1

ACMM Newsletter

AustrAliAn Centre for MiCrosCopy & MiCroAnAlysis

AustrAliAn Centre for MiCrosCopy & MiCroAnAlysis

feAtured MiCrogrAph

in this issue

01 fArewell to prof. CoCkAyne

04 AdditionAl seM MiCrosCopes hitAChi s-4500 feg seM

Zeiss AurigA fiB-seM

05 new Multiphoton MiCrosCope leiCA sp5 ii

06 ACMM introduCtory Courses

07 AlexAndre lA fontAine in south AMeriCA

08 plAnt sCienCe BlossoMs

09 dr MAnAsA gudheti Visiting reseArCh fellow

10 MiCrosCopy And MACrosCopy in the usA

fArewell to prof. dAVid J. h. CoCkAyneBy AllAn Jones

Earlier this year, we were all saddened to hear of the passing of the iconic former director the Electron Microscope Unit (EMU), which was renamed the ACMM in 2010.

issue 29 MArCh–April 2011

Confocal micrograph of ondontoblasts; green shows nestin, an intermediate-filament protein, and blue shows nuclei. The scale bar is 20 µm. Image by Ramin M. Z. Farahani from the Institute of Dental Research, Westmead Millennium Institute.

For those of us who worked and studied with David and remember him well, there are not enough words to express our respect for one of the giants of microscopy or our sadness that he is no longer here to guide and nurture our learning in the fields of knowledge that he was so passionate about all his academic life. But life, as they

say, moves on and there are new generations of young and energetic microscopists who now must find their way to discoveries in microscopy without David’s great per-sonal insight and guidance. It is thus timely that, in this issue of the ACMM Newslet-ter, we pause to reflect on David’s contributions to microscopy in general, but more particularly on his for-mative role in creating what is now arguably recognised as one of world’s great microscopy centres—the

ACMM or Electron Microscope Unit (EMU) as it was known in David’s time—here at the University of Sydney.

It certainly is hard to imagine the EMU here at Sydney without Da-vid’s strong leadership, which drove it forward to achieve a world-class

2

reputation over the 26 years that he was its director. David Cockayne became director of the EMU in 1974, after the first director, Dr Drummond, retired. In the many years that followed, he led the EMU – which had been established in 1958 – from its adolescence, through a variety of suc-cesses and through occasional trials and tribulations as the face of the higher-education sector changed radically, until the unit attained a confident maturity as a respected and important part of the University of Sydney.

David was born in London, England, and lived there until aged eight when his family migrated to Australia. The middle of three children born to John Cockayne and his wife, Ivy (nee Hatton), David soon found himself adjusting to the new climate and lifestyle in seaside Ocean Grove and then suburban Geelong, near Melbourne. David’s natural inclination to scholarship eventually led him to studies in physics at the University of Melbourne where he received a Bachelor of Science in 1965 and a Master of Science in 1966. Both degrees were conferred with First Class Honours and he received a number of scholarships and awards along the way, including the Daniel Curdie Scholar-ship in Physical Sciences, the Professor Kernot Scholarship, the Dixon Research Scholarship and a Commonwealth Postgraduate Award. In 1965, he was awarded a Com-monwealth Scholarship, which allowed for him to read for a doctorate at Magdalen College in the University of Oxford, UK. He was followed to Oxford by his fiancée, Jean, whom he married in the UK in 1967. From 1966 to 1969, he was a postgraduate student in Oxford’s Department of Metallurgy, working with the celebrated electron microscopists Dr (now Professor) Mike Whelan and Professor (now Sir) Peter Hirsch. It was during this time that David, with fellow student Ian Ray, invented the ‘weak-beam’ technique, the crowning achievement of his doctoral research (DJH Cockayne, ILF Ray & MJ Whelan, 1969. “Investigations of dislocation strain fields using weak beams”, Philosophical Magazine, 20, pp. 1265–1270).

The weak-beam method was a new TEM technique devel-oped to allow more accurate imaging of small defects, such as dislocations, in crystalline materials. The innovation of David’s approach was to orient the crystal so that its planes of atoms were not strongly reflecting, which was exactly the opposite approach to normal TEM imaging of crystals. Then a dark-field image of the crystal formed from a Bragg-reflected beam has extremely low background brightness so that the image appears dark. However – and here’s the clever bit – the lattice distortion around the dislocation brings the neighbouring atomic planes close to the Bragg-reflecting condition and, therefore, these parts of the image are bright on the dark background of the surrounding crystal. In this way, the weak-beam technique allowed the position of dislocations and other defects to be accurately determined to within approximately 1 nm and provided an image that was usually simple to interpret without resorting to guesses at the defect structure or without requiring image simulations. Having invented this promising new technique in his doctoral research – a significant achieve-ment in itself – David set out to put weak-beam imaging on a firm practical and theoretical footing during a subsequent research fellowship at Oxford. He further developed the electron-scattering theory underlying the method and finessed the experimental approaches to producing optimal images under weak-beam conditions.

Shortly after this period of great achievement, in 1974 in fact, David Cockayne took up the directorship of the EMU at the University of Sydney, and so began the transforma-tion of the fledgling service unit, which at that point was tucked away under the stairs in what is now the Pharmacy Building, into a major research hub that is one of the jewels in the crown of Sydney’s research infrastructure. David oversaw many momentous changes in the EMU, beginning with the move of the unit to new rooms in the Madsen Building in 1981, which dramatically expanded the space available. Later, with support under the ARC ‘Key Centres’

Staff of the EMU, Key Centre and NWG Macintosh Centre, 1999. Professor Cockayne is in the centre of the back row, fourth from left.

3

scheme, the research side of the EMU was formally split off, in partnership with researchers from the School of Physics, as the Australian Key Centre for Microscopy and Microanalysis; but both entities occupied the same space in Madsen and David was director of both. At this time, he also introduced a formal coursework program so that students could take a diploma or master’s degree through the Key Centre. This program continues even today, well after the ARC seed funds have been exhausted, and it grew from David’s belief that anyone could learn to use electron microscopes and, with appropriate training, could generate world-class research outcomes. This degree program had grown from a seed that David had planted early in his tenure as director.

Very early on, David believed and sup-ported the idea that, as far as possible, users should do their own work, and that the previous approach, in which users sat by while the unit’s staff operated the instruments, was costly in staff resources and did not give the training appropriate to research degrees. He emphasised that, with correct training, there should be no concern that instruments might be damaged by users, and this proved to be the case. Indeed, this major theme of the EMU – providing sound training to produce safe, indepen-dent self-operators – has spread into the wider Australian electron microscopy community, and, perhaps with even more impact, into Asian facilities where the unit’s staff have run training programs over many years. To achieve this, David made the EMU’s internal training programs more structured and focussed, with emphasis on the training of users to become skilled and independent practitioners. This approach made it possible for the EMU to support more users, because they were less reliant upon the availability of staff support, but it also drove the EMU courses inexorably towards becoming not just training courses but true academic units of study, that were research-driven and comprehensive in their depth and breadth.

During this time, David’s research covered a broad range of topics, but particularly focussed on the study of strain relief by misfit dislocations at interfaces in semiconductor heterostructures. He also branched out into a rather differ-ent area – the study of amorphous materials by electron diffraction. With Prof. David McKenzie, from the School of Physics, he developed a powerful electron diffraction technique within a transmission electron microscope to determine accurate radial distribution functions (giving the average distributions of atoms around a central atom) from small volumes of amorphous material, orders of magnitude smaller than possible with X-rays or neutrons due to their small scattering cross-sections.

David’s abiding interest in defects continued during his years as director, resulting in numerous publications through the 1990s and beyond. One focus was the determination of the critical thickness in semiconductor heterostructures, which comprise layers of different semiconducting compounds in which the sizes of the crystalline lattices do not exactly match. This lattice mismatch leads to strain in the layers and this is relieved by formation of ‘misfit dislocations’ once

the layers reach a critical thickness. His team quantified critical thicknesses in different systems, and explored the geometries and motion of the misfit dislocations, providing vital understanding for designers of semiconductor heterostructures. From there, they began to explore small semiconductor islands or ‘quantum dots’ in which the electrons are confined by the small dimensions of the struc-ture so that the dot behaves very much like a single large atom, with the corresponding quantisation of energy levels. Quantum dots are of considerable scientific interest because of their potential use in optical electronics (‘optoelectronics’)

as well as in putative quantum computers. David and the team used a variety of electron microscopy techniques to characterise the shapes, stability and strain-relief behaviour of quantum dots.

Throughout his time in Sydney, David blended world-class science with a genuine service ethos as director of the EMU. He argued that the role of every staff member was unique, and that each had an important role to play in achieving the overall success of the unit. In the same way, he argued that the EMU should not try to emulate a department, but should identify its role in the research and teaching activity of the University, and should then deliver excellence. This paradigm had a profound impact on the EMU, in keeping user support in the forefront of the unit’s activities while maintaining a strong research program that was the envy of many a department head.

David continued as director of the EMU and Key Centre until the start of 2000. In 1999, he indicated his intention to resign and return to the University of Oxford, where he had done his doctorate and since spent occasional sabbaticals. As soon as he had made his decision, he quickly called a meeting of staff so that they could learn of it directly and not through the grapevine. The news was a shock to the staff of the unit and to the wider University community

– probably as much a shock as when, soon after, he was elected a Fellow of the Royal Society (FRS) in recognition of his contributions to science. In January 2000, David departed Sydney to become Professor of the Physical Examination of Materials in the Department of Materials at Oxford. There, he continued the great themes of his research career: providing new insights, mentoring a new research team and building on the foundations of lifetime of achievement that at his retirement, in 2009, put him at the pinnacle of the microscopy world and saw him awarded the Massey Medal of the Institute of Physics.

David is survived by his wife Jean, children Sophie, Tamsin and James and granddaughter Zoe.

throughout his time in sydney, david blended world-class

science with a genuine service ethos as director of the eMu.

4

workshops

Environmental SEM of Biological Samplestime: 9am–1pm Thursday 19 Mayplace: Rooms 236, ACMM, Madsen Building (F09, Level 2)rsVp: Errin Johnson (errin.johnson@sydney.edu.au) by 13 May

This workshop aims to introduce users to our Environmental SEM (ESEM) platform on the Quanta 200. ESEM utilises a specialised detector and vaccuum system that enables im-aging under low-pressure conditions. This allows untreated, fully hydrated biological specimens to be imaged at high resolution. The workshop will comprise a one-hour lecture on the theory, practise and applications of ESEM, followed by discussion time. We will have a short morning tea break, before moving to the microscope for a demonstration of ESEM and low vacuum SEM. Participants are encouraged to bring a sample to image during the demonstration session and should mention what type of sample they would like to bring in their RSVP.

hitachi s-4500 feg seMThis is a second-hand SEM that we have acquired in order to increase our capacity for high-resolution imaging. It is a cold field-emission gun (FEG) SEM, designed specifically for imaging sub-micron scale features. We are currently finalising the training documentation, and expect to begin user training straight after the Easter break. The instrument is currently located in the refurbished room 126 (opposite the lift).

Zeiss Auriga fiB-seMThis new, top-of-the-range focused ion beam (FIB) SEM, with an extensive range of optional accessories, was delivered at the start of April. Installation has begun in room 126, and will continue until after Easter. This will be followed by a period of some weeks in which the relevant technical staff will build up necessary expertise on the instrument, as well as running through a series of performance checks. It is likely to be late May at the earliest before it comes online for the first users, so we ask all of the potential users (and supervisors!) to remain patient.

news froM the seM seCtionIn the past month, we have taken delivery of two additional microscopes to complement our existing SEMs.

SEM masterclass on STEM imagingtime: 2pm–5pm Tuesday 21 Juneplace: Room 236, followed by Lab 125,ACMM, Madsen Building (F09, Level 2)rsVp: Patrick Trimby (patrick.trimby@sydney.edu.au) by 7 June

With the arrival of the new Auriga, we now have two scan-ning transmission electron microscopy (STEM) detectors on our SEMs. Although these are unable to achieve the extreme resolution of TEMs, they are ideally suited for preparatory imaging of a range of TEM samples, both from the life and physical sciences.

This half-day masterclass will be dominated by practical sessions and participants will be encouraged to bring their own samples. Limted places so get in quick!

5

users stArt enJoying new Multiphoton MiCrosCopeBy renee whAn

Thanks to $720,000 in LIEF funding awarded to a multidisciplinary team, led by our own A/Prof. Filip Braet, we are delighted to be able to offer researchers the latest in multiphoton microscopy and correlative imaging.

A new advanced light microscopy plat-form has come though the ACMM’s doors. The Leica SP5 II confocal and multiphoton microscope was installed in late October 2010. Over the last couple months the members of the ACMM Light and Optical and Biospeci-men Preparation teams have been busy putting each of the components of the 5D correlative-microscopy platform through their paces. The idea here is for you to be able to image your live cells then image the same “dynamic” event later at high resolution in the TEM. Sounds easy enough? Well that’s what the ACMM staff and Leica are working on behind the scenes – making it easy for you, our user base. To find out some more about the equipment that came in the successful LIEF bid, see our previous newsletter (volume 28, pages 1–2).

Besides being part of the correlative-microscopy platform, the multiphoton is an advanced microscope that is al-ready helping many researchers within the university to see their samples in a new light. The reasons for this are threefold. Firstly, the microscope is equipped with a new Spectra-Physics Mai Tai DeepSee™ Ti:Sapphire femtosecond pulsed laser, specialised objectives and external non-descanned detectors. This means that we are happily imaging more than 300 microns deep into thick specimens without any signal drop-off. Secondly, the system is equipped with a resonance and galvanometer scanner, so that we can now image around three times faster at higher resolution than ever before (e.g., a 512 x 128 pixel array every 15 ms). Thirdly, the system is equipped with time-correlated single-photon-counting (TCSPC) fluorescence-lifetime imaging (FLIM). This means that we can measure the lifetime of a fluorophore faster and more accurately than before

at various excitation wavelengths. There are a number of research groups in the university that have been enjoying the capabilities of the microscope. The first example is an image was taken by PhD candidate Anna Waterhouse, who is supervised by Prof. Tony Weiss at the School of Molecular Bioscience. The work is part of a multidisciplinary collaboration that also involves Dr Martin Ng at the Heart Research Institute and RPAH, and Prof. Marcela Bilek at the School of Physics. A novel stent with a special tropoelastin coating was implanted into the arteries of rabbits for seven days. The thick curved specimens (around 5 mm in height and millimetres thick) were then longitudinally bisected and examined, with endothelial cell–cell

junctions (green), actin (red) and nuclei (blue) labelled (image 1a); the transmit-ted light image (image 1b) shows the stent location in the tissue.

The second example was taken by PhD candidate Juan Pablo Matte, supervised by Dr Brian Jones from the Faculty of Agriculture. In the process of characterising the indeterminate meri-stems of an Arabidopsis mutant (image 2), they have found periclinal division in a zone (endodermis) that only should show anticlinal division. Wow! Why can they see this? The depth of imaging at high resolution is key here, because previous studies with confocal alone had such a significant drop off-on in signal as they imaged through dense plant tissue that previous images did not gain the resolution required.

The third example comes from an ongoing collaboration between Prof. Cris Dos Remedios from the Faculty of Medicine and A/Prof. Filip Braet (ACMM). Honours student Amy Li has been investigating how the second harmonics of myosin (image 3b) might aid in measuring spontaneous oscil-latory contraction, or ‘SPOC’, in stri-ated human heart tissue. By using the resonance scanner to image as fast as possible, we are finding that increased

1a

1b 2

6

contrast in between sarcomeres in the second-harmonic image compared to the bright-field image (image 3a) will likely lead to better quality data analysis.

So if you think your sample might ben-efit from multiphoton imaging, please contact lab manager eleanor.kable@sydney.edu.au or another member of the light and optical team.

ACMM Courses

Course title semester 1 semester 2

Introductory Microscopy & Microanalysis

March 7–17 July 25 – August 4

Biological Specimen Preparation, TEM & SEM

March 21–25 August 8–12

Operation of the Scanning Electron Microscope

April 4–8 August 15–19

Operation of the Transmission Electron Microscope

April 11–15 August 22–26

Stereology April 18–19 September 19–20

Image Analysis April 20–22 September 21–23

Fluorescence Microscopy Techniques

May 2–6 N/A

Introduction to Confocal Microscopy

May 9–13 September 12–16

Materials Specimen Preparation, TEM & SEM

May 16–19 August 29 – September 1

TEM of Crystalline Materials May 23–26 September 5–8

Microscopy of Biomolecular Processes

N/A October 10–21

Nanostructural Analysis of Materials

N/A October 17–27

Light Microscopy Workshop TBC* TBC*

Research Methodology N/A July 28 – October 13, runs for 9 Thursdays between 4–6 pm**

*Check website for exact information.**Proposed day and time; check website closer to date for exact information.

Need to use microscopy in your research? Thinking about applying a new form of microscopy for the first time? Want to get a better understanding of the fundamentals of microscopy and microanalysis?

Then you should consider registering for one or more of the ACMM courses. There are courses to teach you how to prepare samples efficiently, and in others you can learn about the theory and practice of different microscopy techniques. Forthcoming courses for this year are summarised in the table to the right. Further details about each course and the costs for attending can be found on our website: sydney.edu.au/acmm/courses_training/short_courses/index.shtml

Wolfgang Becker (right), one of the owners and designers of the Becker and Hickl TCSPC system, spent a fine Saturday in November with Carola Thoni (left) and Jose Quevedo (middle) from Leica optimising the system for us.

3a

3b

7

MAn (And his MiCrosCope) Vs. wild

Join us as we catch up with former ACMM staff member Alexandre La Fontaine on his journeys in South America, where he is combining his love of adventure with his love of microscopy.

Until recently, Alex was Atom Probe Engineer here in the ACMM, where he helped users get the most from their atom-probe experiments and kept the two Imago local-electrode atom probes singing. With occasional forays into scanning probe microscopy and with considerable expertise in electron microscopy too, Alex certainly knew his way around a microscopy lab and was a good comrade to have at your side when scientific characterisation was the order of the day. During 2010, however, Alex at last succumbed to the call of the wild and, having finished up his role in centre during December, he and his partner, Aude, set off for a year’s adventure in South America, Asia and other parts of the globe. Apart from getting to travel around this amazing planet and getting to know the locals, Alex, with some support from the ACMM, will also be transfer-ring his passion for microscopy to local school children. We’ll check back with Alex periodically throughout the year to hear how his trip is progressing, but for now here’s our first communiqué from the intrepid man himself:

I am travelling around the world for a year because I want to learn from other cultures and countries, and I want to learn more about myself as well. I have taken with me a small opti-cal microscope and a laptop in order to visit schools wherever I will be, schools that do not have access to this kind of educational tool, where I will do a sort of ‘school open day’ in coordination with the teacher for children between 10 and 12 years old. Furthermore, I am a guest scientist for a US foundation called Science House that has a project, MicroGlobalScope, in which they send two optical microscopes to selected schools all over the world (check www.microglobalscope.com). I have been asked to find around six schools in South America and six in India and Asia that can benefit from the MicroGlobalScope programme. Thanks to the optical microscope

and the laptop generously offered by ACMM, I can approach teachers or community-based volunteers and show them the educational power of such a tool ...

One month ago, we flew to Santiago de Chile, the capital of Chile. Then we travelled towards the south of the country, reaching the region of the lakes (Puerto Varas, a 12-hour bus ride south of Santiago) where we stayed two weeks. It’s a really cool place with volcanoes and lakes and small isolated villages dotted around. One of the highlights during those two weeks was a trek I did with Aude in a stunning, remote location called the Cochamó Valley. This place has got one of the world best granite rocks for climbing, or ‘escalade’ as we call it in French, and climbers (totally crazy guys) come there from all over the world to try the peak: to escalade one peak they have to sleep in the middle of the rock, hanging in the void (I told you they were crazy). So you can imagine our trek! Extremely hard, seven hours through muddy forest to reach a refuge, then the next day a trek to go up a peak. It was 40 metres of vertical rock to climb using a rope... I almost fell, and if you do so, it’s bye-bye. Anyway, sweat, blood and blisters were the rewards for our exertions, although the view was impressive. Then it was back to Puerto Varas where I initiated contact for the microscope project with a school teacher and a community leader. They will work with me on the MicroGlobalScope project and have already found a suitable village. More details to follow...

After this nice stop in Puerto Varas, we decided to go the hard way and not to take a plane to reach the south of Patagonia … not many tourists or gringos do this and now I understand why. The name of the road (sorry, the track) is la Carretera Austral. Six days of minibuses full of locals, no security whatsoever, tracks even worse than

in outback Australia, cliffs everywhere, conductor totally crazy, sleeping in villages so remote they only have potatoes to eat ... It was a pure local Chileno experience! These people are rough, but when we spent a bit of time talking to them, they started to open up and share their stories. Eventually we managed to get to the end of the road in Chile (no more road, only lakes and glaciers). Then we had to trek two days to go through the Argentinean border. Amazing experience!

Hasta luego, Alex

A relaxed Alex during the centre’s Golden Jubilee celebrations at the end of 2008.

Alex explains atom probe tomography to Dr Karl Kruszelnicki during the Sleek Geeks visit to the ACMM in 2010.

8

This workshop, organised by the ACMM in conjunction with the School of Biological Sciences (SOBs) and the Faculty of Agriculture, Food & Natural Resources (FAFNR), was the first of its kind at Sydney. It aimed to address the major research questions in the plant sciences that our research community are tackling, and highlight potential ways in which microscopy and micro-analysis can enable this research. We also hoped to encourage networking between FAFNR, SOBs and the ACMM, and to identify capability gaps that ought to be addressed to move forward our aspirations in the area.

The workshop was opened by Prof. Jill Trewhella, Deputy Vice-Chancellor (Research), who spoke about the importance of plant science research in devising targeted approaches to large-scale issues, such as climate change and sustainability. Specific strategies involving agricultural and plant cell-biology research, as facili-tated by microscopy and microanalysis, were highlighted by Dean of FAFNR Prof. Mark Adams, Head of SoBs Prof. Robyn Overall, and ACMM Director Prof. Simon Ringer.

We also heard from a diverse range of University of Sydney researchers about their current plant- and soil-science projects. Topics included the charac-terisation of a drought-resistant Ara-bidopsis mutant, investigation of the sub-cellular localisation of viral defence proteins, and optimisation of transgene expression in soybean nodules. These talks featured a variety of microscopy and microanalysis techniques, which were further showcased in presenta-tions by ACMM staff. These ranged from our capabilities in biological specimen preparation, through to light and electron microscopy, with a par-ticular focus on how these techniques can be applied to plant specimens. Exciting high-end techniques, such as multiphoton, environmental SEM, TEM tomography and microCT, were also discussed.

The workshop was a success on all fronts. The large turnout, of over 60 people, was extremely encouraging and we hope that the networks and momentum generated from this work-shop can be carried forward this year. To this end, the ACMM has established a Plant & Soil Science Microscopy Group that meets on a monthly basis. These meetings aim to provide a forum in which plant researchers can present their work and discuss microscopy techniques and issues relating to it. Each meeting consists of two 30-minute presentations followed by a discussion over cheese and wine. For more information, please contact Errin Johnson (errin.johnson@sydney.edu.au).

Prof. Mark Adams (FAFNR) spoke about strategic outlooks for agricultural research at Sydney.

Prof. Simon Ringer discussed the past, present and future of plant science microscopy at the ACMM.

plAnt sCienCe BlossoMs At the ACMMBy errin Johnson

The Frontiers of Plant Science and Microscopy Workshop was held at the ACMM on 29th November 2010.

Dr Deborah Barton (SoBs) showed some brilliant correlative work on microtoubule arrays.

Prof. Robyn Overall (SoBs), Prof. Geoff Wasteneys (University of British Columbia) and Prof. Jill Trewhella (DVC-R).

Dr Patrick Trimby (ACMM), Victor Lo (School of Chemical and Biomolecular Engineering) and Delfine Cheng (ACMM).

9

Manasa joined us from the Department of Physics and Astronomy at the University of Maine, where she is a research scientist. Her background is in chemical engineering, but during her PhD and postdoctoral work Manasa ended up learning a lot about microscopy, physics, lasers, optics, data analysis, cell culture and molecular biology. After she made an initial ap-proach to Prof. Simon Ringer in 2009, it became obvious that her research interests matched nicely to those of our very own A/Prof. Filip Braet.

In conversation, Manasa explained: Both of us are looking at the patho-genesis of disease. I’ve been looking at it in terms of the influenza virus and Filip has been looking at it in terms of cancer, but we are both doing so at the nanoscale. In the cell membrane, there are specialised areas called rafts and we wanted to study these to see what function they have in disease. We are also interested in the actin cytoskeleton, as, of course, is Filip. In the past, it was thought that it was just the protein or the lipids that were important in the rafts, but it doesn’t appear so clear cut any more. The rea-son that there is so much controversy as to how big are these rafts are is that they are below the diffraction limit of the light microscope, but this is how most people are studying them. If you want to study the dynamics, you can’t use electron microscopy and this is where the super-resolution microscopy that I have been working on comes in.

On a cell membrane where everything is so tightly packed together, you can’t really resolve below 200 nm with light microscopy. In super-resolution microscopy, there are two ways to get around this limit, which bring the resolution right down – that makes a big difference. The first is to physically shrink the point-spread function so it

physically occupies a smaller space and the second is to control the sample to get around the problem. The second is what I do.

Fluorescence photoactivation localisa-tion microscopy (FPALM) has a lateral resolution of 20–40 nm, which is a significant enhancement over the 200-250 nm diffraction-limited resolution obtained with widefield and confocal microscopy. FPALM localises single molecules and reconstructs a complete picture to achieve resolution below the diffraction limit in live biological samples. First, the biological structure of interest is tagged with photoactivat-able fluorophores that are initially in a dark state. Then a sparse subset of fluorophores is activated (usually with a 405 nm laser) to a bright state. Single-molecule positions are imaged by using a highly sensitive camera and the fluorophores are subsequently photobleached. By repeating this cycle several times, information about a large number of closely positioned molecules is obtained, which is then used to reconstruct a high-resolution composite image. The sample prep is just like light microscopy so you can do it all on live cells.

Manasa gave a fascinating seminar on this topic while she was here, inspiring many of the audience to think about the possibilities that super-resolution

super-(CritiCAl-resolution)-hero hits the ACMMBy Jenny whiting

With expertise in super-resolution microscopy and an interest in correlative microscopy, Dr Manasa Gudheti had long been keen to visit the ACMM. Thanks to the University of Sydney’s International Visiting Research Fellowship scheme, Manasa was able to spend two months working with Filip Braet late last year.

Dr Manasa Gudheti (far right) enjoys some ACMM hospitality.

microscopy can offer. However, the focus of her visit was to learn new skills, and she continued: I was particularly interested to learn more about correlative microscopy while I was in Sydney and the fact that this is also Filip’s interest is perfect. I would really like to correlate super-resolution microscopy with EM. For me it is important because when you just do fluorescent spectroscopy you are specifically tagging only certain things, which means that you can miss out on the overall context whereas when you do EM you get more of an overall picture of how your molecule is part of the whole. You can do live-cell imaging, then fix the sample and do EM – it helps to corroborate what the new technique is telling you.

I’ve had a wonderful time at the ACMM; I’ve learnt loads, especially about good spec. prep. and TEM. The wide array of research that is going on and the variety of instruments is amazing, and everybody is really friendly and easy to work with. There are lots of collaborative projects that we can do together in the future and Filip is so knowledgeable – he is a real microscopy guru.

We hope to see Manasa again soon and keep up with her developments in correlative and super-resolution microscopy.

10

For almost the entire year, I was based mainly at the University of Wisconsin, Madison, where my husband, Prof. Scott Kable, was enjoying his sabbati-cal as a Fulbright scholar.

In the first half of the year, I attended several seminars and workshops. The University of Wisconsin had just installed its FEI Titan aberration-corrected microscope and there was a one-day meeting by the Midwest Microscopy and Microanalysis Society (M3S) in conjunction with this installa-tion. There were some very interesting talks given over a large range of topics, from nanotubes to multiple labelling of proteins.

After attending a workshop on nothing but TEM, I then attended a Frontiers of Cellular Imaging Workshop at Marquette University, Wisconsin. This workshop had talks and hands-on practicals from industry and research-ers and ranged from advances in fluorophore technology to live-cell imaging.

In July, while travelling around the US, I visited the Centre for Nanoscale Systems at Harvard University in Boston for a day. Dr David Bell (another ex-pat) hosted me. It was great to see the Zeiss TEMs and the fabulous clean rooms they have. It was also interest-ing to learn about how they run their booking and charging facilities, not to mention to see the lab design, as it is a relatively new building.

From September to mid-December, I joined Prof. Jim Weisshaar’s research group at the University of Wisconsin, Madison. The attraction for joining this group was to get back into a microscope lab and to catch up on high-resolution light and optical tech-niques. It was great to focus my brain to really understand these techniques.

This reminded me of my days at Cornell University where the microscopes were built and adapted by the students, which is not something you can do in a multi-user facility.

During the year, I also joined forces with A/Prof. Paul Rigby, a colleague from the University of Western Australia node of the AMMRF, to visit labs and attend conferences together. Following the Microscopy and Microanalysis conference in Portland, Oregon, we were invited to present talks at the head office of Invitrogen Molecular Probes in Eugene, Oregon, and at Thermo Fisher in Pittsburgh, Philadelphia. Invitrogen is a leading company that develops and sells most of the fluorophores that are used in both of our laboratories. Paul and I gave a joint talk on the AMMRF and on our respective roles in teaching and re-search to approximately 90 Invitrogen staff. Following this, we had a tour of the facility and its laboratories, as well as individual meetings with various sec-

Prof Paul Rigby (UWA) and Ellie Kable with Mike Ignatius of Invitrogen, after Paul and Ellie had given their talk to the Invitrogen team in Eugene, Oregon.

MiCrosCopy And MACrosCopy in the usABy ellie kABle

During 2010, our laboratory manager, Ellie Kable, spent an extended time in the USA travelling for pleasure and work. Ellie returned to the ACMM in January this year and has agreed to give us the lowdown on her experiences.

tions heads, where we discussed how the relationship between Invitrogen Life Technologies and the AMMRF nodes might be better strengthened and enhanced. Thermo Fisher produce instrumentation focussed on high-content screening and the talk on the AMMRF model and operation given by Paul and myself was very well received. Both of these meetings laid the foundations for greatly improved interactions with these companies in the future.

One of the highlights of our trips was the opportunity to spend a week work-ing with Prof. Simon Watkins (who is a member of the AMMRF’s International Technical and User Advisory Group) in Pittsburgh, Philadelphia. Here Paul and I honed our skills on multi-colour TIRF and live-cell microscopy as well as see-ing how Simon’s very successful Center for Biologic Imaging at the University of Pittsburgh (http://www.cbi.pitt.edu/) was run and operated.

11

AustrAliAn Centre for MiCrosCopy & MiCroAnAlysis

for More inforMAtion ContACt

t +61 2 9351 2351f +61 2 9351 7682e acmm.info@sydney.edu.ausydney.edu.au/acmm

The Kable family tours the amazing Monument Valley, which lies on the border between Arizona and Utah.

The Kable family near the beginning of the North Rim trail in the Grand Canyon. After 7 hours we had made it halfway down and back.

From seeing things on microscopic scales for fun at work, the family and I visited natural and man-made vistas at amazing varieties of scales: from sea level at Key West to 13,000 ft at Teluride; from vast vistas of land at Monument Valley and the Grand Canyon (see photographs at left) to the amazing cities of Washington DC and NYC; from whale watching on the east coast to misty sunsets on the west coast and much more in between. It was a truly wonderful year of educa-tion in so many areas.

So now back on deck and rested, I am a more informed laboratory manager, and I can’t wait to use some of my new found microscopy knowledge for our user community along with implement-ing some great ideas for running and improving our microscopy facility.

grAnts AdViCeFor those of you applying for ARC and/or NHMRC grants this year, don’t forget to incorporate ACMM access costs into your requested budget(s). Details on how to do this can be found on our website, www.sydney.edu.au/acmm. Follow the “Advice to ARC/NHMRC Applicants” under the Quick Links on the front page. Good luck.