris. To complete my mas-ter’s degree, I have to do an internship in another country; so now here I am in Sydney, working on the twinning deformation of pure titanium. My project is supervised by Dr Gwénaëlle Proust, from the School of Civil Engineering and from the ACMM, who I would like to thank for giving me this opportunity. My work on the project is to write a code that will extract quantitative data regarding twinning deformation from electron backscattered diffraction (EBSD) scans, so that we can determine precisely the role of twin-ning in the deformation of titanium. I have also had training in using EBSD in a scanning electron microscope, in order to understand how to prepare samples for analysis and how to use the instrument and the software.
“I chose to come to Sydney and the ACMM because I would like to discover a new part of the world, and also because I have not picked an engineering specialty yet. By doing my internship in civil engineering at the University of Sydney, I am taking the first step in what I want to do. Moreover, the facilities at the ACMM are amazing. I could never have expected to work on these kinds of instruments at my uni-versity. Plus the people are very friendly and helpful when something is not working for you. It’s the same impression that I have for the University of Sydney and for Australia in general. It has always been a dream to study in a university so great and now it has happened.”
Joffrey dréMont
“I am twenty-two-year-old engineering student from Toulouse, a large city in the south of France. Having obtained A-levels in maths, science and biology, I did a two-year technical degree in civil engineering. I am now completing a sandwich course in the
CESI-Angouleme Engineering School. As my course requires that I undertake a three-month international internship, I’ve come to the ACMM, where I am working with Dr Baptiste Gault on a study of light alloys. In particular, we are examin-ing the partitioning of magnesium in lithium-rich precipitates in aluminium alloys. I began by learning to make atom probe tips of the alloys, which I have recently run, and now I have my first results from which to make a 3D reconstruction of the alloy nanostructure. In a few days, I will start working with PhD student Vicente Aruallo-Peters on his different alloys to characterise the early stages of precipitation of the T1 phase in an 2198 AlCuLi-based alloy. This alloy has been developed mainly for aeronautical applications and was heat treated at 155°C for periods of less than two hours.
“I chose the ACMM for two reasons: first of all, I wanted to go to an English-speaking country so that I can learn to speak English really well and, secondly, I wanted to work in a laboratory to explore the world of research. At the ACMM, I have addressed these two reasons and discovered a lot more. The centre is a place where the people are kind, helpful and smiling, and where it is possible to talk about everything and anything with anyone. For me now, English is still difficult to understand – people talk very fast – but I am making progress! The University of Sydney is a very good place, it’s very big and the buildings are beautiful.”
Above L-R: Interns Gregoire Cleren, Laure Aschehoug, Nicolas Martin.
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g’dAy froM JAkArtA
Former ACMM researcher Dr. Leigh Stephenson has travelled far north to Indonesia as an Australian Youth Ambassador for Development (AYAD), part of the government’s Australian Volunteers for International Development program. He left on the 22nd July 2011 and will be working in a science museum for a year. Here he corresponds directly from the tropics.
Hello everyone. Apa kabar? So let me fill you in a little …
For six years starting in June 2005, I studied and work in the fantastic environment afforded by the University of Sydney and the friendly, cosmopolitan staff in the yellow sandstone Madsen Building. I was young and innocent back in the day, when a PhD student was a rare sight and the group of us barely filled one office. What fun was had back then! And there were fewer microscopes as well. And they ran on steam.
Seriously now, I valued my time as a PhD student in what was then the ‘EMU’. For five years, under the supervision of Prof. Simon Ringer and Dr Michael Moody, I helped improved atom-probe data analysis. After that, I worked on an ARC-funded research project on characterisation of third-generation solar cells that were made by our col-laborators at the ARC Photovoltaics Centre of Excellence. I am very pleased that Dr Waikong Yeoh is taking over my ACMM role in this research.
Recently, I decided to embark to Indonesia where I have a twelve-month AYAD assignment as a Science Park Market Research Officer at Pusat Peragaan Iptek (PP-IPTEK) in Jakarta. Now I’m living on the edge of that huge city, a city that ranks amongst the largest in the world. Unofficially, there are about twenty-million folk here, squeezing into an
area smaller than Sydney. I still cannot imagine how, although the sprawl continues far beyond the lines on the map.
The work is a different tack to my usual bread-and-butter. At the science museum, I will help establish monitoring and evaluation methods for the various educational programs, galleries and scientific exhibits. A large part of my year will also involve helping my new colleagues to build the capacity of their organisation and to develop their knowledge and skills to enable them to continue to implement these methods long after I have returned to Australia.
People here are lovely if you go out of your way to try and converse. I’m having a great time learning the language, but also I’m busy firing water rockets, solving mathematical puzzles, growing a beard, planning my holidays to exotic locales, roller-blading through deserted exhibition halls and making new friends. Still – I miss home. A little. To work at the ACMM was a privilege, and I’ve been lucky to know you all.
Alright, I must sleep now. The mosquitoes will eat me alive unless I retreat underneath my net.
Salam,Leigh
PS The weather is quite warm here … you should come visit!
Research Methodology July 28 – October 13, 2hrs per week, Thursdays 2–4 pm
* Two-day lecture course in the principles of light and laser microscopy, covering specimen preparation, fluorescence microscopy and confocal microscopy. Please email Ellie Kable ([email protected]) to obtain the registration form for this special short-course.
Here’s just a quick reminder about ACMM training courses that are running this semester. There are still places available in most courses, so get in fast.
To register, just follow the link from the short-courses website if you are an existing user of the facility. If you’re new to the centre, you will need to register as a user on the ‘Accessing the ACMM’ webpage before you can register to attend a course. Remember that, if you miss out on a course you want to attend this year, the next courses will be run in Semester One of 2012. The latest information about each course and the costs for attending can be found on our website: sydney.edu.au/acmm/courses_training/short_courses/index.shtml
