Think Small, Win Big
An image from the Nikon winning video of neurodevelopment in zebrafish embryos [Haynes/He/Nikon Small World]
In September this year, a video of zebrafish embryo growing its elaborate nervous system won the 2018 Nikon Small World in Motion competition.
Created by Dr Elizabeth Haynes, a biologist from the Halloran Lab, University of Wisconsin Madison, and microscopist, Jiaye "Henry" He, from the Huisken Lab, Morgridge Institute for Research, both from the US, the winning video reflects time lapse footage of 16 hours and uses custom light sheet microscopy to capture the entire zebrafish embryo in 3D, at a high temporal resolution.
Their collaboration began when the Haynes and colleagues from the Halloran Lab were looking for ways to capture the intricacies of zebrafish embryo growth over time.
Haynes knew light sheet microscopy could work well, and also knew of the Huisken Lab, which develops light sheet methods to image the early development of model organisms, including zebrafish.
"We had been doing CRISPR screens on zebrafish but really wanted to find a higher-throughput method of imaging," she says. "I had been following the Huisken Lab's microscopy work, heard they were coming to Madison and got very excited."
Dr Elizabeth Haynes from the Halloran Lab and Jiaye "Henry" He, from the Huisken Lab, work together to study zebrafish embryo development. [Haynes/He]
Haynes and He quickly joined forces, and set to work on embryo imaging using He's multidirectional selective plane illumination microscope (mSPIM).
Providing an evenly illuminated focal plane with a relatively thin light sheet, the instrument has a high axial resolution and can image entire embryos.
As He points out, light sheet microscopy, in general, is geared towards long-term imaging of live organisms.
"Compared to point scanning methods such as confocal microscopy, light sheet systems are more efficient in terms of optical sectioning and detection which means we don't over-illuminate a sample with the excitation laser," he says. "So our systems reduce phototoxicity and photobleaching... and I have yet to encounter a live sample that I’ve managed to photobleach during our time-lapse experiment."
"I am also really interested in image analysis guided microscopy, so I analyse the data coming from the microscope right away, and retune the instrument's parameters to take better images," he adds.
Henry He's multidirectional selective plane illumination microscope developed for imaging zebrafish. [Huisken Lab]
He's multidirectional plane illumination microscope is designed specifically to image zebrafish, and has a vertical configuration with a small water immersive chamber with a precisely controlled sample environment.
According to both Haynes and He, conventional gel-mounting techniques restrict embryo growth, yet this water chamber allows the zebrafish to develop 'naturally' and is just the right size for the sample to be maintained within the microscope's field of view.
"As a microscopist, I've always mounted samples in gel, as you don't want to have to hunt for it while imaging," points out He. "But it was only when working with biologists that I realised how damaging placing the sample in a soft gel can be."
Beauty and scale
Indeed, in the past, Haynes and colleagues have produced short ten minute confocal microscopy movies of microtubule and vesicle dynamics in cells within developing embryos mounted in gel.
However, as the biologist highlights, embryos don’t make large changes in body size or shape in such short time-frames, and longer time periods could restrict growth.
"Light sheet imaging, with the sample immersed in water, has changed this and our images have been beautiful," she says.
"We can get so much information from imaging the entire embryo and we have this incredible juxtaposition of scale," she adds. "During twenty hours of light sheet imaging we can see the individual axons and individual filopodia on growth cones yet we can also see the entire elongation of the body of the embryo... seeing everything in concert means we're now seeing detail that we never thought to look for before."
He concurs, saying: "When imaging in just water, nothing constrains the sample and you can see it curling up in a very natural way in the chamber."
"And it is the differential in scale that is jaw-droppingly beautiful... the entire sample is around 1mm in size but the smallest dynamic feature that we are resolving is of the order of hundreds of nanometres," he adds.
"I never cease to be delighted with watching embryos develop and seeing the kind of details that this microscopy brings." Elizabeth Haynes, Halloran Lab
So where next for the researchers? Haynes' research stems from a desire to better understand the kinesin light chain genes in zebrafish.
Kinesin comprises two heavy chains and two light chains, and transports cargo along microtubules. The heavy chain provides the motor activity while the light chain binds to the cargo.
“There are many genes in the kinesin light chain and their individual roles are poorly understood,” says Haynes. “If we can learn what changes in axon growth may occur when different kinesin light chain genes are perturbed, we can better understand their functions in the development of neurons, and their potential roles in neurodegenerative disorders such as Alzheimer's disease.”
2018 Nikon Small World in Motion Competition - First Place [Nikon]
Clearly, the latest collaboration and ensuing imagery can only help. He, for one, is convinced that the close collaboration between a microscopist and biologist, as is the case for himself and Haynes, has accelerated progress.
"This interdisciplinary collaboration means we've really been able to tune everything to make our optics and instruments function not only as expected but also as required for the biologist," he says.
However, the researchers' collaboration is far from over. He now intends to multiplex his microscope so Haynes can image many embryos sequentially, accelerating analysis.
Indeed, in the Nikon video, the researchers were only taking one 3D image a minute, a sufficient frequency to capture embryo development dynamics. However, capturing this single image took only seconds leaving the mSPIM to 'rest' for some 50 seconds before the next image was generated.
"We want to use this resting time and multiplex the imaging so we can image multiple samples, one after another, in these gaps," says He. "So we're now building a mid-throughput microscope that can image around 25 embryos sequentially at the same temporal resolution as our winning video, giving us a much higher throughput."
As part of this, the researchers will 3D print a custom zebrafish chamber that uses an aqueous media in which the embryo can still extend its body without any intrusive forces being applied.
And Haynes can barely wait. "I never cease to be delighted with watching embryos develop and seeing the kind of details that this microscopy brings," she says. "When you have the luxury of looking at everything, as we have here, then you start wondering, how exactly are those tiny details contributing to development?"
"We have so many questions that we want to ask now and I could probably work on projects stemming from this for years and years, and still not be done," she adds.