A microscope for life
Low-light microscopy pioneer, Dr Philippe Laissue, has built a novel light sheet instrument to gently image coral. Rebecca Pool finds out more.
When Philippe Laissue, Biologist and Director of the Bioimaging Facility at the UK-based University of Essex, was first asked to look at coral, his knee-jerk reaction was to use a laser scanning confocal microscope.
Fascinating structures emerged but as he puts it: "I could only look at a sample once, after that it would just be fried."
After tinkering with lower light levels and environmental control, he switched to widefield imaging and was soon watching coral polyps emerging from their protective limestone skeletons.
The results were better yet despite Laissue's best deconvolution efforts, he simply couldn't resolve the corals' intricate 3D structures.
"I realised that if I could combine the 3D [resolution] of a confocal microscope with the low light of widefield microscopy, then that that would give me the best of both worlds," he highlights. "And I knew the only way to do that was with light sheet microscopy."
At the time, commercial light-sheet set-ups and the open access light sheet microscopy platform, OpenSPIM - used to image model embryos - weren't compatible with Laissue's large coral fragments.
Reflectance image of coral skeleton. Scalebar 200 micron. [Laissue]
All options were too phototoxic and sample preparation for the open access selective plane illumination microscope involved mounting live embryos in low viscous media within polymer tubes. Clearly this was never going to work for reef-building corals.
"A coral sample needs to grow as if it was on the reef, so to accommodate this, sample size and photo-toxicity, I realised that I would have to build my own microscope," says Laissue, "As a biologist without a physics background, this was a little intimidating."
Still, using OpenSPIM as a starting point and drawing on many other open-source online projects, including Micro-Manager Open Source microscopy software, Laissue set to work.
"Given the size of the coral fragments, I knew that I needed a wide light sheet and eventually figured out that a rotating mirror would work for this," he says. "I remember climbing into a skip and gutting a laser printer to get that and then finding out it worked really well and gave a very wide light sheet."
Laissue soon received a Royal Society Industry Fellowship to develop a lightsheet illumination module, using funds to source optomechanical components, breadboards, lenses and more from Thorlabs and other suppliers.
As he puts it: "These funds gave me the ability to try out many different parts which was so very crucial, and prototyping with affordable components was such an important first step."
Left side: Using a conventional microscope (a Confocal Laser Scanning Microscope), a coral polyp is fully retracted due to the intense light. Right side: The polyp has fully emerged in the gentle illumination of a custom-made light-sheet fluorescence microscope. The scalebar shows about a hair’s breadth. [Nature Methods vol.14, 2017].
With his prototype module developed, Laissue joined forces with design engineers from UK-based manufacturer of scientific instruments, Cairn Research, to build what is now known as the Large Selective Plane Illuminator (L-SPI).
Recently commercially available, the module can operate with any microscope, single-mode fiber laser source, and scientific camera to enable light sheet imaging of large, unwieldy samples.
According to Laissue, instrument development has focused on specimen comfort, and as such, has a relatively 'chunky' lightsheet'.
Philippe Laissue working on the prototype version of the coral light-sheet microscope.
But as the researcher asserts, submicron resolution is achievable when water immersion objectives are used for detection.
"I have seen details within, for example, the endosymbiotic algae in coral tissue," he says. "I can also see the dynamics of the tissue structure itself as it attaches and detaches from the skeleton."
And as Laissue also highlights, working with Cairn Research design engineers made a huge difference to his light sheet microscopy development.
"Cairn has been great with the miniaturisation of components, reducing overall overhead and we have also worked with a further company to integrate very fast piezo-stages," he highlights. "Also, our biggest sample chamber is actually six by six by two centimetres in size, which is just huge for microscopy, and ideal for coral colonies."
But its not all about coral. Laissue spent last Summer at Woods Hole Oceanographic Institute, Massachusetts, US, investigating which other species can be imaged with the L-SPI.
Four-sided reflectance imaging of a bumblebee. The first LSPI illuminated from top and right (teal coloured), while the second imaged from bottom and left. Scalebar 1 mm. [Laissue]
As he points out, cephalopods are now the subject of widespread research, including RNA editing and neurology, and with this in mind, he and colleagues imaged a squid embryo in an eggshell.
"The whole embryo was left entirely intact; and for me it is so important to leave any sample as undisturbed as possible," he says.
Laissue and colleagues have also imaged photosenstive aquatic invertebrate bryozoans and are also looking at Arabidopsis and Nicotania plants. "Plants are very interesting but a real challenge as they are notoriously difficult to image dynamically," points out Laissue.
Reflectance imaging of ghost moth (Trioda sylvina) with short lamellate antennae and missing proboscis. [Laissue]
And the researcher is also excited about using the device as part of a fluorescence in situ hybridisation set-up to analyse river sediments.
"None of this is about phototoxicity but we can scan large areas with this a lot faster than we could with a spot scanning confocal [microscope]," he says. "This is the beauty of light sheet microscopy; it can be adapted in so many different ways."
But throughout light sheet microscopy development, reducing phototoxicity has remained at the forefront of Laissue's mind.
As he puts it: "At a very basic level I am fascinated by the interaction of light with living matter and what it can do to in the context of different cell types."
Indeed, as part of his live imaging-based studies of corals at the University of Essex's Coral Reef Research Unit, in the School for Biological Sciences, Laissue has endeavoured to minimise light-induced damage to samples during observations. And in July last year, he published a paper, 'Assessing phototoxicity in live fluorescence imaging', in Nature Methods.
As Laissue highlights, many conventional fluorescence microscopes use illumination at cell-damaging levels, which has led to inconsistent and even misleading results.
So to tackle this, his paper highlights the problem of phototoxicity in biology and provides guidelines to assessing photodamage.
Crucially, for the researcher, such publications raise awareness amongst biologists that light isn't harmless, causes photodamage to any cell under observation and is a serious constraint in live imaging and many conventional microscopy methods.
"Scientific journals are devoting more editorial space to the dangers of phototoxicity, and peer reviewers increasingly demand controls to ensure that the described behaviour is not chiefly photo-induced," he points out.
Large Selective Plane Illuminator from Cairn Research: The L-SPI is designed to operate with any microscope, single-mode fibre laser source or scientific camera.
Given the rising awareness of phototoxicity, Laissue now hopes his L-SPI, and light sheet microscopy in general, will be increasingly used to image biological samples.
"I think light sheet microscopy still has a way to go before it becomes widespread but I think it's going to get there," he says. "In twenty years' time, I believe you will find light sheet microscopes in many laboratories where you would have originally found confocal instruments."
And in the meantime, Laissue intends to continue studying corals using L-SPI. As he asserts, the method has allowed him to study the organisms under continuous illumination for twelve hours, without stress.
"I find these corals endlessly fascinating and can now look at them in a way that has never been done before," he says. "And if this also helps to raise awareness of our massively endangered coral reefs, then all the better."