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Real life under the microscope

It’s been a long time since Anton van Leeuwenhoek studied bacteria under one of the first light microscopes: 333 years, to be precise. But the fascination of watching life unfold under a microscope has never faded.

Beyond conventional light microscopy, electron microscopes gave us glimpses of the fine structure of sub-cellular organelles like nuclei and mitochondria, as well as detailed 3D images of surface structures. But these were static and lifeless, only able to be viewed after the cell was chemically fixed, frozen and sectioned. Like a freeze-frame from a movie, you could only tell what state the cell was in at the moment of preservation: not what came before or after.

What researchers have really wanted to see, in super-fine 3D detail, is how whole living cells move and interact. How they grow, signal or sense their environment, passing messages from the inside to the outside of the cell and from one cell to another. But to see this type of action at the sub-cellular level in living cells, we needed technology that could break through the light diffraction barrier, which causes very small objects to blur. And that’s just what the brand-spanking new microscopes at Monash Micro Imaging (MMI) can do.

This is the relatively new world of super-resolution fluorescence microscopy, or nanoscopy. As part of the centralised Monash Technology Research Platforms, MMI has been able to commission two of the latest microscopes that allow dynamic, super-resolution cellular imaging of living cells.

Using lasers focused into incredibly small points of light, the ‘STED’ nanoscope can resolve tiny structures down to about 40–80 nanometres. That’s way smaller than a cell’s nucleus, which comes in at around 10,000 nanometres; it’s even smaller than a mitrochondrion, about 1,000 nanometres across.

Professor Trevor Lithgow’s group at Monash University’s Biomedicine Discovery Institute (BDI) were the first to have some fun with nanoscopy. Using a technique called single molecule localisation microscopy, they’ve watched a bacterial membrane-transport protein complex in action. Proteins destined for the bacterial cell surface are assembled inside the complex, known as the beta-barrel assembly machinery, or BAM, before being inserted into the outer membrane.

“Using the MMI’s super-resolution microscopes we have, for the first time, watched as the BAM complex swells while these cell-surface proteins are being made, and collapses back as they exit,” says Professor Lithgow.

The second new microscope, called a lattice light-sheet microscope, captures very high resolution images of living cells in 3D on camera as they move, grow and interact.

“The phenomenal thing about this microscope, is that instead of using a single laser point to scan the cell, we now scan a sheet made up of light strips through the cell,” explains Associate Professor Ian Harper, Director of MMI. “Just imagine strips of light, a bit like a venetian blind, making up a lattice. We’re able to sweep the lattice through the cell to make a 3D image, taking just fractions of a second.”

Making all this possible was the technical and intellectual collaboration between staff at MMI and the BDI, which led to successful national infrastructure grant bids and university strategic funding.

“This really was a brilliant result of the meeting of the minds,” stresses Professor Lithgow. “The outcome was far greater than any of us could have managed separately.”