Feature: Super resolution microscopy breaks the light barrier

Optical microscopy has undergone something of a revolution over the past decade, so say Professor Leann Tilley and Associate Professor Trevor Smith, from LaTrobe and Melbourne University, respectively.

It started several years ago with the introduction of a spectrum of new fluorescent molecules, which have now been complemented by super resolution microscopy, a completely new optical technology and instrumentation that is enabling biologists to address important questions as never before.

The problem with microscopy was long considered to be its very reliance on light. Ever since 1873, when German physicist Ernst Abbe suggested that there was a limit to the resolution achievable from a microscope, it was believed the wavelength of light placed a practical limit of around 250 nanometres (nm) as the smallest distance between two features in a sample that could be resolved. Yet, unfortunately for microscopy devotees, many features of interest are well below 250 nm in size.

“This is the size of a small bacterium or a large virus or mitochondrion,” says Tilley. “So if you want to look inside a cell into the deepest darkest recesses, into the organelles, then you have to do better than that. Up to now we have only had electron microscopy – with a 40-fold higher resolution – to turn to, but then you have all the associated issues with the biological specimen preparation that is needed to avoid beam damage.”

Then, in a series of startling advances over the last decade, this diffraction limit barrier has been well and truly broken. “This new technology doesn’t break the laws of physics, it just sort of bypasses them using specialised and very clever optical strategies,” says Smith, who is an expert in x-ray and laser science at the University of Melbourne.

Light entertainment

Tilly and Smith hosted a half-day workshop on super resolution microscopy covering the latest developments in the field at OzBio2010 on October 1, supported jointly by the ARC Centre of Excellence for Coherent X-ray Science (CXS) and OzBio2010. Six experts in the field will cover different aspects of this rapidly-advancing field of microscopy.

“Our combined interest in super resolution optical microscopy comes from the work that Trevor and I do through the ARC Centre of Excellence,” says Tilley, who is a cell biologist working on malarial pathogenesis at La Trobe. The CXS’s charter is to bring physicists, chemists and biologists together to develop fundamentally new approaches to probing biological structures and processes by combining the latest in imaging technology, structural and molecular biology, and laser science.

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According to Tilley, their original philosophy was to take serious cell biologists and mix in some hardcore physicists and chemists to address tough biological issues using their combined expertise, along with the latest technologies in x-ray imaging, rather than having all of them work separately at the interface.

“Since then we have branched out to do pretty much any kind of high-end imaging technique that is available or can be developed. The newish field of super resolution microscopy and our interest in it fits within that general scheme of trying to get biologists to talk to physicists, and then the chemists like Trevor bridge the gap – he can speak both languages.”

Tilley and Smith also belong to the recently established Cellular Nano-Imaging Consortium (CNIC), which is an affiliation of scientists with interests in super resolution optical microscopy managed under the auspices of the ARC Centre of Excellence.

The idea of CNIC is to bring together institutions and researchers with cross-disciplinary interests in new imaging capabilities, by providing on-line access to information about the techniques and the resources currently available, principally in Victoria although not exclusively. As part of this, the CNIC plan includes organising workshops and conference sessions like that being held at OzBio in October.

“These instruments are very expensive and it makes sense to move the technology forward in Australia by facilitating an environment where the instruments are run and maintained by experts, but also that there are expert users on hand to provide the ancillary equipment and advice that people need to do internationally competitive research in this rapidly moving field,” says Smith.

Most of the new super resolution microscopy approaches fall under three main technique areas. The first is Structured Illumination (3D-SIM), which is the modality that Smith and Tilley are concentrating on in their own work.

The second is based on the ‘pointillist’ idea, whereby a location image is built up from photoactivatable proteins imaged one at a time. The Photo-Activated Localisation Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) microscopes fit into this category (see ‘A matter of perspective’ in January/February 2010 issue, page 32).

The third is called STED or Stimulated Emission Depletion microscopy. This is the so-called doughnut technique that relies on interfering light beams that illuminate the sample with an outer and an inner ring resulting in photoexcitation only of the fluorescence molecule in the hole of the doughnut (see ‘Lord of the Z ring’ in May/June 2010 issue, page 36).

“Then there are variations on those and other techniques are coming along as well,” says Smith. “In fact, the appearance of some new instrument or technique variation is almost a monthly event at the moment.”

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Full spectrum

“There are, of course, pros and cons with each different technique or instrumentation, and not all of these new techniques are suited to all fluorophores or all samples,” says Smith. “The major commonality of course is that they are all expensive to buy commercially, or not even available as yet commercially.”

Through the OzBio workshop and CNIC’s other activities, Tilley and Smith want to get the message out that there are a lot of different techniques becoming available, and someone needs to know enough about each of them to make an educated decision on the way to go with a given application or sample.

“The super resolution microscopy workshop at OzBio will target as many of these choices as we can,” says Tilly. “We want to pull together a range of people who either currently own such an instrument or have access to one, either bought or built, and match them up with those scientists who want to know more about and/or use such an instrument.

“On the speaker list, Katharina Gaus, from the University of New South Wales, will cover PALM, Cynthia Whitchurch, from the University of Technology Sydney, and I will cover the 3D SIM, Trevor will cover STED and the more technical aspects of 3D SIM, and then Guy Cox, from Sydney University, will give an overview of the field at present. Finally, we have Sam Hess from the University of Maine, who co-invented the STORM technology. Most of all, it should be a lot of fun.”

Focus on 3D SIM

The super resolution technique that Tilley and Smith are concentrating on currently in their own work is 3D-Structured Illumination Microscopy – Tilley as an enthusiastic user with all the questions to answer and Smith to help answer them by building his own 3D SIM.

“Engineering efforts like Trevor’s not only help cell biologists like me stay at the cutting edge in imaging, but it also enable Australia to be involved in the development and progress of these technologies,” says Tilley.

Structured Illumination Microscopy illuminates the sample with a periodic excitation pattern that interacts with small features in the sample to generate Moiré images. The subsequent reconstructed images have increased resolution over conventional optical microscopy in both the xy and z planes. It is suitable for multi-colour imaging and 3D sectioning can be used to obtain images of whole cells.

“SIM has the advantage of working with virtually any wavelength range and most commonly used fluorophores can be used,” says Smith. “Mainly because of this, it seems to be the method getting picked up most rapidly for biological applications.”

Tilley is very excited about some of the results she is achieving using the new 3D SIM instruments in Trevor’s lab and in the Microscopy Facility of Cynthia Whitchurch at UTS, where a commercial 3D SIM instrument has been installed. Tilley is imaging cells she has looked at many times before by conventional confocal or widefield microscopy.

“I am suddenly seeing things for the first time. For example, one of the intracellular structures that we always saw as a single blob (technical term) by confocal imaging now appears as a doughnut-shaped structure, indicating the presence of sub-compartments.

We are seeing features of cellular architecture in higher detail and getting new information about protein location and how they are organised within organelles…and therefore new clues about protein function. We are truly amazed at the amount of new imaging information we are collecting with the same old samples.”