In new work, the first microscope to concurrently monitor movement and neuron activity in zebrafish larvae without interfering with their behaviour has been developed by researchers in Germany and Portugal. To enable wider use in the research community, the neurobehavioural tracking microscope uses off-the-shelf components and is open source. The collaboration has demonstrated potential applications with studies of spontaneous behaviour and larvae response to stimuli including a neuroactive drug (Nature Methods 14 1079).

Called NeuBtracker, the fluorescence microscope is specifically designed to study zebrafish larvae, eyelash-length vertebrates that are widely used in neuroscience research. As genetic model organisms, they provide a simplified, special case of neuron behaviour, explained senior author Gil Westmeyer of Helmholtz Zentrum München and the Technical University of Munich.

"[In zebrafish larvae] we have a chance to understand how specific circuits of brain cells give rise to certain specific behaviours. It's very difficult to do that with more complex brains at this point in time," said Westmeyer.

Larvae transparency also means that optical techniques such as fluorescence microscopy can visualize any structure in the body, including the entire brain, without invasive procedures. In mice, in contrast, optical techniques can only access small sections of the brain. In a further advantage, drugs can be conveniently administered via the water that the zebrafish larvae are contained in. The larvae also have no blood-brain barrier, enabling easier administration of neuroactive compounds.

Simultaneous tracking

NeuBtracker works in combination with genetically-encoded sensors that target specific cells in the larvae and whose fluorescence is optically stimulated. In their study, the researchers used the GCaMP6s protein illuminated by a blue LED. The calcium sensor detects large spikes in the element that occur upon neuron activation.

The microscope tracks larvae behaviour and neuron activity using two cameras. An infrared camera with an extended, static field-of-view (FOV) locates the larva and tracks it as it moves. A second, scientific CMOS camera images the fluorescence in a smaller region-of-interest (ROI). A dynamic FOV follows the moving ROI like a spotlight, guided by the output of the infrared camera that controls a pair of galvanometric mirrors.

The dynamic FOV sidesteps the need to continually reposition the dish containing the larvae, as required by previous neuroimaging devices, thereby avoiding mechanical stimulation of the larvae. It also removes the need to restrain the larvae, enabling their natural behaviour to be observed.

Using NeuBtracker, first author Panagiotis Symvoulidis, Westmeyer and co-authors successfully imaged spontaneously swimming larvae and larvae exposed to olfactory, pharmacological and visible light and infrared stimuli.

Future developments

In separate work, the researchers incorporated a moving stage into the device. The adaptation is a step towards high-throughput automated drug testing, where individual larvae are positioned in multiple wells on a single assay plate. The plate is held stationary while the microscope moves above it, enabling sequential imaging of the larvae.

Drug screening studies of larvae using NeuBtracker could prove particularly valuable as an early step in drug development, enabling the formulation of hypotheses that can be tested in more complex organisms such as mice, Westmeyer told medicalphysicsweb.

Despite its name, NeuBtracker is not limited to neurological imaging in the brain. In future, it could also be used to image physiological systems and processes such as the cardiovascular and immune systems and metabolic processes.

"There are many fluorescent sensors, so anything that can be expressed in a zebrafish can potentially be studied with NeuBtracker," said Westmeyer.

In the long term, the researchers are keen to extend NeuBtracker to enable photoacoustic imaging (Optoacoustics sees neurons light up). In doing so, the greater penetration of photoacoustic imaging could be exploited to potentially image mice and larger, non-transparent adult zebrafish, making longitudinal studies from larvae to adulthood possible.