A microscopic view of life

Materials World magazine
29 Oct 2019

A new super high-speed microscope enables scientists to image living samples and neuron activity in real-time. Ceri Jones reports.

SCAPE 2.0 is the latest version of a microscope that operates at ultra-fast speeds to image living tissue, such as the beating heart of a worm. The system has also captured neurons firing, near-isotropic imaging of a free-moving Caenorhabditis elegans worm, and blood flow and calcium transport in a zebrafish – all in real-time.

Full results of the project, led by Columbia University, USA, Zuckerman Institute Biomedical Engineer, Dr Elizabeth Hillman, are in the paper, Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0, published in Nature Methods.

Swept confocally aligned planar excitation (SCAPE) 2.0 is 30 times faster than the first iteration in 2015. The team spent four years refining the technique and integrated a high-speed intensified camera in order to bring the system up to 300 volumes per second, capturing images at 18,000 frames per second with a 1.2GHz pixel rate.

The device is ideal for observing delicate tissues such as brain, retina and tumour tissues. ‘We want to look at the response of tumours to radiation therapy, by being able to look quickly at cell signalling and blood flow dynamics and understand physiological response within any form of tissue that is not just a two-dimensional tissue culture,’ Hillman told Materials World. ‘We're also developing a medical version to do imaging of human tissue during surgery to guide surgeons.’

According to Hillman, SCAPE 2.0 would also support physical fields such as examining bubble migration behaviour in solids and liquids, film thicknesses, and geometry and construction of microsystems like computer chips without needing to deconstruct them first.

Getting up to speed

Speed is imperative to successfully imaging moving, living organisms.SCAPE 2.0 uses light sheet technology whereby a beam of light illuminates an entire horizontal plane of the sample, for the camera to focus on that section and take an image. The light is shifted to the next section and the camera moved and refocused, to form a stackable series that builds up a 3D image.

However, moving both light sheet and camera introduced mechanical coupling issues, and restricted the maximum speed of the process. To overcome this, the team relayed the image via the mirror, positioned at an oblique angle, so both camera and sample could be stationary. Also, with this technique, the system would only require one lens.

‘The mirror in the back causes the sheet to scan sideways across the sample, so each image is like a depth lateral scan moving in an orthogonal direction. That lets us move the sheet as quickly as we want. And if we wanted to do 10 volumes a second, we would just move the mirror 10Hz, which is very simple,’ Hillman said.

‘We also image the light coming back from the sheet off of the same mirror, so the mirror is moving the sheet but also maintaining the focus of the camera. The camera is stationary, streaming images as fast as it can while the mirror just goes back and forth at between 10-300Hz.’

Hillman explained how the light sheet circumvents problems with point scanning because doing a 400 x 400 pixel image at once gives you 160,000 times longer to integrate the signal on each pixel. The technique also uses a far lower level of light than comparable microscopes.

‘When you're coming in from the top of your sample with a focus spot, you're inadvertently blasting and bleaching the tissue above and below the plane you're looking at. If you were to try to do 3D imaging of 100 planes, it is basically like imaging every single one a hundred times. With the sheet, it is only illuminating the plane you're imaging – it is way more photo efficient, easier to detect signals for equivalent pixel rate or equivalent data streaming.’

Overcoming limitations

Hillman’s team has helped 10 other research groups build their own SCAPE 2.0 system, which has sparked technological adaptations for specific project needs.

For instance, while one group is decreasing the field of view to maximise resolution for use in sub-cellular imaging, another has taken the opposite path to increase the view size up to 5mm to work with larger live samples, including squid, despite this sacrificing the resolution. However, while the system is versatile, there are still many areas for refinement.

‘The biggest limitation is depth into the sample. If it's a living thing, where you can't make it transparent, then the depth to which you can image is the limiting factor at the moment for us,’ Hillman said. ‘But if we use longer wavelengths as we go to the key photon system, we can extend that depth.

‘We can take immediate advantage of any improvement in camera sensitivity, and the other area is indicators. In the last few years, people worked hard on making fluorescent indicators really bright and stable. Any time we get more photons, we can do more with that.’

Leica Microsystems has licensed the SCAPE 2.0 technology and technique, and is currently developing a commercial system.