Microscopy Mission

Editorial

Rebecca Pool

Friday, June 16, 2017 - 13:30
Image: Professor Michelle Peckham uses a vast array of light and electron microscopy methods to investigate molecular motors and more [James Aegerte].
 
In Autumn last year, Professor Michelle Peckham was named the new President of the Royal Microscopical Society. Renowned for her research breakthroughs in myosin, muscle and more, Peckham was unanimously voted in by the RMS Council.
 
As she tells Microscopy and Analysis: "This is all about making sure microscopists have a voice and somewhere to go to share experiences, and receive help and expertise."
 
"There's so much fantastic microscopy going on and I want to make a case for the importance of microscopy wherever and whenever possible," she adds.
 
Peckham first came across microscopy when completing her PhD in Physiology at University College London. Interested in both biology and physics, physiology was apt and she was using microscopy to prepare muscles for her research into muscle energetics.
 
However, her passion for microscopy grew when she moved to King's College London in 1985 to work with biophysicist, Malcolm Irving. Here, she was using a specialised form of light microscopy - birefringence - to investigate muscle cross bridge orientation, considered very novel at the time.
 
"Put muscle in a polarising microscope and it shifts the light in such a way, it looks very beautiful," she says. "But you can also use this birefringence to measure the alignment of proteins in the muscle, and this is where I really got interested in microscopes and started using them as an analytical tool."
 
3D PALM image of an entire adult heart muscle cell (cardiomyocyte). The fluorescent protein used was mEos3 fused to the Z-disc protein α-actinin, which is found at the end of muscle sarcomeres.
 
From here, Peckham moved to the University of California, San Francisco, to continue investigating muscle cross bridges, using fluorescence polarisation. And a year later, in 1988, she returned to the UK, and the University of York, to study insect flight muscle.
 
At the time, the York group led by biologist Professor David White, were mutating the contractile proteins in the flight muscle of Drosophila to assess how this affected crossbridge kinetics. As Peckham highlights: "This was so tricky; I was taking the very small flight muscle out of this model organism and seeing how well the muscles worked after introducing the mutations."
 
Still, two years on, the results had been published in Nature, and importantly, Peckham was now very interested in using molecular biology to manipulate proteins. With a Royal Society University Research Fellowship in tow, she headed to King's College London to study the cell and molecular biology of muscle development in mammal models, using live cell imaging.
 
"Drosophila is a great genetic system but it's not as complex as a mammalian cell, and if you want to model muscle disease to understand how humans work, you really have to use a model system closer to humans," she says. "I needed this so I chose cultured muscle cells as they are pretty much identical to what you would see in a human."
 
Image of a cancer cell, in which siRNA was used to silence the myosin Myo9b. Magenta: actin and green: non-muscle myosin 2A. Image was taken using a Zeiss Airyscan LSM880 confocal microscope.
 
At the time, the first commercial confocal microscopes were taking off, and crucially, biologists had just started to use green fluorescent protein as markers in molecular studies. Peckham was using live cell imaging to investigate muscle cell behaviour in cultured cells and confocal microscopy to investigate the associated cytoskeleton.
 
"This was so exciting as you could now actually label a protein in a cell and see what happened to it," says Peckham. "Everyone jumped on that bandwagon including us and I would head to the University College London branch of the Ludwig Institute for Cancer Research to use a confocal microscope."
 
Research at King's continued for seven years, during which time Peckham also worked alongside Graham Dunn, using a novel method - Digitally Recorded Interference Microscopy with Automatic Phase Shifting - to investigate cell crawling behaviour.
 
According to the researcher, the technique pioneered by Dunn was 'fantastic' for label-free live cell imaging, but specialised, and as such, was never widely used. What's more, by 1997, Peckham had also been offered at lecturing position at the University of Leeds, which she accepted.
 
From here, Peckham's research flourished as she adopted and developed an ever-wider range of imaging methods to study muscle, molecular motors and the cytoskeleton.
 
Image of a cultured cell, expressing eGFP-tagged non-muscle myosin 2A (in green). Endogenous non-muscle myosin 2A is in magenta.  Image was taken using a Zeiss Airyscan LSM880 confocal microscope.
 
At the time, many fellow researchers were fixated on investigating so-called muscle myosin - the motor protein best known for its role in muscle contraction - but Peckham saw her research branching out into another avenue.
 
"What's really fun about muscle cells is that to form a muscle fibre, many, many cells have to fuse," she points out. "So I got interested in this, the changes that would take place in the cytoskeleton, and importantly, what all the different non-muscle myosins were doing."
 
"Even today, I look back now and still see all these different myosins in all the different organisms and they are all so weird and wonderful," she adds. "We've now spent years imaging these and looking at them under electron microscopes, and are still not running out of interesting questions to ask."
 
Laboratory life
In the years that have followed, Peckham has used myriad methods to unravel many mysteries surrounding muscle formation. From fluorescence and electron microscopy to atomic force microscopy as well as X-ray crystallography, nuclear magnetic resonance and other biophysical approaches, her laboratory has developed new ways to better investigate the structure and function of motor-related proteins in cells.
 
More recently, Peckham and colleagues have developed super resolution methods including PALM/STORM as well as an instant structured illumination microscope (iSIM) for imaging the cytoskeleton in live and fixed cells.
 
According to Peckham, the team's PALM/STORM system provides nearly 10 nm resolution and has been used to image key muscle structures and primary cilia. Meanwhile, the iSIM captures very sharp, high-resolution images at fast frame rates in live cells.
 
"Super-resolution microscopy has been challenging as you have to prepare your specimens very carefully to get meaningful images, particularly with STORM and PALM," says Peckham. "I think people can be a little scared to take up this new technology, but we've been making these instruments easier to use and now we have researchers looking at viruses and even physicists looking at quantum dots."
 
And instrument construction continues. Right now, Peckham and colleagues are also building a total internal reflection fluorescence (TIRF) microscope to image single molecules in live and sick cells while an undergraduate student is also constructing a light sheet microscope.
 
"This really is just as a fun thing to do but I am interested in light sheet microscopy and would really like to have a lattice light sheet microscope at some point," says Peckham. "This really would be a nice toy to play with and would be useful to a lot of researchers."
 
3D STORM image of a dividing cell, stained for tubulin. Image taken using the home-built PALM/STORM system at Leeds (courtesy of Ruth Hughes).
 
Indeed, over time, correlative microscopy is becoming increasingly important to life in Peckham's laboratory, and right now, collaborators at the University of Leeds are  acquiring a new cryo-focused ion beam (cryo-FIB) to circumvent the issues of imaging thick cells in an electron microscope.
 
"What we would really, really like to do is image cells using super-resolution methods and then take those same cells and look at them with cryo-electron microscopy," she explains. "We could than look at the molecular organisation and link it back to what we've seen in the super resolution microscope."
 
But throughout the drive for instrument and method development, Peckham's passion for myosin and the cytoskeleton remains a constant.
 
"I think we're always going to be interested in the cytoskeleton of these cells in some way, shape or form, so we'll always be trying to integrate imaging in the light microscope while carrying out experiments with other techniques," she says.
 
And for Peckham, sharing experiences remains key. "Microscopy has so much potential now and there are so many different approaches and techniques," she says. "If you haven't got what you want, someone else will have so why not just go and talk to them?"
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